TITLE OF THE INVENTION

IMMUNE-REGULATORY LI-KEY PEPTIDE VACCINES FOR PROPHYLAXIS AND LONG-TERM PROTECTION AGAINST SARS-COV-2 INFECTION AND COVID-19 DISEASE

TECHNICAL FIELD

The instant invention relates to immune-regulatory compositions for preventing and treating viral infection and related disease in mammalian subjects. In specific aspects the invention relates to methods for regulating an immune response to prevent or treat infection by a respiratory virus, such as a coronavirus or influenza virus, including SARS-CoV-2, in human subjects.

BACKGROUND OF THE INVENTION

Coronaviruses are enveloped, positive-sense single-stranded RNA viruses that infect a wide range of avian and mammalian species, including humans. At least four human coronaviruses (hCoVs) are endemic, "common cold" viruses (HCoV-OC43, HCoV-229E, HCoV-HKUl and HCoV-NL63) that have circulated in human populations for at least centuries, causing only mild respiratory disease in most subjects.

Recent zoonotic outbreaks of "novel" coronaviruses have raised heightening alarms among epidemiologists worldwide. Three such outbreaks have caused high morbidity and fatality rates in human populations, often resulting in pneumonia-like symptoms referred to as "Severe Acute Respiratory Syndrome" (SARS), a type of Acute Respiratory Distress Syndrome (ARDS). The first SARS coronavirus, SARS-CoV, emerged in Guangdong China in 2002. A subsequent strain, MERS- CoV, appeared in Saudi Arabia in 2012. Recently, the SARS-CoV-2 ("COVID-19") coronavirus was identified in Wu Han City China in 2019.

SARS-CoV, MERS-CoV and SARS-CoV-2 all "jumped" to humans from other mammalian species within the last 20 years. Horseshoe bats are considered the primary reservoir for all of these novel coronaviruses, while the intermediate hosts who transmitted the virus to humans have been identified as the masked palm civet for SARS-CoV, the dromedary camel for MERS-CoV, and the Malayan pangolin for SARS-CoV-2 (Lam et al., 2020). The high pathogenicity and airborne transmissibility of SARS-CoV and MERS-CoV raised concerns about the potential for another coronavirus pandemic many years before the current SARS- CoV-2 ( COVID-19) pandemic struck. Despite these alarms, in many parts of the world preparations for the instant COVID-19 pandemic were sadly under-resourced. Since December 2019 the COVID- 19 outbreak has spread rapidly among human populations worldwide. The SARS-CoV-2 virus is extremely contagious via airborne transmission through expirated droplets, and to a lesser extent by contact transmission. Early during the COVID-19 pandemic it was determined that that the SARS- CoV-2 virus is productively shed and transmitted by pre-symptomatic and asymptomatic carriers. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. As of December 27, 2020, over 81 million cases of COVID-19 have been reported worldwide, with nearly 1 .8 million deaths among roughly 220 countries. The worst hit country is the United States, with over 19.5 million infections and 341,000 deaths.

COVID-19 disease is moderate or asymptomatic in a majority of infected persons, however about 20% of infected subjects develop severe symptoms. COVID-19 morbidity and mortality are particularly high among the elderly, immunocompromised and subjects with underlying pulmonary disease, heart disease, diabetes, cancer or other serious health conditions. The clinical course of COVID-19 pneumonia exhibits a broad spectrum of severity and progression. In some patients, shortness of breath (dyspnea) develops in a median of 8 days after the onset of illness (range of 5-13 days), while in others, respiratory distress is absent. Roughly 3-30% of patients require intensive care. Severely ill patients who present with shortness of breath and hypoxemia can rapidly progress to life- threatening ARDS, with the most severe cases (15-30% of hospitalized patients) developing pulmonary or multiple organ dysfunction or failure, sepsis, shock, vasculitis, thromboses, and/or coronary complications within a matter of days (Tu et al., 2020).

Severe pathogenesis in ARDS involves hyper-inflammation of the lungs mediated by massive infiltration and proliferation-of neutrophils and macrophages in the lung parenchyma. These inflammatory effector cells invade the pulmonary vasculature, alveolar septa and alveolar airspaces causing oxidative stress, proteolysis and phagocytic destruction of endothelial and epithelial barriers, with attendant vasculitis, hypercoagulation and lung fibrosis, among other inflammatory tissue and organ injuries. Other common sequelae of severe COVID-19 disease include a major decline in lymphocytes, especially natural killer (NK) cells, in the blood, and atrophy of the spleen and lymph nodes with declining lymphocytes in lymphoid organs. A distinct hyperinflammatory condition has been identified in a small subset of children infected with COVID-19, known as multisystem inflammatory syndrome in children (MISC). This Kawasaki-like disease is characterized by fever, rash, red eyes, dry or cracked mouth, redness in the palms of hands and soles of feet, and swollen glands, correlated with inflammation and swelling of blood vessels, attended in some cases by toxic shock syndrome (TSS). As of October 1 , 2020, the number of MISC cases in the US surpassed 1,000.

Excessive inflammation in COVID-19 disease is associated with so-called "cytokine storm syndrome" (CSS) (also known as "cytokine release syndrome" or CRS). CSS is characterized by an excessive, uncontrolled release of pro-inflammatory cytokines, chemokines and other inflammatory factors, leading to excessive and potentially life-threatening inflammation. CSS has previously been documented in a variety of infectious diseases, rheumatic diseases, autoimmune disorders and in certain subjects undergoing tumor immunotherapy.

CSS in the case of infection begins in a focal infected area, but can rapidly spread throughout the body. Severe COVID-19 patients with CSS exhibit elevated pro-inflammatory cytokine profiles, similar to observations for SARS-CoV and MERS subjects. Huang et al. (2020) report that a diverse array of pro-inflammatory cytokines and other inflammatory factors are elevated in patients with serious COVID-19 disease. Among 41 COVID-19 inpatients (13 ICU, 28 non-ICU), they observed increased levels of interleukin (IL)-1B, IL-1RA, IL-7, IL-8, IL-9, IL-10, fibroblast growth factor (FGF), granulocyte-macrophage colony stimulating factor (GM-CSF), IFNy, granulocyte-colony stimulating factor (G-CSF), interferon-y-inducible protein (IP10), monocyte chemoattractant protein (MCP1 ), macrophage inflammatory protein 1 alpha (MIP1A), platelet derived growth factor (PDGF), tumor necrosis factor (TNFa), and vascular endothelial growth factor (VEGF). In ICU patients, levels oflL-2, IL-7, IL-10, G-CSF, 1P10, MCP1 , MIP1A, TNFa were higher than in the non-ICU patients (Huang et al., 2020; Conti et al., 2020],

Inflammation is ordinarily an adaptive response, evolved to combat injury and defend against foreign substances and pathogens. However, hyperinflammation as in CSS and ARDS can be extremely harmful. Cytokine induction of macrophages and neutrophils appears to play a central pathogenic role in COVID-19-related hyperinflammation. Pro- inflammatory cytokines such as interleukin-1 (IL-1) play a fundamental role in tissue inflammation and fibrosis. IL-1 activates macrophages to perform phagocytosis on infected cells, and these activated cells in turn release additional inflammatory cytokines. Other pro-inflammatory cytokines implicated in clinical progression of COVID-19 disease include interferon-alpha (IFNa), tumor necrosis factor (TNF), IL-8, IL-6 and others (Ho et al., 2003; Auyeung et al., 2005; Chousterman et al., 2017; Chen et al, 2006).

The COVID-19 pandemic, with its high attendant morbidity and fatality and dire economic impacts, has created an urgent need for innovative prophylactic and therapeutic tools to prevent and treat SARS-CoV-2 infection and COVID-19 disease. Efforts to develop useful vaccines and therapeutics against SARS-CoV-2 have been frustrated by the novelty and complexity of the viral genome, and its complex, unpredictable etiology. SARS-CoV, MERS-CoV, and SARS-CoV-2 all have large single-stranded, positive-sense RNA genomes (27-32 kb, encoding 6-10 genes). The structural proteins include spike (S), envelope (E), and membrane (M) proteins, making up the viral coat, and the nucleocapsid (N) protein that packages the viral genome (all translated from subgenomic RNAs, some undergoing glycosylation in the host Golgi apparatus to form glycoproteins). A replication- and transcription-related gene is translated into two large non-structural polyproteins by overlapping open reading frames (ORFs) translated by ribosomal frameshifting.

Current vaccine candidates against SARS-CoV-2 focus primarily on the spike (S) glycoprotein, which mediates binding and fusion of the virus to host cells to permit intracellular colonization. The S protein is primed by a host serine protease and is recognized by host cellular receptors (angiotensin-converting enzyme-2 (ACE2) receptors for both SARS-CoV and SARS-CoV-2 viruses, dipeptidyl peptidase 4 (DPP4) for MERS-CoV). The varied etiology and epidemiology of SARS coronaviruses relates in part to differences between individuals and populations in ACE2 receptor expression levels, and possibly also to ACE2 structural differences between individuals. Children and younger individuals generally express lower levels of ACE2 receptor, which may contribute to their relative resistance to severe COVID-19 disease. Several ACE2 genetic variants have been identified in human populations that may further impact viral infection and pathogenicity. However, no significant variation has been found among cognate ACE2 residues recognized by the SARS-CoV-2 S protein, indicating the virus exploits a highly-conserved attach ment/entry site, which correlates with the rapid spread of SARS-CoV-2 across continents and among genetically diverse human populations (Cao et al., 2020).

Antigenicity of SARS-CoV proteins for vaccine development varies, depending on the quantity and antigenicity of epitopes within the respective proteins. SARS-CoV structural proteins (spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins) possess higher immunogenicity to elicit T cell responses than nonstructural proteins (Li et al., 2008). The S and N proteins exhibit high immunogenicity to engage both humoral and cellular responses (Bucholz et al., 2004; He et al., 2006). The M and E proteins are also significant structural proteins, anchoring on the envelope membrane surface of viral particles (Armstrong et al., 1984). The M protein comprises a transmembrane glycoprotein composed of a triple-membrane domain spanning about a third of the protein. In SARS-CoV the M protein is the most abundant structural protein in the assembled virion and plays a significant antigenic role in mediating humoral responses (i.e., efficient neutralizing antibody responses) (Pang et al., 2004). The E protein is a small integral membrane polypeptide that forms an ion channel (Pervushin et al., 2009). Inactivation of the E protein results in attenuated virulence through impairment of virion morphology or tropism (DeDiego et al., 2007; 2008).

Cellular immunity also represents an important aspect of immune protection against SARS coronaviruses. Screening of HLA-A*0201 restricted CTL epitopes elicited by SARS-CoV in convalescent subjects has identified immunodominant epitopes from the S protein and the transmembrane domain of the M protein restricted by HLA-A*0201 (the most common allele among HLA-A2 subtypes) (Zhou et al., 2006; Liu et al., 2010). Related CTL epitope mapping studies for the SARS-CoV M protein revealed 2 CTL epitopes that elicit strong responses in enzyme-linked immunospot (ELISpot) assays, and in human leukocyte antigen (HLA) tetramer staining of peripheral blood mononuclear cells (PBMCs) from SARS-CoV convalescent subjects, indicating the M protein is a dominant immunogen for both humoral and cellular immune responses.

Numerous concerns have been raised about the general efficacy and safety risks for vaccines and immuno-therapeutics directed against SARS coronaviruses. Conventional vaccines may contribute to adverse inflammatory responses and pose risks for Antibody Dependent Enhancement (ADE) (discussed in detail below). Excessive activation-induced cell death of lymphocytes is another troubling complication of SARS-CoV infection, that might be exacerbated by certain immune- stimulatory treatments. Lymphocytopenia is a prominent diagnostic marker for severe COVID-19 disease. Both T cells and NK cells in patients with severe COVID-19 are seriously reduced, while in critically ill patients, NK cells may be undetectable and memory helper T cells and regulatory T cells profoundly decreased (Hui et al., 2019). Striking COVID-19 autopsy findings reveal that secondary lymphoid tissues are destroyed in severe cases. Spleen atrophy is also commonly observed, correlated with decreased lymphocytes, significant cell degeneration, focal hemorrhagic necrosis, macrophage proliferation and macrophage phagocytosis in the spleen. Similarly, lymph node atrophy and reduced numbers of lymph nodes are observed, along with decreased numbers of CD4 + T cells and CD8 + T cells in the spleen and lymph nodes (Hui et al., 2019). In the lung, diffuse alveolar damage (DAD) is associated with massive infiltration of monocytes and macrophages, moderate levels of multinucleated giant cells, and very few lymphocytes. Most infiltrating lymphocytes found in the lungs are CD4 + T cells. Contributing to this etiological puzzle, COVID-19 virus inclusion bodies are detected in alveolar epithelia and macrophages, even when PCR tests are negative in blood or throat swabs (Zu et al., 2020; De Wit et al., 2016; Chan et al., 2015B). These findings suggest a "primary cytokine storm" induced by viral infection, mainly elicited by alveolar macrophages, epithelial cells and endothelial cells, rather than a "secondary cytokine storm" induced by activated T lymphocytes (Chan et al., 2012; Kanne ct al., 2020). In view of these complex and challenging circumstances, finding optimal targets and resolving safety concerns for T cell-engaging COVID-19 vaccines and immune-therapeutics remain uncertain.

Neutrophils are key pathogenic effectors in COVID-19 disease. Neutrophil numbers and activity are directly associated with CSS and ARDS severity. Neutrophils are the most common type of white blood cell (WBC) in the bloodstream. They are phagocytes which migrate from the blood during the acute phase of inflammation to sites of injury or infection. Neutrophils freely move by chemotaxis from the outset of an infection into and through blood vessels and interstitial compartments, attracted by chemokines (such as Interleukin-8 (IL-8), C5a, fMLP, Leukotriene B4, and H2O2) expressed by activated endothelial cells, mast cells, and macrophages. Once localized, neutrophils express and release additional cytokines, amplifying inflammatory responses by recruitment and activation of other inflammatory cells. Neutrophils considerably outnumber monocyte/macrophage phagocytes, and are generally regarded as the hallmark of early, acute inflammation. Severe ARDS pathogenesis in COVID-19 subjects is marked by massive infiltration of neutrophils into pulmonary capillaries, alveolar septa and alveolar airspaces, coupled with major destructive changes associated with neutrophil activity.

Neutrophil activity is ordinarily beneficial, when properly directed and attenuated. In addition to recruiting and activating other cells of the immune-inflammatory system, neutrophils play a key role in front-line defense against pathogens. Neutrophils have three methods for directly attacking viral and bacterial pathogens: phagocytosis (ingestion), degranulation (release of soluble anti- microbials), and generation of neutrophil extracellular traps (NETs). Neutrophil degranulation releases an assortment of proteins from three distinct types of granules. Azurophilic granules (or "primary granules") release myeloperoxidase, bactericidal/permeability-increasing protein (BP1), defensins, and the serine proteases neutrophil elastase and cathepsin G. Specific granules (or "secondary granules") release alkaline phosphatase, lysozyme, NADPH oxidase, collagenase, lactoferrin, histaminase, and cathelicidin. Tertiary granules release cathepsin, gelatinase, and collagenase.

When neutrophils are overactivated, as in COVID-19 disease, these various enzymes and antimicrobial agents can cause serious damage to tissues and extracellular components, leading to destruction of essential "barrier" components present in blood vessels, lungs, kidneys and other organs, disrupting their structural integrity, pathogenic resistance and normal function. Another pathogenic effect of excessive neutrophil infiltration and activation in COVID-19 disease involves the generation of neutrophil extracellular traps (NETs). NETs are networks of extracellular fibers composed of DNA containing histones and granule-derived enzymes, such as myeloperoxidase (MPO) and elastase (Brinkmann et al., 2004). The process of NET formation by neutrophils (NETosis) has been widely studied. The process starts with neutrophil activation by pattern recognition receptors (PRR) or chemokines, followed by reactive oxygen species (ROS) production and calcium mobilization, which leads to the activation of protein arginine deiminase 4 (PAD-4), an intracellular enzyme involved in the deimination of arginine residues on histones (Li et al., 2010). Ordinarily NETosis mediates capture and killing of pathogens; however, increasing evidence has shown that NETs arc implicated directly in pathogenesis of several diseases (Brinkmann et al., 2004; Colon et al.,

2019). Various studies describe a causal role for NETs in tissue damage to vital organs during sepsis (Colon et al., 2019; Czaikoski et al., 2016; Kambas et al., 2012). During experimental and clinical sepsis, NETs are found in high concentrations in the blood, and are positively correlated with biomarkers of vital organ injuries and sepsis severity. Disruption or inhibition of NETs using recombinant human DNase (rhDNase) or PAD-4 inhibitors markedly reduces organ damage, especially in the lungs, and increases survival of severe septic mice (Colon et al., 2019). NETosis is now a well-documented correlate of severe COVID-19 pathology, associated with CSS (Mehta et al.,

2020), ARDS (Lai et al., 2020) and microthrombosis (Magro et al., 2020; Dolhnikoff et al., 2020). Vasculitis and thrombosis coupled with endothelial damage are also prominent features in severe COVID-19 patients. Many critical ill COVID-19 patients have vasculitis, often with gangrene at the extremities. Autopsies reveal that pulmonary blood vessels associated with the alveolar septa are congested and edematous, with infiltration of monocytes and their macrophage progeny within and around the blood vessels. Small vessels show hyperplasia, vessel wall thickening, lumen stenosis, occlusion and focal hemorrhage. Hyaline thrombi of microvessels are found in severe cases (Zu et al., 2020; Hui et al., 2019; Chan et al., 20I 5B). Vascular and alveolar damage appear to result in part from direct infection and injury of epithelial and endothelial cells by the virus, as well as from hyper- inflammatory pathogenic impacts of neutrophils and macrophages.

Severe COVID-19 patients frequently exhibit hypercoagulation, as is common in childhood cases of multisystem inflammatory syndrome in children (MISC). Common sequelae of these conditions include prolonged prothrombin time and elevated levels of D-dimer and fibrinogen. Many of the most severe COVID-19 patients (71.4% of non-survivors) develop overt Disseminated Intravascular Coagulation (D1C) (Tang et al. 2020). A high proportion of acro-ischemia is also observed in deteriorating patients with COVID-19, indicating a hypercoagulable status before the onset of overt DIC. Several factors contribute to coagulation disorders in COVID-19 patients. Persistent inflammation in severe and critical COVID-19 patients acts as an important trigger for the coagulation cascade. Certain cytokines, including IL-6, activate the coagulation system and suppress the fibrinolytic system. In the setting of COVID-19, pulmonary and peripheral endothelial injury is also a primary inducer of hypercoagulation. Undoubtedly, hyperinflammatory activities of neutrophils and macrophages contribute to endothelial as well as epithelial injury. Endothelial cell damage strongly activates the coagulation system via exposure of tissue factor and other pathways. Over- aggressive immune and inflammatory responses may in turn be exacerbated by dysfunctional coagulation, with these processes acting in a sort of feedback loop toward an uncontrolled endpoint. Emergence of antiphospholipid antibodies in COVID-19 patients also appears to intensity coagulopathy (anti-cardiolipin and anti-β2GPl antibodies have been detected in COVID-19 patients) (Zhang et al., 2020).

Viruses and bacteria may also directly attack and destroy elements of the host immune system or inflammatory control machinery. As noted above, severe COVID-19 disease is marked by profound reductions in numbers of lymphocytes, though it is uncertain whether NK cells and other lymphocytes can be directly invaded and destroyed by SARS-CoV viruses. Since both SARS-CoV and SARS-CoV-2 appear to principally infect cells via the angiotensin converting enzyme 2 (ACE2) receptor (apparently absent on lymphocytes), these essential immune cells are likely falling victim to CSS destructive mechanisms, particularly activation-induced cell death.

A variety of anti-inflammatory medications have been proposed as therapeutic candidates for COVID-19 treatment, for example non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, among others. Also contemplated are immunosuppressants, pro-inflammatory cytokine antagonists (such as IL- 6R monoclonal antibodies, TNF inhibitors, IL-1 antagonists, janus kinase (JAK) inhibitors, etc.) and other immunomodulatory agents to reduce pulmonary and systemic inflammation. Despite the large armamentarium of available anti-inflammatory agents, none of these are without uncertainties regarding their efficacy and potential for adverse side effects in the contexts ofCOVID-19 disease. Anti-inflammatory therapy for COVID-19 disease patients presents fundamental risk/benefit concerns in terms of whether and when to treat subjects with an anti- inflammatory regimen. These fundamental questions remain under intense debate, with no consensus in sight. A principal concern is that anti-inflammatory medications may delay or impair beneficial immune anti-viral defenses, and/or concurrently increase risk of secondary infection, particularly in subjects facing pre-existing immune system impairment, or impairment mediated by the virus itself. Other questions arise in the case of biological agents targeting pro-inflammatory cytokines, which may only inhibit a narrow range of inflammatory factors and fail to curb CSS generally. Alternatively, anti-inflammatory drugs may act too broadly and impair beneficial immune functions. For example. JAK inhibitors may exert potent anti-inflammatory effects, while at the same time impairing crucial immune mechanisms mediated by INF-a. Yet another fundamental confounding question relates to the optimal time window for anti-inflammatory treatment, which may be critical in COVID-19 disease. Severe patients often show an extended period of moderate symptoms, followed by abrupt deterioration 1 2 .weeks after symptom onset, after which anti-inflammatory therapy may be unable to achieve a favorable response.

SARS-CoV-2 shows a tropism for, and actively replicates in, the upper respiratory tissues (Wolfel et al., 2020). Like SARS-CoV, SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its main receptor for cellular entry, which is broadly expressed in vascular endothelium, respiratory epithelium, and at least some alveolar monocytes and macrophages (Lu et al., 2020). Tropism for upper respiratory tissue facilitates the extraordinary transmissivity of SARS-CoV-2, via continuous pharyngeal shedding of the virus even when symptoms are minimal and restricted to the upper respiratory tract. Later in the disease course, SARS-CoV-2 resembles SARS-CoV in terms of viral replication advancing to the lower respiratory tract, followed by extensive attack in severe cases against the lungs and other target organs that express ACE2 (including heart, kidney, gastrointestinal tract and distal vasculature). The extent and duration of viral spreading correlates with clinical deterioration, mainly occurring in the second week following disease onset. However, disease progression in severe cases is not solely attributable to direct viral spread and damage, but additionally involves immune-mediated inflammatory injury induced by the virus. The two distinctive features of severe and critical patients during this stage are progressive increase of inflammation, and hypercoagulation.

There is no doubt that immune-mediated inflammation plays a key role in severe SARS-CoV-2 pathogenesis, as has been shown for SARS-CoV. The progression ofCOVID-19 involves a continuous decrease in lymphocyte count, with significant elevation, infiltration and hyperactivation of neutrophils, coupled with a broad elevation of inflammatory markers (including C-reactive protein, ferritin, interleukin (lL)-6, IP-10, MCP1 , MIP1A, and TNFa). Reduced lymphocyte count and elevated levels of ferritin, IL-6 and D-dimer were reported in various studies to be directly associated with increased mortality ofCOVID-19 patients. Mechanisms underlying progressive lymphocytopenia in severe and critical COVID-19 patients remain unclear, though decreases in B cells, T cells, and natural killer (NK) cells are all more prominent in severe cases. Other studies have reported increased levels of CD8+ T-cell activation (measured by proportions of CD38 and HLA-DR expression) despite a reduction in CD8+ T-cell count. Lymphopenia was also an important feature of SARS-CoV-2 disease progression, and declines of both CD4+ and CD8+ T lymphocytes correlate with pathogenic radiographic changes. ACE2 levels in infected cells and tissues of COVID-19 patients are correlated with severity of ARDS, and are directly associated with hyper-elevation of pro-inflammatory cytokines mediating CSS and ARDS. Although neutrophils do not appear to express ACE2, and likewise a majority of activated macrophages in COVID-19 diseased lungs are ACE2-negative, the role of ACE2 in COVID-19 hyper- inflammation is clear. Studies by Li et al. (2020) show that ACE2 is directly involved in viral- mediated dysregulation of host immune responses, cytokine expression and inflammation. More specifically, Li and colleagues report that high expression of ACE2 correlates with intensity of both innate and adaptive immune responses, B cell regulation and cytokine secretion, as well as hyper- inflammatory responses induced by IL-1 , IL- 10, IL-6 and IL-8. Thus, immune and inflammatory dysfunction that mediates CSS and ARDS is broadly linked to high expression of ACE2. Additionally, high expression of ACE2 correlates with increased expression of genes involved in viral replication, and alteration of the transcriptome of SARS-CoV-2-infected epithelial cells (enhancing viral entry, replication and assembly). T cell activation and inflammatory responses mediated by T cells are also induced by SARS-CoV-2 alteration of the transcriptome in infected cells. Increased levels ofIL1β, IFN-γ, IP 10, and MCP1 in patients infected SARS-CoV-2 appear linked to the hyper-activation and exhaustion of T-helper- 1 (Th1) cell responses. ACE2 also indirectly mediates activation of neutrophils. NK cells, Th17 cells, Th2 cells, dendritic cells and TNFa secreting cells, contributing to CSS and ARDS. High ACE2 expression in pulmonary tissue correlates with levels of cytotoxic activation of macrophages, and with neutrophil inflammation, along with bias toward a Th2- dominated immune response (Li et al., 2020). Intriguingly, ACE2 expression is progressively upregulalcd during SARS-CoV infection.

Direct infection of lymphocytes by SARS-CoV has been reported, however rapid reduction of lymphocyte counts in COVID-19 patients is primarily attributed to two mechanisms; redistribution of circulating lymphocytes, and depletion of lymphocytes through activation-induced apoptosis or pyroptosis. No viral gene expression has been detected in peripheral blood mononuclear cells (PBMCs) of patients with COVID-19, and the normal viral transporter, ACE2, is evidently not expressed on these cells. Wang et al. (2020) suggest that T lymphocytes may be more permissive to SARS-CoV-2 than to SARS-CoV, through ACE2-independent endocytosis. Other reports suggest upregulation of apoptosis, autophagy, and p53 pathways in PBMCs of COVID-19 patients (Xiong et al.. 2020). while others report that NK and CD8+ T cells are exhausted in COVID-19 patients through overexpression of NK.G2A (Zheng et al., 2020).

The foregoing discussion reveals that complex immune and inflammatory dysregulation begins very early during COVID-19 disease. The clinical course of SARS-CoV-2 infection may be conceptually divided into three phases: viremia phase, acute phase (pneumonia phase) and severe or recovery phase. Patients with competent immune systems, without serious risk factors (old age, major co-morbidities, etc.) may generate effective and adequate immune responses to suppress the virus in the first or second phase, without manifesting immune dysfunction and hyper-inflammation symptoms of CSS, ARDS or MISC. In contrast, patients with immune dysfunction or other risk factors may fail the initial phase and progress to severe or critical disease. Certain pathogenic changes ofCOVID-19 may be preventable or reversible through timely intervention, particularly in mild and moderate cases, however the complexity and refractory nature of this disease presents many obstacles to safe and effective use of therapeutics, heightening the importance of vaccine development.

Studying the original SARS-CoV coronavirus, Zhao et al. (2008) reported that the viral nucleocapsid (N) protein potentiates TGF-β to mediate hyper-inflammation, while disabling a critical activity of TGF-p for inducing apoptosis in mature immune and inflammatory ceils. The SARS-CoV N protein specifically induces expression of plasminogen activator inhibitor-1, while suppressing Smad3/Smad4-mediated apoptosis of human peripheral lung epithelial (HPL) cells. The hyper- activating effects of the SARS-CoV N protein on TGF-β inflammatory cascades is Smad3-specific. N protein associates with Smad3 and promotes Smad3-p300 complex formation while interfering with complex formation between Smad3 and Smad4 (Zhao et al., 2008). These findings indicate that SARS coronavirus N proteins can activate certain pathways and mechanisms of TGF-β -mediated hyper- inflammation, while at the same time blocking pro-apoptotic TGF-β activation, resulting in lifespan prolongation and hyper-activation of pathogenic inflammatory host cells. This TGF-β re- programming mechanism is believed to contribute to the extreme elevation in numbers of activated macrophages and neutrophils in the lungs ofCOVID-19 subjects presenting with severe CSS and ARDS. Whereas activated macrophages and neutrophils ordinarily undergo cell death or apoptosis after performing their anti-viral functions (phagocytosis and degranulation), SARS-CoV-2 apparently hyper-act ivates and extends the lifespan of these cells through N-protein suppression of TGF-p- induced apoptosis.

It is not unprecedented that SARS coronaviruses misappropriate and reprogram normally beneficial inflammatory cells and processes in COVID-19 disease. There are many examples of molecular strategies implemented by viruses and bacteria to disable, misdirect and appropriate host immune and inflammatory processes to benefit the pathogen. In the case of neutrophils, molecular interactions between these key inflammatory effector cells and pathogens can profoundly alter neutrophil proliferation and longevity. Both viral and bacterial pathogens are capable of either accelerating neutrophil lysis after phagocytosis, or prolonging neutrophil lifespan. Chlamydia pneumoniae and Neisseria gonorrhoeae have both been shown to prolong neutrophil lifespan by inhibiting neutrophil apoptosis. Other pathogens prolong neutrophil lifespan both by blocking apoptosis and by inhibiting phagocytosis-induced cell death (PICD). On the other end of the spectrum, certain pathogens redirect neutrophil cell fate after phagocytosis by accelerating cell lysis and/or apoptosis, contributing to tissue necrosis.

It is now clear that SARS-CoV viruses dysregulate normally beneficial immune-inflammatory functions of neutrophils, causing these critical cells to be destructively miscued and overactivated. Elevated levels of neutrophils predict worse outcomes in COVID-19, and the role ofNETs appears particularly critical. In one recent study, Zuo and coworkers (2020) reported that increased infiltration of neutrophils into capillaries of the lungs and overexpression ofNETs correlated strongly with severity of viral pneumonia/ARDS in COVID-19 patients. Sera from patients with severe COVID-19 exhibit elevated levels of cell-free DNA, myeloperoxidase (MPO)-DNA, and citrullinated histone H3 (Cit-H3). indicating elevated NET levels. Cell-free DNA levels also correlated strongly with acute inflammatory phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels and observed prothrombic (blood clot forming) effects ofNETs. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation than those capable of breathing unassisted. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. These data reveal that high levels of neutrophils and NETs in patients with severe COVID-19 likely contribute significantly to CSS, ARDS and attendant vasculitis and thrombus formation in these patients.

Other reports show more generally that macrophages and neutrophils are both readily infected by COVID-19. It is increasingly apparent that these invasive migratory cells serve as a "trojan horse" vector to convey the virus to vulnerable tissues and organs, where these cells mediate inflammatory and pathogenic effects that fundamentally increase viral replication and spread. The SARS-CoV-2 virus infects monocytes/macrophages directly, then suppresses the anti-viral Interferon (1FN) response of these cells. Since macrophages and dendritic cells (DCs) act as antigen presenting cells (APCs), infection and IFN-impairment of these cells by SARS-CoV-2 impairs host adaptive immune responses against the virus. At the same time. SARS-CoV-2-infected monocytes/macrophages produce large amounts of pro-inflammatory cytokines and chemokines, contributing to local tissue inflammation and CSS. The destructive impacts of neutrophils (likely infected, activated and immortalized by the SARS-CoV-2 virus), are even more fundamental to disease severity and progression. SARS-CoV-2 infection of macrophages and neutrophils results in congestion and impairment of the host's circulation, and degradation of epithelial and endothelial barriers and alveolar compartments (through vasculitis, pulmonary fibrosis, NET deposition, and thrombosis). In severe cases, these effects culminate as ARDS, attended by extreme pulmonary pathogenesis, blockade of circulation and gas exchange, hypoxemia and eventual lung/heart failure. These severe, highly morbid and often fatal, impacts are consistent with a novel (zoonotic) generalist virus, as opposed to the attenuated impacts of a specialized virus long-coevolved with its host.

To properly assess immunological management strategies for COVID-19, both innate and adaptive immunity processes must be considered. Innate immune responses against viral infections rely heavily on interferon (IFN) type I and its downstream cascade, controlling viral replication and inducing the adaptive immune response. To mount this primary response, innate immune cells must recognize invasion by the virus through detection of pathogen-associated molecular patterns (PAMPs). For RNA viruses such as coronavirus, PAMPs include genomic RNA and replication intermediates recognized by endosomal RNA receptors, TLR3 and TLR7 and the cytosolic RNA sensor, RIG-I/ MDA5. This leads to activation of a downstream signaling cascade mediated by NF-KB and IRF3. In the nucleus these transcription factors induce expression of type I IFN and other pro-inflammatory cytokines. Type I IFN activates the JAK-STAT pathway, whereafter J AK1 and TYK2 kinases phosphorylate STAT1 and STAT2. STAT1 and STAT2 form a complex with IRF9, which is translocated to the nucleus to initiate transcription of IFN-stimulated genes (ISGs) under the control of IFN-stimulated response clement (ISRE)-containing promoters. Ordinarily, this type I IFN innate response is able to suppress viral replication and dissemination at an early stage in healthy individuals. However, in both SARS-CoV and MERS-CoV, type I IFN responses to viral infection are suppressed. Both SARS-CoV and MERS-CoV employ multiple strategies to interfere with type I IFN production and signaling, in a manner closely associated with disease severity (Channappanavar et al., 2017). SARS-CoV impairs type I IFN induction by interfering with downstream RNA sensors directly or indirectly, for example by degrading RNA sensor adaptor molecules MAVS and TRAF3/6 and inhibiting IRF3 nuclear translocation (Kindler et al., 2016). MERS-CoV utilizes similar strategies and further mediates repressive histone modification (Kindler et al., 2016). Once type I IFN is secreted, both SARS-CoV and MERS coronaviruses are able to inhibit downstream IFN signaling by decreasing STATI phosphorylation (De Wit 2016). Viral proteins involved in this modulation type I IFN innate responses include both structural proteins (such as M and N) and non-structural (ORF) proteins.

In severe cases of SARS-CoV and MERS-CoV. delayed or impaired type I IFN response compromises early viral control and contributes to hypcrinflammatory pathogenesis. Successful viral repl ication mediates hyperproduction of type 1 IFN, causing excessive recruitment, activation and pro- inflammatory cytokine production by neutrophils and macrophages. Emerging data for SARS-CoV-2 reveal that innate immune responses play a critical role in determining protective versus destructive host responses. SARS-CoV-2 appears to induce delayed type I IFN response and attendant loss of viral control in early infection, overwhelming at-risk individuals with underlying diseases such as diabetes and cardiovascular disease. The observed resistance of children to severe COVID-19 disease is believed to be attributable in part to their ordinarily very strong innate immune function.

Adaptive immune responses against COVID-19 are especially critical to determining safe and effective vaccine strategies. Generally, Th1 type T cell immune responses play a dominant role in adaptive immunity against viral infection. The cytokine microenvironment generated by antigen presenting cells dictates the direction of T cell responses (i.e., ThI versus Th2). Helper T cells orchestrate the overall adaptive response, while cytotoxic T cells are important for direct killing of virus-infected cells. Humoral immune responses, especially production of neutralizing antibodies, play a protective role by limiting infection at later phases and preventing future reinfection.

SARS-CoV infection induces seroconversion as early as day 4 after onset of disease, and is detectable in most patients within I d days. Long lasting specific IgG and neutralizing antibodies have been reported up to 2 years after infection in the case of SARS-CoV (Liu et al., 2006). MERS-CoV seroconversion is seen by the second or third week of disease onset. For both SARS-CoV and MERS- CoV. delayed and weak antibody responses are correlated with severe outcomes (Liu et al., 2017). Comparatively limited serology details are known for SARS-CoV-2. In one study, a patient showed peak specific IgM at day 9 after disease onset, and switched to IgG by week 2 (Zhou et el., 2020). Interestingly, sera from 5 patients with COVID-19 showed significant cross-reactivity with SARS- CoV. but reportedly not against other coronaviruses. Sera from all five patients were able to neutralize SARS-CoV-2 in in vitro plaque assays, suggesting all had successful humoral responses (Zhou et al., 2020). Yet to be determined is the critical question of how antibody kinetics (specificity) and titer may correlate with disease severity and risks of reinfection and ADE.

T cell responses in SARS-CoV infection have been extensively investigated. One study using 128 convalescent samples reported that CD8+ T cell responses are more frequent, with greater magnitude, than CD4+ T cell responses. Virus specific T cells from subjects with severe disease tended to be central memory type, with a significantly higher frequency of polyfunctional CD4+ T cells (expressing IFNy, TNFa, and IL-2) and CD8+ T cells (expressing IFNy, TNFa and degranulated state), compared to those of mild-moderate disease subjects. Strong T cell responses correlated significantly with higher neutralizing antibody, while elevated serum Th2 cytokines (IL-4, IL-5, IL- 10) corresponded to more severe outcomes (Li et al., 2008). In MERS-CoV studies, 70% of T cell responses were directed against structural proteins (spike, envelope, membrane, and nucleocapsid). An early rise of CD8+ T cells correlated with disease severity, and at the convalescent phase a dominance ofTh1 helper T cells was observed (Shin et al., 2019). In an animal model, memory CD4+ T cells specific for a conserved epitope between SARS-CoV and MERS- CoV protected against lethal challenge from both viruses (Zhao et al., 2016). Cumulative evidence therefore suggests that Th1 type T cell responses arc important to successful control of SARS-CoV and MERS- CoV, and likely also for SARS-CoV-2, while excessive CD8+ T cell responses may be associated with lung pathogenesis.

More recent studies on SARS-CoV-2 clarify the roles of T cell responses in clearing virus and determining the course and severity of COVID-19 disease. T cell responses against SARS-CoV-2 are presumptively initiated by respiratory professional antigen-presenting cells (APCs) that engulf and process viral antigens, as shown for SARS-CoV. Because production and activity of type 1 interferons (IFN-ls) that induce and enhance antiviral T cell responses is suppressed in SARS-CoV-2 infection, especially in severe cases (Blanco Melo et al., 2020; Fladjadj et al.. 2020), impaired or dysregulated T cell immunity is predicted to accompany severe SARS-CoV-2 infection, as seen in SARS-CoV-1 infection (Channappanavar et al.. 2016). Evidence indicates that dysregulation leading to hyper- activation of antigen-specific T cells elicited by SARS-CoV-2 contributes significantly to hyper- immune-inflammatory pathogenesis in severe cases (Tay et al., 2020; Anft et al., 2020). Blocking this T cell dysregulation is the objective of current, successful treatment of severe COVID-19 subjects with the immunosuppressant dexamethasone (Horby et al.. 2020). Recent data for SARS-CoV-2 reveal that patients recovered from COVID-19 develop virus- neutralizing anti-S immunoglobulin (Ig) titers (Nisreen et al., 2019). Since T cell help is required to generate high-affinity IgG antibodies, S protein-reactive T cell immunity must also be well developed in these convalescent patients (Sette et al., 2008; Bachmann et al., 1997). Further studies reveal that SARS-CoV-2 S protein-reactive T cell responses are present in a diverse array of patients with moderate, severe, and critical COVID-19 disease (Anft et al., 2020; Braun et al., 2020). Significantly, SARS-CoV-2 S-protein- reactive CD4+ T cells increase with disease progression (Weiskopf et al., 2020). These findings correlate with prior reports for Dengue virus, which show that dominant T cell responses toward viral antigens are associated with immunopathogenesis in severe cases (Duangchinda et al.. 2020). For COVID-19, it has been proposed that anti-SARS-CoV-2 cell T cell responses may be more predictive of severe outcomes than antibody testing (Sekine et al., 2020; Gal lais et al., 2020), however much research remains to be done in order to clarify these relationships. COVID-19 T cell immunity was explored further in a recent study by Thieme et al. (2020). T cell responses against SARS-CoV-2 proteins (S, M, and N proteins) were evaluated in COVID-19 patients suffering a range of clinical manifestations during acute disease and at recovery. Peripheral blood mononuclear cells (PBMCs) from subjects classified with moderate, severe, recovered critical, and deceased critical COVID-19 disease were analyzed and compared to unexposed PBMC samples (collected and preserved pre-COVID-19). The PBMC samples were stimulated with overlapping peptide pools (OPPs) spanning the SARS-CoV-2 S, M, and N proteins, and antigen-reactive T cell responses were detected by intracellular staining with flow cytometry. Activation markers were CD154 and CD137 for CD4+ T cells and CD 137 in combination with interleukin (IL)-2, IFN-g, tumor necrosis factor a (TNF-a). and/or Granzyme B (GrB) in CD8+ T cells. From these assays all three of the S, M, and N OPPs induced SARS-CoV-2-reactive CD4+ and CD8+ T cell responses (CD4+ T cell responses were detected in 56 of 65 samples, and CD8+ T cell responses were detected in 33 of 65 samples, against at least one. of the three proteins). Within the 56 CD4+ responsive samples, M-protein OPPs induced a detectable CD41 T cell response in the highest number of samples (M = 45, N = 36, S = 42). Within the 33 CD8+ responsive samples, the S-protein OPP was dominant (S = 26, N = 14, M = 13). While SARS-Cov-2-reactive T cells of both classes were also induced by SARS-CoV-2 OPPs in PBMC samples from unexposed donors, the incidence and level of T cell responses were significantly lower in unexposed donors, confirming the specificity of SARS-CoV-2 peptide stimulation in COVID-19 patients.

The magnitude and functionality of T Cells directed against S, M, and N Proteins shows different patterns for CD4+ and CD8+ T cells (Thieme et al., 2020). The M protein OPP induces the highest frequencies of reactive CD4+ T cells. Additionally, compared to S- and N-reactive CD4+ T cells, M-reactive CD4+ T cells express cytokines and effector molecules IL-2, IFN-g, TNF-a, and GrzB more frequently (the N OPP induces the lowest cytokine/effector responses). This pattern was not found in CD8+ T cells. T cells reactive to S, N, and M-proteins did not show a strong correlation in CD4+ and CD8+ T cell-restricted immunity.

While defective switching between innate and adaptive immunity appears to be correlated with unfavorable outcomes in COVID-19 disease (Cameron et al., 2007), T cell responses of both CD4+ and CD8+ cells (reactive to S. M, and N proteins) are reported to be similarly robust in critical COVID-19 subjects compared to moderate and severe subjects. This similarity is also reported for polyfunctional T cells expressing more than one cytokine or effector molecule (a hallmark of protective immunity in viral infections). Expression of IFN-g, TNF-a, IL-2, and IL-4 cytokines, and the effector GrzB, was analyzed in parallel to differentiation stage phenotyping. Both quantity and functionality of these markers for T cell immunity were similar, or even higher, in patients with critical COVID-19 disease compared to moderate and severe cases. Cytokine expression of bifunctional CD4t T cells was dominated by IFN-g, TNF-a, IL-2. Antigen-reactive IFN-g-, 1L-2-, and TNF-a-producing CD4 t T cells constituted over 70% of trifunctional CD4+ T cells. The majority of polyfunctional CD8+ T cells produced the cytotoxic effector molecule GrzB, most commonly in combination with IFN-g and TNF-a.

Thieme and colleagues noted that their experiments did not determine the prospective role of T cell polarization in immunopathogenesis. Previous reports suggest that T helper 2 (TH2)-dominated T cell responses are associated w ith increased immunopathology in SARS-CoV disease models (Deming et al., 2006; Yasui et al.. 2008). Immunodominance of T cell responses toward certain peptides has been associated with immunopathology in flaviviruses (Duangchinda et al., 2010; Screaton et al., 2015; Reynolds et al.. 2018; Mongkolsapaya et al., 2006). In the Thieme et al. study, the relative composition of T cell responses against S-, M- and N-proteins in COVID-19 patient samples appeared mostly uniform across different disease severities. Based on the association between polyfunctionality and stage of phenotypic differentiation of T cells. Thieme et al. compared frequencies of CD4+ and CD8+ T cells w ith effector memory (TEM)/TEMRA phenotypes among moderate, severe and critical COVID-19 subjects. T he presence of S-, N-, and M-reactive T cells with advanced differentiation phenotypes early after diagnosis suggested preexisting cellular immunity, consistent with detection of SARS-CoV-2 -cross-reactive T cells in unexposed donors in this and other studies (Braun et al., 2020; Grifoni et al., 2020). Few unexposed donors showed detectable polyfunctional T cells. The memory composition of SARS-CoV-2-reactive T cells in unexposed donors resembled that of COVID-19 patients, w ith the exception of fewer CD4+ and CD8+ TEM and more central memory (TCM) and naive (TNAIVE) cells.

Thieme and colleagues (2020) further evaluated the role of T cell immunity in SARS-CoV-2 viral clearance. In two patient groups screened as "cleared" and "uncleared" of virus, there was no significant differences in detectable T cell responses. Frequencies of SARS-CoV-2 protein-reactive CD4+ and CD8+ T cells, frequencies of polyfunctional CD4+ T cells, and ratios between initial and follow-up samples, did not differ significantly between cleared and uncleared groups. Patients who failed to clear virus achieved a comparable magnitude of SARS-CoV-2-reactive T cell immunity compared to cleared patients at the initial time point tested. Higher titers of neutralizing antibodies were actually observed in patients who did not successfully clear the virus, compared to patients who cleared the virus.

With regard to disease severity, Thieme and coworkers did not find a correlation between SARS-CoV-2-reactive T cell immunity and improved disease status in COVID-19 patients. The magnitude of S-, M-. and N-reactive T cells measured at the peak of severe COVID-19 disease did not significantly differ from corresponding measurements directly after patient recovery. T cell data for recovering critical patients were also comparable in magnitude and functionality to S-, M-, and N- reactive T cell immunity measured at the initial visit for patients who later experienced critical or lethal outcomes. These data suggest that other cell subsets, or cell subsets located at other sites (e.g., infected organs), are responsible for antiviral control and hyper-inflammatory disease manifestation. Critical patients appear to have a generally higher level of SARS-CoV-2-reactive T cells as compared to the non-critical controls, but critical patients are likely further along in their infectious course, with more time for T cell proliferation. In summary, patients with critical COVID-19 demonstrate equal, or even slightly higher, frequencies of CD4 + and CD8+ T cells reactive to S. M, and N OPPs, indicating that critical COVID- 19 patients maintain robust cellular immunity following infection. The slightly higher magnitude and functionality of T cell responses observed in critical COVID-19 cases may reflect a longer or more severe infection course, with a stronger immunogenic environment corresponding to a greater viral burden and inflammatory bystander activation. Yet another explanation may be that SARS-CoV-2 triggers overexpression of circulating chemokines, possibly interfering with chemotaxis of T cells to accurately target, infiltrate and sequester in infected tissues (Laing et al, 2020).

Poly functional T cells are generally regarded as a hallmark of protective immunity. IFN-g- and TNF-a-coproduction by SARS-CoV-reactive CD4+ and CD8+ T cells indicates an effector/memory phenotype and long-term protection (Wherry et al., 2004; Channappavar et al., 2014). These cells can contribute substantially to immunopathogenesis, consistent with a reported correlation between T cell proliferation, IFN-g-induced protein- 10 (IP- 10) levels, and COVID-19 disease severity (Laing et al.. 2020).

The identification of differentiated SARS-CoV-2-reactive T cells in unexposed donors raises serious questions about the role of preexisting immunity in COVID-19 disease. A recent study by Braun and colleagues also found SARS-CoV-2-reactive T cells with effector phenotypes in SARS- CoV-2-naive subjects, which were shown to be cross-reactive against common cold coronaviruses (Braun et al.. 2020). In this study, CD4 + T cells reactive against the S protein of SARS-CoV-2 were identified in PBMCs of 83% ofCOVID-19 patients, and in 35% of SARS-CoV-2-unexposed subjects. Anti-SARS-CoV-2 S CD4+ T cells from unexposed donors were primarily reactive against C -terminal epitopes of the SARS-CoV-2 spike protein, w hich exhibit greater sequence similarity with spike proteins of human endemic coronaviruses (hCoVs) compared to N-terminal epitopes. Spike-reactive T cell lines generated from SARS-CoV-2-nai've donors responded similarly to the C-terminal region of spike proteins of hCoVs (hCoV-229E and hCoV-OC43). as to those of SARS-CoV-2. These findings indicate that SARS-CoV-2 spike-protein cross-reactive T cells (predominately exhibiting a Th1 memory phenotype) are widely present in SARS-CoV-2-natve subjects, likely elicited by previous encounters with endemic human coronaviruses. The presence of spike-protein cross-reactive T cells in a high fraction of the general population has important implications for the design and safe use of COVID-19 vaccines. The biological role of pre-existing cross-reactive T cells in naive subjects infected with SARS- CoV-2 remains unclear. It is reasonable to presume that the presence or absence of these cells contributes to the divergent clinical manifestations of COVID-19. Some have suggested that resistance of children and young adults to symptomatic SARS-CoV-2 infection may be due to a higher level of pre-existing, cross-reactive T cells, possibly due to a higher frequency of transmissive inter- person contacts causing more frequent infections by hCoVs. Alternatively, depending on the frequency of infection and duration ofT cell immunity elicited in adults against endemic hCoVs, older adults may accumulate higher levels of cross-reactive memory T cells in an age-dependent fashion. On the basis of epidemiological data, it is estimated that adults contract an hCoV infection on average once every two-three years. Protective antibodies may wane mid-term, but memory T cell immunity appears to be very long lasting. For SARS-CoV specifically, memory T cell responses targeting the virus have been reported 1 1 years post-infection (Callow et al., 1990; Ng et aL, 2016).

SARS-CoV-neutralizing antibodies are associated w ith COVID-19 disease recovery, and these have been detected as long as 12 months after disease onset (Li et al., 2008). The longevity of neutralizing antibody responses against SARS-CoV-2 remains unknown. Although antibodies against hCoVs in general are known to wane within months after infection, hCoV reinfection is typically accompanied by low-level and short-lived virus shedding with only mild symptoms of short duration, indicating humoral-independent residual immunity (Callow- et al., 1990). Understanding the extent to which SARS-CoV-2-specific humoral and cellular immunity mediates durable protection against reinfection is of critical importance. It is equally important to determine whether prior infection and pre-existing cross-reactive immunity may contribute to hyper-immune and -inflammatory responses seen in severe COVID disease.

Effective vaccines and therapeutics against SARS-CoV-2 must take into account the critical role of hyper-inflammatory activation of macrophages and neutrophils by this virus. These mechanisms enhance viral replication and spread w ithin the lungs and mediate extreme pathogenesis in severe cases. Neutrophils evidently lack ACE2 receptors, and only a small percentage of monocytes/macrophages in the lungs ofCOVID-19 patients reportedly express ACE2 (Zhu et al. (2020). Nonetheless, both neutrophils and macrophages are infected by SARS-CoV-2, and infected macrophages contain the SARS-CoV-2 nucleoprotein (N) antigen, correlated with upregulated expression of pro-inflammatory cytokines (Feng et al., 2020). If ACE2 is minimally expressed or absent in macrophages and neutrophils, these key target cells may express other cognate receptors utilized by the virus, or another cellular entry mode may be utilized.

In the latter context, the phenomenon of "antibody-dependent enhancement" (ADE) has been proposed to explain certain aspects ofCOVID-19 infection and disease. The specter that prior exposure to SARS-CoV-2 infection, to another hCoV. or to a SARS-CoV-2 vaccine might increase infection risk and/or disease severity through an ADE mechanism, poses major concerns for vaccine safety. The ADE model has been explored for SARS-CoV, where it has been reported that circulating antibodies actually enhance infection and hyper-inflammatory activation of FcyR-expressing cells (including macrophages, DCs and neutrophils). According to this ADE model, circulating antibodies from a prior coronavirus exposure may bind to a newly-infecting, related virus and be actively taken up along with the virus by mononuclear phagocyte system (MPS) cells (including monocytes and their differentiated progeny, macrophages and DCs) and neutrophils via their Fcγ receptors. This uptake mechanism could explain how these cells, that do not substantially express ACE2, receptors are found to be extensively colonized by SARS-CoV-2.

FcyRI expression by neutrophils is strongly upregulated in the presence of inflammatory cytokines, such as interferon-y (IFN-y) or granulocyte colony-stimulating factor (GCSF), reaching up to 20,000 copies per cell (Fanger el al., 1989: Chen et al., 2012; Perussia et al., 1983; Guyre et al., 1990; Repp et al., 1991). This receptor upregulation activates neutrophils to high capacities for efficient binding of monomeric IgG (Perussia et al., 1983), phagocytosis of IgG-opsonized bacteria and viruses (Schiff et al., 1997), and elevation of ROS production induced by FcyRI cross-linking (Akerley et al., 1991 ). FcyRI upregulation also enables neutrophils to efficiently trigger antibody- dependent cytotoxicity (ADCC) (Repp et al.. 1991 ). As a consequence, neutrophil FcyRI expression is strongly correlated with infection state and disease progression in a diverse array of pathogenic inflammatory conditions (Davis et al., 2005; Strohmeyer et al., 2003; Song et al., 2008).

Regulatory interactions between neutrophils and IgG immune complexes (ICs) are central to inflammation, allergic reactions, and autoimmunity . Soluble ICs require primed neutrophils to efficiently trigger external ROS production and degranulation, while insoluble ICs can activate unprimed neutrophils, leading to intracellular ROS production, degranulation, and sustained liberation of inflammatory mediators, such as IL-8 and leukotriene B4 (LTB4), that drive neutrophil-mediated inflammation (Fossati et al., 2002; Mayadas et al., 2009). Neutrophils express distinct FcyR subtypes. FcyRIlIB has a primordial role in homeostatic removal and recycling of soluble ICs within the vasculature. FcyRlIA engages soluble ICs in tissues to induce pro-inflammatory processes, including NETosis (Chen et al., 2012). Engagement of either neutrophil FcyR by deposited ICs leads to neutrophil accumulation and activation of neutrophil inflammatory effector functions (Tsuboi et al., 2008). These characteristics and mechanisms place neutrophils and macrophages in the virtual eye of a prospective ADE storm, significantly complicating COVID-19 immunotherapy and vaccine development.

ADE was first demonstrated in an in vitro model of West Nile fever virus infection. In this model Fc receptor-mediated West Nile infection was blocked using anti-FcR IgG or Fab fragments (Peiris et al., 1981 ). ADE has since been documented for a wide range human and animal viral infections, including human immunodeficiency virus (HIV) and Ebola virus, however the fundamental clinical significance of these findings remains uncertain (Takada et al., 2003).

SARS-CoV-2 shares a high degree of genetic and protein similarity with other human coronaviruses (hCoVs), including common cold coronaviruses as well as the first two SARS viruses, SARS-CoV and MERS-CoV. Similar to what has been shown for Dengue, pre-existing immunity against other coronaviruses may predispose individuals to SARS-CoV-2 infection, or to develop more severe COVID-19 disease upon re-infection. Increased susceptibility to infection by SARS-CoV-2 may be facilitated by circulating, cross-reactive antibodies elicited or potentiated by the prior exposure. While there is limited evidence about the potential of anti-SARS-CoV-2 antibodies to mediate ADE, several studies of SARS-CoV indicate that circulating anti-SARS-CoV antibodies can enhance infection of cells expressing Fey receptors. In order to safely develop antibody-based therapeutics and vaccines to control SARS-CoV-2 infection, it is therefore critical to assess whether anti-SARS-CoV-2 antibodies have the capacity to mediate ADE, and if so to determine the precise molecular mechanisms and potential role of FcyRs in this process. If ADE is involved in SARS-CoV infection and pathogenesis, this mechanism may fundamentally determine the course and severity of disease mediated by hyper-activation of neutrophils and macrophages.

As noted above, SARS coronaviruses are known to infect immune cells such as T cells (Gu et al., 2005), macrophages (Cheung et al., 2005; Yilla et al., 2005), monocytes (Yilla et al., 2005) and dendritic cells (DCs) (Law et al., 2005). SARS-CoV-2 has been shown to induce phenotypic and functional maturation of mononuclear phagocyte system (MPS) cells, and to upregulate MHC class II and costimulatory molecules on monocyte-derived DCs (Tseng et al., 2005). MPS cells include DCs, macrophages and other monocytes, which are all similar with regard to ontogeny, location, function and phenotype (Guilliams et al., 2014). MPS cells account for the majority of immune cells in the lung microenvironment of severe COVID-19 patients (Liao et al., 2020), and SARS-CoV-2 is believed to utilize MPS cells as trojan horse vectors to disseminate the virus within the host and establish systemic infection (Park, 2020) (as described for hantaviruses by Raftery et aL, 2002; 2020). Neutrophils are another primary inflammatory cell type infiltrating the lungs, alveolar airspaces, and pulmonary vessels in COVID-19 patients, and are also infected, activated and used as transport vectors by SARS- CoV-2.

High ADE vaccine risks for coronaviruses may be expected based on experiences with feline infectious peritonitis virus (FIPV), a highly virulent feline coronavirus prevalent in both wild and domestic cats (Vennema et al., 1990; 1998). Immunization against FIPV paradoxically increases disease severity following later virus challenge of convalescent vaccinees. In vitro infection of macrophages by FIPV was enhanced by non-neutralizing monoclonal antibodies against the spike viral protein, and this phenomenon occurred even in the presence of neutralizing antibodies. Pretreatment w ith protein A prevented ADE (Hohdatsu et al., 1991 ). As many as 50% of cats passively immunized with anti-FIPV antibodies developed severe disease (peritonitis) when challenged w ith the same FIPV serotype (Takano, 2019). An attenuated FIPV virus vaccine is currently available in several countries for intranasal delivery, but this vaccine is deemed controversial by experts, both in terms of safely and efficacy.

ADE has been described in the greatest detail for flaviviruses, including the human Dengue virus, which resembles COVID-19 in the broad sense that it exhibits a wide range of clinical disease susceptibility and severity (ranging from asymptomatic to severe symptomatic or fatal disease). This extreme clinical variability points to individual immune factors affecting disease susceptibility and severity. The Dengue virus is transmitted to humans in tropical areas by mosquitoes, triggering fever, headache, vomiting, arthromyalgias and skin rash. Severe forms manifest as Dengue haemorrhagic fever and Dengue shock syndrome, which mostly affect younger persons. Dengue fever causes 100 million new infections and 40,000 deaths annually (Roth et al., 2018). There are four serotypes of Dengue virus, all known to elicit protective immunity. While homotypic protection is long-lasting, cross-neutralizing antibodies against different serotypes are short-lived and may only persist up to 2 years (Montoya et al., 2013). These cross-reactive neutralizing antibodies initially decrease odds of symptomatic secondary infection, which odds increase as the titer of these antibodies diminishes following primary infection (Katzelnick et al., 2016). Subsequent infection with a different Dengue serotype often runs a more severe course. In this circumstance, non-neutralizing antibodies predominate and bind to the Dengue virions, acting as a cloaking transporter for the virus to mediate infection of phagocytic cells via their Fc receptors. Thus, heterotypic antibodies at sub-neutralizing titers mediate ADE in persons infected with a serotype of Dengue virus that is different from the first infection. Protection against all Dengue diseases occurs when antibody titers are very high, but the hazard of severe Dengue disease increases inversely with protective antibody titers (about 8 times higher in children having the lowest levels of residual neutralizing antibodies) (Katzelnick et al., 2016). Dengue viral load and incidence of severe disease are highest in persons with low-intermediate titers of antibodies elicited by a prior Dengue infection (Endy et al., 2004; Waggoner et al., 2020).

The most worrisome aspect of ADE was reported during Dengue vaccine development trials in Asia and Latin America for the first recombinant live-attenuated, tetravalent Dengue vaccine (who.int., 2020). Safety of this vaccine was called into question by follow-up data showing that hospitalization of children 3 years post-vaccination was higher in vaccine recipients than among controls (Hadinegro et al., 2015). The proposed explanation is that Dengue vaccination mimics primary infection, whereafter waning immunity exposes vaccinated children to increased ADE risk. Further analyses showed that, while the vaccine protected previous Dengue sufferers against severe disease upon subsequent Dengue re-infection, the risk of severe clinical outcome was actually increased by vaccination among seronegative persons not previously exposed to the virus (Sridhar et al.. 2018). Following these reports a Strategic Advisory Group of Experts convened by the World Health Organization (WHO) concluded that only Dengue seropositive persons should be vaccinated when Dengue control programs include vaccination, and Dengue vaccination should not be performed on children under the under the age of 9 years (who.int. 2020).

ADE is emerging as an increasingly relevant obstacle to comprehensive vaccine strategies for COVID-19. With regard to the original SARS virus, SARS-CoV, antibodies elicited by a vaccine candidate against this virus have been shown to enhance infection of B cell lines, in spite of protective responses, in a hamster model (Kam et al., 2020). The mechanism of infection enhancement was shown to be dependent on expression of Fey receptors. Notably, SARS-CoV virion uptake in this model did not exploit the ACE2 pathway (Jaume et al., 201 1). Further in vitro studies using a human promonocyte cell line showed that SARS-CoV was neutralized by concentrated antisera against the spike protein, but higher dilutions not only failed to prevent infection but actually facilitated it, inducing higher levels of promonocyte infection and apoptosis. In a macaque model of SARS, post infection increases in levels of anti-spike IgG skewed wound healing responses of lung-infiltrating macrophages towards a proinflammatory profile (Liu et al., 2019). These authors reported similar observations in human patients deceased of ARDS. These effects are believed attributable to the interaction of anti-SARS-CoV antibodies complexing with virus and mediating cellular uptake via Fc receptors into macrophages, triggering cytokine upregulation and pathogenic inflammatory responses.

ADF, in COVID-19 has also been proposed to account for a relatively high severity of COV1D- 19 cases observed in China compared with other regions of the world (Tetro et al., 2020). Prior infection of Chinese subjects with other coronaviruses, including SARS-CoV, may have primed and predisposed these subjects to develop more severe COVID-19 disease when infected with SARS- CoV-2. In this context, high cross-reactivity of antibodies against the spike proteins of SARS-CoV-2 and SARS-CoV have been reported, but these cross-reactions are rarely cross-neutralizing (Lu et al., 2020). Priming may also have originated from other bat coronaviruses, considering that several recent zoonotic cross-overs to humans (of SARS-CoV, MERS and SARS-CoV-2) may have occurred previously with other coronavirus strains in clinically silent form. The noted resistance of younger persons to severe COVID-19 disease may be in part due to their relatively limited exposure to prior coronavirus infections, limiting their titers of cross-reactive antibodies capable of associating with SARS-CoV-2 to mediate ADE.

A majority of patients who recover from mild COVID-19 develop neutralizing antibodies (Wu el al.. 2020). Antibody response profiles of patients who develop severe disease have yet to be fully characterized. It remains unclear what associations may exist between levels and specificity of humoral responses and COVID-19 disease severity. It is predicted, however, that a large majority of antibodies elicited by COVID-19 infection are expected to be non-neutralizing. Thus, for COV1D-19- recovered individuals, to the extent non-neutralizing antibodies are present in the circulation after effective neutralizing antibody titers decline, re-infection will present uncertain risks of ADE. Likewise, it remains uncertain whether treatment ofCOVID-19 patients with convalescent plasma will pose elevated risks of ADE upon subsequent reinfection. The same is true for anti-SARS-CoV-2 vaccines comprising whole viral proteins or large subunits that elicit large panels of non-neutralizing antibodies, along with a waning minority of neutralizing antibodies. At some point, after protective antibodies are diminished, vaccinated individuals may actually be at elevated risk of infection and more severe disease consequences through ADE than un-vaccinated persons.

There is currently a frantic global push to develop anti-SARS-CoV-2 vaccine agents to prevent or reduce SARS-CoV-2 infection and COVID-19 disease. Numerous SARS-CoV-2 vaccine candidates are currently in development and clinical testing. In general, anti-viral vaccines may comprise live-attenuated, recombinant, or recombinant chimeric viruses, inactivated or killed viruses, immunogenic subunits of a virus, viral vector-delivery vaccines, and DNA- or RNA-based genetic vaccines.

Very soon after the outbreak of COVID-19, in early January of 2020, the genome sequence of SARS-CoV-2 was published. Among the earliest-developed vaccine candidates for SARS-CoV-2 were Moderna's and Pfizer-BioN Tech's mRNA vaccine candidate. These vaccines comprise a synthetic mRNA encoding a SARS-CoV-2 spike (S) protein construct. After intramuscular injection, these vaccines direct the host cellular machinery to produce the encoded S protein to elicit an antiviral immune response directed against the native S protein of SARS-CoV-2. Unlike conventional vaccines (e.g.. made from inactivated or dead pathogen, live-attenuated virus, or small immunogenic viral subunits), Moderna's and Pfizer's mRNA vaccines do not require use, handling or patient exposure to any potentially infective form of the SARS-CoV-2 virus, The simple structure of nucleic acids obviates problems of incorrect folding and other complications that can occur with recombinant protein-based vaccines. Additionally, genetic vaccines have lower costs of production than many conventional vaccines. However, the amount of mRNA vaccine that is delivered, and the optimal timing and route of administration are uncertain factors that can influence efficacy of these vaccines. Both Moderna's and Pfizer's mRNAhave been reported to be highly effective (above 90%) for eliciting protective immune responses in test subjects, and both vaccines are likely to pass final efficacy and safety review to be among the first vaccines mobilized in the US for emergency use authorization. Important drawbacks to these vaccine platforms include instability of the vaccines during storage, with Pfizer's construct requiring long-term storage at -94 °C, and Moderna's at -4 °C. 1'his severely limits the ability to nationally and globally distribute these vaccines in a timely fashion, and increases deployment costs substantially. mRNA vaccines are also rapidly degraded in the body, indicating that multiple dosing may be required for effective vaccination.

A number of adenoviral vector-based DNA vaccines against SARS-CoV-2 are also in development. Among these, Oxford University has engineered ChAdOxI nCoV-19, a non-replicating adenovirus vector encoding a recombinant SARS-CoV-2 S protein, which is presently in clinical trials in the US (NCT04324606). The non-replicating nature of the adenovirus vector makes this vaccine relatively safe in children and vaccinees with underlying diseases. Adenovirus-based vectors exhibit a broad range of tissue tropism that covers both respiratory and gastrointestinal epithelia, the two main sites that express ACE-2 receptors rendering them vulnerable for SARS-CoV-2 infection. This broad tropism, however, increases certain risks of toxicity and inflammation associated with the use of adenoviral vectors. The latest generation of high-capacity adenoviral vectors are devoid of viral genes, and thus have significantly improved safety profiles over earlier vectors, and also exhibit more prolonged transgene expression (likely sufficient for vaccine use). Historically, adenoviral vectors have exhibited toxic and inflammatory side effects, which are substantially reduced if not eliminated with modern, replication-incompetent vectors. Finally, there is a low theoretical risk of malignancy with adenoviral vectors, due to their ability to integrate randomly into the genome of host cells. Two additional viral vector DNA vaccines in development, from AstraZeneca and Johnson & Johnson, utilize a common cold human adenovirus as a vector to direct expression of the SARS-CoV-2 S protein w ithin body of vaccinated subjects.

Two other leading SARS-CoV-2 vaccine candidates are protein vaccines from Novavax and a Sanofi-GlaxoSmithKline. These vaccines use recombinant SARS-CoV-2 S protein injected directly into the body. The synthetic spike protein is grown in insect cells, which is slower and more costly than some other technologies. Both the Novavax and Sanofi-GSK vaccines require an adjuvant, a chemical agent that boosts the immune response. Adjuvants can sometimes cause pain or swelling at the injection site. An advantage of these protein vaccines is that they can stored at regular refrigerator temperatures (36-46 °46 making them easier to ship than some other leading candidates.

Yet another vaccine strategy for COVlD-19 employs a stabilized subunit vaccine. Enveloped viruses like SARS-CoV-2 require fusion of the viral membrane with the host cell membrane for infection. This process involves a conformational change of the viral S glycoprotein from a pre-fusion form to a post-fusion form. Although pre-fusion glycoproteins are relatively unstable, they are able to elicit strong immune responses. The University of Queensland is developing a stabilized subunit vaccine against SARS-CoV-2 based on molecular clamp technology, which allows recombinant viral proteins to stably remain in their pre-fusion form. Previously applied to influenza virus and Ebola virus, molecular clamp vaccines have proved their capacity to induce neutralizing antibodies. They are also reported to be potent after two weeks at 37 °C.

Nanoparticle-based vaccines represent an alternative strategy to incorporate and present antigens to vaccinate at-risk subjects. Through encapsulation or covalent functionalization, nanoparticles can be conjugated with antigenic epitopes to mimic viruses and elicit antigen-specific lymphocyte proliferation and cytokine production. This technology is permissive of mucosal vaccination through intranasal or oral routes to stimulate immune reactions at the mucosal surface and systemically. Novavax, Inc. is producing a nanoparticle-based anti-SARS-CoV-2 vaccine using antigens derived from the viral S protein stably expressed in a baculovirus system.

A common uncertainty inherent to each of the foregoing SARS-CoV-2 candidate vaccine platforms relates to their long-term safety profiles. This is by necessity lacking in all cases, given the recent appearance of the virus. Safety concerns are particularly manifest for SARS-CoV-2 vaccines, in view of the complex, serious and confounding nature ofCOVID-19 disease etiology. With this reality in mind, none of the foregoing SARS-CoV-2 vaccine candidates can be said to have been tested and proven to obviate serious risks of long-term side effects, such as ADE.

The use of convalescent plasma from COVID-19-recovered patients has been encouraged based on recent studies of patients with severe COVID-19 disease (see, e.g., Duan et al., 2020). In the uncontrolled trial of Duan and coworkers, administration of plasma containing high titers of SARS- CoV-2 neutralizing antibodies was reportedly effective against SARS-CoV-2 based on clinical, biochemical and radiological parameters, with prompt viral suppression and no severe adverse effects observed, This rescue treatment is also untested for likely ADE consequences that may pose serious risks to "long-haul" or reinfected patients.

A different kind of rescue treatment for COVID-19 patients is based upon the use of manufactured neutralizing antibodies directed against the SARS-CoV-2 virus. Development of specific viral surface epitope-targeting neutralizing antibodies is considered a promising approach to prevent or treat COVID-19 infection. AbCellera (Canada) and Eli Lilly and Company (USA) are co- developing a functional antibody, LY-CoV555 (bamlanivimab) designed to neutralize SARS-CoV-2 in infected patients. For this purpose. Lilly screened over 5 million immune cells from an early U.S. patient recovered from COVID-19, to identify more than 500 promising anti-SARS-CoV-2 antibody sequences. Further development yielded the monoclonal antibody LY-CoV555, which binds the ACE2 receptor binding domain (RBD) of the SARS-CoV-2 S protein, to potently neutralizes SARS- CoV-2 infectivity. Vir Biotechnology, Inc., ImmunoPrecise, Mount Sinai Health System, and Harbour BioMed (HBM) are also developing monoclonal antibodies that bind and neutralize SARS-CoV-2. This strategy is best exemplified by Regeneron's monoclonal antibody cocktail REGN-COV2. This antiviral biologic employs two monoclonal antibodies directed against the SARS-CoV-2 spike protein, designed to block the virus from entering host cells (Hansen et al., 2020). REGN-COV2 was also developed by screening a large panel of antibodies reactive against the spike (S) protein RBD. isolated from humanized mice and convalescent COVID-19 patients. From this panel, neutralizing antibodies pairs were selected that do not compete for binding to the SARS-CoV-2 S RBD. These antibody pairs were further screened against a SARS-CoV-2 pseudovirus expressing the spike protein to eliminate antibody candidates against which the pseudovirus readily developed escape mutations. The optimal antibody pair cocktail, REGN-COV2, is now in phase III trials, having been shown to clinically reduce viral load and associated symptoms in infected COVID-19 patients with mild-moderate disease. Treatment benefits have been most pronounced in patients at an early disease stage who fail to mount their own effective immune response (i.e„ patients who remain seronegative or low seropositive for an extended period after infection). REGN-COV2 is thus contemplated as a rescue substitute for patients with deficient anti-COVID immune responses, similar to the therapeutic use of convalescent plasma. Neither convalescent plasma nor monoclonal antibody treatment yields a long- lasting protective benefit against subsequent SARS-CoV-2 infection. Recently the National Institutes of Health (N1H) scrapped a clinical trial of Eli Lilly's anti-SARS-CoV-2 antibody LY-CoV555 for hospitalized COVID-19 patients. Investigators stopped enrolling patients in the study after finding that LY-CoV555 was unlikely to improve outcomes in the hospitalized patient population. Whether any anti-SARS-CoV-2 monoclonal antibody therapy will be of clinical value for hospitalized patients with advanced COVID-19 disease remains to be determined.

Considering the limitations of these and other COVID-19 immunologic rescue treatments, and in view of the limited armamentarium of other effective therapeutics for COVJD- 19, the goal of effectively resolving the COVID-19 pandemic will require new and more effective vaccines. Because SARS-CoV-2 has so deeply penetrated human populations, it must be regarded as a future "endemic" hCoV. meaning individuals will re-encounter the virus long after initial outbreaks wane. Thus, effective vaccines will necessarily induce potent, long-lasting immunity, while at the same time minimizing the risks of ADE in long-term COVID-19 vaccine safety. Resolving this safety concern is urgent in view of the viral-transmissive and pathogenic roles of macrophages and neutrophils in COVID-19 disease progression, (involving ACE2-independent infection of these cells by SARS-CoV- 2). This trojan horse infection and spreading strategy of SARS-CoV-2 is not subject to blockade by convalescent serum or anti-ACE2 monoclonal antibodies, such as Regeneron's REGN-COV2, and will likewise be refractory to the majority of current COVID-19 vaccine candidates targeting the S protein-mediated ACE2 viral uptake pathway. At the same time, these vaccines that are predicted to elicit extensive non-neutralizing antibody production (comparable to the antibody makeup of convalescent serum), presenting considerable ADE risks, especially after neutralizing antibody titers wane and treated subjects become vulnerable to re-infection. This is true for all vaccine candidates that deliver or direct expression of whole viral proteins, or large protein subunits, which are predicted to induce overbroad immune responses and elicit excessive non-neutralizing antibodies and memory T cells that confer minimal or no protective benefits while substantially increasing the likelihood of adverse, ADE-related impacts in cases of re-infection and "long-haul" disease.

In view of the foregoing, critical needs exist in the art for more effective tools and methods to combat SARS-CoV-2 infection and related consequences of COVID-19 disease. Primary among these objectives is the goal of obtaining safe and effective vaccines to prevent or reduce SARS-CoV-2 infection and COVID-19 disease. Useful vaccines must be carefully designed and tested to avoid unintended impacts, such as hyper-activation of inflammatory targets and pathways, or antibody dependent enhancement (ADE) of infection and/or inflammation, that could exacerbate hyper-immune and hyper-inflammatory responses contributing to Cytokine Storm Syndrome (CSS), Acute Respiratory Distress Syndrome (ARDS), multisystem inflammatory syndrome in children (MISC), Extrapulmonary Systemic Hyperinflammation Syndrome (ESHS), and vascular congestive and thrombotic conditions associated therewith, including Disseminated Intravascular Coagulation (DIC), thrombosis and stroke. SUMMARY OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention achieves the foregoing objects and satisfies additional objects and advantages by providing innovative COMPLETE VACCINE ^{I M} compositions and methods, employing li-Key- SARS-CoV-2 antigenic peptide hybrid vaccines, to potently prevent or reduce SARS-CoV-2 infection, and discretely regulate immune responses in vaccinees to avoid hyper-immune and -inflammatory responses associated with natural SARS-CoV-2 infection. According to the novel vaccine design and construction methods of the invention, multi-targeting and multi-functional li-key peptide vaccines are provided that selectively regulate balanced immune and inflammatory responses in vaccinated subjects. In certain aspects of the invention. Ii-Key-SARS-CoV-2 antigenic peptide hybrid vaccines are effective as multi-targeting and multi-functional immunogens to: 1 ) Stimulate broad T cell immune responses (both CD4+ and CD8+); 2) Enhance or exclusively target Th1 T cell responses (disaffecting, negating or attenuating Th2 T cell responses); 3) Stimulate a B-Cell (neutralizing antibody) response; 4) Invoke long-term antigen-specific memory immunity against SARS-CoV-2; and/or 5) Provide a broad-spectrum of protection against SARS-CoV-2 effective for vast majority (at least 85%-95%) of human subjects.

The invention achieves these surprising anti-SARS-CoV-2 effective vaccine results in compositions and methods that further serve to negate or minimize adverse side effects that attend natural SARS-CoV-2 infection, and that are also likely to follow administration of current lead vaccines against SARS-CoV-3 (at least in cases of natural SARS-CoV-2 infection following vaccination and waning of protection), including: 1 ) Antibody dependent disease enhancement (ADE); 2) Hyper-immune and hyper-inflammatory conditions associated with SARS-CoV-2; and 3) Cytokine storm syndrome (CSS) associated with severe COVID-19 disease following SARS-CoV-2 infection.

The invention provides for rapid production and deployment of vaccines, including anti- SARS-CoV-2 vaccines, using an li-Key peptide vaccine development strategy based in part on computational vaccinology tools and methods to identify safe and immunologically relevant SARS- COV-2 targets. Within these aspects of the invention, exemplary Ii-Key-SARS-CoV-2 Complete Vaccine ^TM hybrid peptide constructs incorporate T-cell and B cell SARS-COV-2 epitopes to specific activate cell-mediated and humoral immune effector cells, with resulting immunologic memory. The subject Ii-Key-SARS-CoV-2 vaccines can be rapidly evaluated for safety and efficacy, for example using ex vivo human trials. In illustrative embodiments. Ii-Key-SARS-CoV-2 peptide vaccines are tested against convalescent COVID-19 patient cells, serum or other samples, to identify optimal constructs to generate neutralizing, rather than non-neutralizing antibodies, activate the beneficial Th1 responses as opposed to pathogenic-associated Th2 responses, avoid ADE and CSS responses, and achieve long-term memory immune priming and induction.

The discoveries presented herein offer powerful new tools for rapid design, construction and deployment of a wide range of li-key peptide vaccines, effective against a diverse array of current and future viral pathogens. The resulting vaccine products are fast and economical to produce, and exceptionally stable for long-terms storage.

Ii-key peptide vaccines and methods of the invention selectively regulate immune and inflammatory target cells, response mechanisms, pathways and signals in immunized subjects, to elicit focal and potent anti-viral immune responses, while minimizing pathogenic hyperimmune and hyperinflammatory responses associated with severe illness and death in COVID-19 patients. The vaccine compositions and methods of the invention regulate specific immune and inflammatory functions to disrupt viral infection and pathogenicity and facilitate viral clearance, while disaffecting, inhibiting or negating hyper-immune and -inflammatory targets, mechanisms, pathways and signals that contribute to disease progression.

By virtue of the highly directed and discretely active immunological effects of Applicant's li- key peptide vaccines, incidence of SARS-CoV-2 infection and viral burden in virus-exposed subjects are minimized, and serious COVID-19 disease manifestations, including critical disease and death, are prevented or greatly diminished. The discrete immune and inflammatory regulatory activities of Applicant's li-key peptide vaccines also minimize long-term adverse sequelae predicted for certain conventional vaccines, including insufficient duration of protection, and antibody dependent enhancement (ADE)

In one aspect of the invention, Ii-Key-SARS-CoV-2 prophylactic peptide vaccines are provided according to a.novel, computational and experimental rational design and construction method, generally comprising the following steps:

I ) An initial pool of candidate vaccine peptides comprising known or predicted immunogenic epitopes with antigenic sequence identity or similarity to a SARS-CoV-2 antigenic epitope, is provided. In exemplary embodiments, computational vaccinology tools are employed for epitope analysis and selection, to identify, select and characterize members of the initial candidate peptide pool, using selective and screening algorithms applied to a known target viral genome, nucleic acid sequence, or amino acid sequence of a putatively antigenic viral protein, protein functional domain, protein antigenic region, or candidate epitope;

2) Candidate peptides within the initial pool (each having been selected computationally to comprise one or more putative antigenic epitope(s) with presumptive antigenic sequence identity or sequence similarity to one or more SARS-CoV-2 predicted antigenic epitope(s)) are further screened and optionally modified to improve immunogenicity, enhance immunogenic function or immune-regulatory selectivity, and/or to exclude human sequence overlap, for example to reduce risk of autoimmunity. In exemplary embodiments these further selections and/or modifications are determined by using further computational vaccinology tools and steps.

3) Candidate SARS-CoV-2 antigenic peptides are constructed with li-key components to form a hybrid Ii-Key-SARS-CoV-2 antigenic peptide construct having a typical length of about 20 to 28 amino acids, and generally less than 40 amino acids. The li-Key sequence linked to the antigenic peptide can interact with an allosteric site on MHC class 11 molecules adjacent the antigenic epitope-binding trough, to enhance presentation of the antigenic peptide/epitope by MHC class II molecules for improved antigen-specific helper T-cell stimulation.

4) Candidate Ii-Key-SARS-CoV-2 antigenic peptide constructs are screened to evaluate immunogenic activity and selectivity, for example to validate antigenic activity to induce a neutralizing antibody response. In one illustrative screening design, candidate li-Key- SARS-CoV-2 antigenic peptides are immobilized as an affinity ligand to a solid substrate, matrix or carrier, for example magnetic beads, and the bound peptides are exposed to a positive neutralizing antibody sample (i.e.. a sample known or predicted to contain neutralizing antibodies directed against SARS-CoV-2). In exemplary embodiments the positive neutralizing antibody-containing sample comprises a blood, serum or other biological sample from a recovered COVID- 19 (convalescent) patient. In alternative embodiments the presumptive or known neutralizing antibody-containing sample comprises one or more known, characterized anti-SARS-CoV-2 neutralizing antibodies. The immobilized candidate Ii-Key-SARS-CoV-2 antigenic peptide is incubated with the positive neutralizing antibody-containing sample for a time sufficient to allow selective binding of neutralizing antibodies in the sample to the immobilized peptide. Prospective or known neutralizing antibodies thus determined to bind the Ii-Key-SARS-CoV-2 antigenic peptide "specifically" (e.g.. with a dissociation constant (Ko) indicative of specific antibody-antigen binding, usually in a range of 10 ^-6 to 10 ^-9 ) indicate that the candidate li- Key-SARS-CoV-2 antigenic peptide is "recognized” as an antigen (presumptively for having the same one or more epitope(s) that elicited original production of the cognate neutralizing antibody present in the sample), and is thus presumptively capable of eliciting an immune response in vivo.

5) Prospective or known neutralizing antibodies that specifically bind a candidate li-Key- SARS-CoV-2 antigenic peptide can be separated from the peptide (e.g., using a magnetic separator, coupled with conventional elution) for further characterization. The objective in this step is to confirm that li-Key peptide cognate antibodies (i.e., antibodies from the known or presumptive neutralizing sample that specifically bind a Ii-Key-SARS-CoV-2 antigenic peptide) in fact possess anti-SARS-CoV-2 neutralizing activity. This can be done using any of a variety of viral neutralization assays. In one exemplary protocol, SARS- CoV-2 antigenic peptide cognate antibodies are qualified for positive neutralization activity using a SARS-CoV-2 plaque reduction neutralization test (PRNT). Confirmation of positive anti-SARS-CoV-2 neutralizing activity for a li-Key antigenic peptide-binding antibody from the presumptive or known neutralizing antibody sample allows selection of Ii-Key-SARS-CoV-2 antigenic peptides that will presumptively elicit a neutralizing antibody response (i.e., peptides that comprise a putative antigenic epitope having predicted antigenic sequence identity or sequence similarity to a SARS-CoV-2 antigenic epitope, further characterized by demonstration of specific recognition by antibodies confirmed to possess anti-SARS-CoV-2 neutralizing activity— which peptides are thus qualified as likely competent antigens for eliciting neutralizing antibody B cell responses in immunized subjects). Additionally, this neutralizing validation assay allows for identification and elimination screening of undesired Ii-Key-SARS-CoV-2 antigenic peptides recognized (bound) by non-neutralizing antibodies (i.e., antibodies present in the neutralizing sample that specifically bind the Ii-Key-SARS-CoV-2 antigenic peptide, but which fail to exhibit neutralizing activity when disbound, isolated and tested in a SARS- CoV-2 neutralization assay).

6) An additional or alternate step to refine initial candidate li-Key-SARS-CoV-2 antigenic peptide selection involves screening candidate Ii-Key-SARS-CoV-2 peptides to select for beneficial immune regulatory functionality . In one aspect of the invention, this refinement or screening step selects for Ii-Key-SARS-CoV-2 antigenic peptide that elicit a T cell immune response polarized toward a ThI - versus Th2-type response, for example using an ex vivo T cell activation assay. In one exemplary embodiment, anti-SARS-CoV-2 immune-competent peripheral blood mononuclear cells (PBMCs) are provided from blood samples of COVID- 19 convalescent patients. The SARS-CoV-2 convalescent PBMCs are exposed to different candidate Ii-Key-SARS-CoV-2 antigenic peptides, and ELISpot assays, flow cytometry or other suitable detection methods are used to characterize T cell activation responses elicited by the individual candidate Ii-Key-SARS-CoV-2 peptides. Employing these methods, for example, Ii-Key-SARS-CoV-2 peptides that elicit ThI -type T cell responses are determined by detection of activated cells expressing one or more accepted ThI -positive markers, while those that elicit Th2-type T cell responses are determined by detection of activated cells expressing one or more accepted Th2-positive markers. In certain embodiments, only Ii-Key-SARS-CoV-2 peptides that elicit predominately ThI -type T cell responses are selected and used in SARS-CoV-2 vaccine compositions and methods of the invention, while those that elicit a Th2-biased T cell response are excluded.

7) A further optional screening process may be used to exclude off-target and ADE activities of li-Key-SARS CoV-2 antigen peptides. In one aspect, this involves determining candidate Ii-Key-SARS-CoV-2 peptides that elicit neutralizing antibody responses, without eliciting non-neutralizing antibodies, as described above. In further aspects, li-Key-SARS- CoV-2 peptides are additionally screened to exclude candidates that elicit off-target immune or inflammatory responses that contribute to hyper-inflammation, such as is associated with CSS and ARDS, or another ADE activity or mechanism. In an exemplary assay of this kind, a comprehensive pool of li-Key-SARS CoV-2 peptide-binding antibodies (putatively including both neutralizing and non-neutralizing antibodies) isolated from COVID-19 convalescent blood or serum, is incubated with a suitable ADE assay subject, for example U937 cells (Liu et al. Virologica Sinica (2019) 34:648-661). This assay allows for detection of ADE-contributory off-target responses that correspond to hyper-immune and hyper-inflammatory activation processes associated with CSS and ARDS, including induction of non-neutralizing antibodies.

8) A further optional screening process may be used to select li-Key-SARS CoV-2 antigen hybrid peptides capable of mediating induction of antiviral CD8+ cytotoxic T cells in a mammalian subject (for example by activating CD4+ T cells that drive adaptive immunity by potentiating maturation of CD8+ "killer" T cells). In exemplary screens, candidate li- Key-SARS CoV-2 antigen hybrid peptides are contacted with a CD8+ competent test subject, for example COVID-19 convalescent blood or PBMC sample, and the subject is observed for induction of CD8+ T cell markers (e.g., Granzyme B (GrB)).

According to the computational vaccinology and experimental screening and characterization methods above, a candidate pool of presumptive antigenic peptides predicted to be capable of eliciting an anti-SARS-CoV-2 immune response in a mammalian subject is identified for constructing a panel of candidate Ii-Key-SARS-CoV-2 antigenic peptide vaccines of the invention. In one example that followed Applicants’ computational vaccinology methods and teachings, a candidate pool of 32 presumptive antigenic peptides was identified based on the SARS-CoV-2 envelope (E), membrane (M) and spike (S) proteins. This exemplary candidate pool of antigenic peptides includes: E 1 -15 (MYSFVSEETGTL1VN) (SEQ ID NO: 4): M 13-27 (LKKLLEQWNLVIGFL) (SEQ ID NO: 5); M 32-48 (ISLLQFAYANRNRFLY1) (SEQ ID NO: 6); M 93-107 (LSYFIASFRLFARTR) (SEQ ID NO: 7); M 97-1 13 (IASFRLFARTRSMWSFN) (SEQ ID NO: 8); M 146-160 (RGHLR1AGHHLGRSD) (SEQ ID NO: 9); M 165- 179 (PKEITVATSRTLSYY) (SEQ ID NO: 10); M 175- 190 (TLSYYKLGASQRVAGD) (SEQ ID NO: 1 1 ); M 201 -217 (IGNYKLNTDHSSSSDNI) (SEQ ID NO: 12): S 25-38 f PPA YTNSFTRGVY Y) (SEQ ID NO: 13); S 87- 102 (NEGVYFASTEKSN1IR) (SEQ ID NO: 14); S 140- 156 (FLGVYYHKNNKSWMESE) (SEQ ID NO: 15); S 154-171 (ESEFRVYSSANNCTFEYV) (SEQ ID NO: 16); S 198-212 (DGYFK1YSKHTPINL) (SEQ ID NO: 17); S 239-255 (QTLLALHRSYLTPGDSS) (SEQ ID NO: 18): S 272-288 (PRTFLLKYNENGTITDA) (SEQ ID NO: 19); S 315-329 (TSNFRVQPTESIVRF) (SEQ ID NO: 20): S 338-352 (FGEVFNATRFASVYA) (SEQ ID NO: 21); S 347-364 (FASVYAWNRKRISNSVAD) (SEQ ID NO: 22); S 446-468 (GGNYNYLYRLFRKSNLKPFERDI) (SEQ ID NO: 23); S 483-500 (VEGFNCYFPLQSYGFQPT) (SEQ ID NO: 24); S 492-508 (LQSYGFQPTNGVGYQPY) (SEQ ID NO: 25); S 536-549 (NKSVNFNFNGLTGT) (SEQ ID NO: 26): S 797-816 (FGGFNFSQILPDPSKPSKRS) (SEQ ID NO: 27); S 866-879 (TDEMIAQYTSALLA) (SEQ ID NO: 28); S 895-912 (LQIPFAMQMAYRFNGIGV) (SEQ ID NO: 29); S 920-934 (QKLIANQFNSAIGKI) (SEQ ID NO: 30); S 924-938 (ANQFNSAIGKIQDSL) (SEQ ID NO: 31 ): S 998-1012 (TGRLQSEQ TYVTQQL) (SEQ ID NO: 32); S 1044-1058 (GKGYHLMSFPQSAPH) (SEQ ID NO: 33): S 1 152-1 166 (LDKYFKNHTSPDVDL) (SEQ ID NO: 34): S 1169-1183 (ISGINASVVNIQKEI) (SEQ ID NO: 35), each of which candidate peptide is predicted to be capable of eliciting an anti-SARS-CoV-2 immune response in a mammalian cell or host.

Presumptive antigenic li-Key peptides of the invention, exemplified by the aforementioned 32- member peptide pool, and structural variants thereof that retain antigenic functionality, are useful in the methods and compositions of the invention for constructing Ii-Key-SARS-CoV-2 prophylactic hybrid peptide vaccines. In exemplary embodiments, MHC class II antigen presentation-enhancing hybrid polypeptides are designed and synthesized to incorporate the presumptive antigenic peptide, linked to an N-terminal li-key peptide, for example a li-Key peptide comprising a sequence LRMKLPKPPKPVSKMR (SEQ ID NO: 36) or a natural variant or modified, functionally conserved synthetic structural variant thereof (e.g., a truncated, substituted or addition variant thereof that retains MHC II antigen presentation-enhancing activity). In exemplary Ii-Key-SARS-CoV-2 hybrid peptides, the anti-SARS-CoV-2 antigenic peptide is typically located at a C -terminus of the hybrid peptide. An intervening chemical linker typically is used to flexibly link the N-terminal and C-terminal components of the hybrid peptide, the linker comprising a variable length, flexible chain of up to about 20 amino acids. The linker is optionally selected to have a length of about 4-6 amino acids, and in certain embodiments is constructed to be unable to hydrogen bond in any spatially distinct manner to a MI IC class II molecule. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of direct charging of MHC Class II molecules with li-Key Hybrid antigenic peptides.

Figure 2 a schematic diagram illustrating solid phase synthesis of li-Key hybrid peptide for vaccine use.

FIGURE 3 is a schematic diagram depicting the effector immune cells, cytokines and transcription factors involved in determining Th1 versus Th2 differentiation of CD4+ T lymphocytes.

Figure 4 provides a graphic example of EpiMatrix® protein immunogenicity scoring for SARS-CoV-2 proteins.

Figure 5 illustrates an "EpiBar®" cluster feature relating to antigenic peptides that induce strong T cell responses, reactive with multiple Hl. A alleles, identified using the ClustiMer® system.

Figure 6 graphically illustrates homology scoring of peptide epitopes using the JanusMatrix® human homology scoring system.

Figure 7 depicts immunogenicity profiles for 32 exemplary candidate SARS-CoV-2 antigenic peptides comprising T cell epitope clusters selected according to the staged computational vaccinology screening methods of the invention.

Figure 8 is a schematic illustration of an exemplary ELISpot assay for detecting and characterizing immune cell responses to Ii-KeySARS-CoV-2 antigenic peptide hybrids, to guide rational peptide vaccine design.

Figure 9 is a schematic illustration of a useful CBA assay system for screening li-KeySARS- CoV-2 antigenic peptide hybrids for their potential to elicit pro-inflammatory cytokine expression by responsive immune cells.

Figure 10 graphically depicts results of CBA assays to detect CSS-associated pro- inflammatory cytokines in samples from COVID-191CU patients versus healthy controls.

The present invention may be understood more fully by reference to the detailed description and examples below, which are provided for illustrative purposes and will be understood by skilled artisans to represent non-limiting embodiments of the invention. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

OF THE INVENTION

The instant invention provides novel processes and tools to enable rapid design, construction and deployment of COMPLETE VACCINE™ products, which comprise one or more hybrid synthetic li-Key peptide antigen constructs. These multi-functional, immune-regulatory peptide vaccines are particularly well-suited for rapid pandemic response to novel pathogens and emergent strains of endemic pathogens. The subject hybrid li-Key peptide antigen constructs are short, simple peptides that can be rapidly, economically synthesized in large quantities (hundreds of millions of doses in just a few months), in a stable (including lyophilized) form, amenable to rapid, low-tech, global deployment.

Hybrid li-Key peptide antigen vaccines of the invention are typically provided in the form of a COMPLETE VACCINE™ product, incorporating a plurality of antigenic epitopes to elicit multiple immune-regulatory responses from a plurality of effector cell types in vaccinated mammalian hosts. These COMPLETE VACCINE™ products effectively and safely induce prophylaxis and clearance of virus, and also mediate long-term memory protection, without eliciting pathogenic, hyper-immune or hyper-inflammatory responses. In related embodiments, li-Key peptide antigen vaccines of the invention are crafted and typed to minimize potential for antibody dependent enhancement (ADE) of infection and attendant disease complications believed to be correlates of SARS-CoV-2 and other viral infections in individuals previously exposed to the same virus, or to a structurally related virus. li-Key antigenic peptides have been shown to increase MHC Class II immune responsivity up to 100 times or greater than what can be achieved using corresponding antigenic peptides alone (Humphreys et al 2000). The activity and safety of li-Key peptide vaccines have been proven in Phase I and 2 clinical studies involving over 300 patients or volunteers. Thus, Ii-Key-SARS-CoV-2 prophylactic peptide vaccines and other vaccines within the invention are qualified for ready deployment into human clinical studies. With respect to the ongoing COVID-19 pandemic, li-Key- SARS-CoV-2 prophylactic peptide vaccines offer a rapid path to global protection for human populations presently suffering from this dire public health and economic scourge.

One novel aspect of the invention employs state-of-the-art computational algorithms and databases to direct rapid development, construction and deployment of multi-potent li-Key-SARS- CoV-2 peptide vaccines to protect against SARS-CoV-2 infection, disease progression and community transmission. Proprietary computational tools are designed and implemented for accelerated design of proteome-derived, epitope driven vaccines, based on genetic and proteomic sequencing of target pathogens, including viruses. The exemplary Ii-Key-SARS-CoV-2 prophylactic peptide vaccines described herein can be multi-functionally designed and constructed to include class I, class II, and/or B-cell epitopes of SARS-CoV-2, to activate cellular (T cell) and humoral (antibody) immune responses, thereby creating a COMPLETE VACCfNE ^TM that is effective for balanced, selective immune regulation and acute and long-term protection against SARS-CoV-2/COVID-19.

Coupled with Applicants' computational vaccinology methods and tools for mapping and selecting candidate SARS-CoV-2 antigenic peptides, a novel selection and characterization paradigm employing staged in vitro and ex vivo studies to rapidly, economically and sensitively refine selection and characterization of li Key-SARS-CoV-2 antigen hybrid peptides. Using these methods and tools, vaccine candidate li Key-SARS-CoV-2 antigen peptides are rapidly characterized and selected for their discrete activities to elicit cellular and/or humoral immune activation having specific target and safety profiles. In exemplary aspects, novel li Key-SARS-CoV-2 peptide vaccines are designed and constructed to induce protection from SARS-CoV-2 infection primarily or exclusively via Th1- activated effector targets, without inducing pro-inflammatory Th2-responsive targets. Alternatively or additionally, the subject li Key-SARS-CoV-2 peptide vaccines can be constructed to primarily or exclusively induce neutralizing antibody responses, with minimal or no stimulation of non- neutralizing antibodies, to minimize antibody-dependent enhancement (ADE). In yet additional aspects, vaccine compositions can employ multiple li Key-SARS-CoV-2 peptides, or single hybrid peptides comprising multiple antigenic epitopes, that each has a discrete immunogenic target and/or functionality. In this manner li Key-SARS-CoV-2 peptide vaccines can diversely affect and regulate multi-functional immune responses in the host. In one embodiment, multiple li Key-SARS-CoV-2 peptides, or a multi-epitope peptide construct, can be designed, constructed and administered to generate discrete, non-competing neutralizing antibodies that target multiple, sterically-exclusive neutralization sites on the SARS-CoV-2 virus (for example multiple, spatially discrete targets on the SARS-CoV-2 spike ( S) protein receptor binding domain (RBD) and/or membrane fusion domain).

Ii-Key-SARS-CoV-2 candidate vaccine peptides are functionally screened and optimized for vaccine use according to novel selection and characterization protocol that includes detailed immune- regulatory activity profiling. In one aspect of this profiling methodology, Ii-Key-SARS-CoV-2 candidate vaccine peptides are tested against anti-SARS-CoV-2 immune-competent blood samples from convalescent COVID-19 patients, to evaluate T-Cell and antibody responses elicited by the hybrid peptides. Exemplary T-Cell assays include ELISpot assays to select for peptides that elicit CD4+ Th1 responses, and optionally to screen out peptides that elicit CD4+ Th2 responses. Other exemplary profiling assays validate candidate Ii-Key-SARS-CoV-2 antigenic peptides that can elicit CD8+ cytotoxic T cell responses. Additional profiling assays screen candidate Ii-Key-SARS-CoV-2 antigenic peptides against convalescent COVID-19 serum (or a defined panel of known anti-SARS- CoV-2 neutralizing antibodies) to validate immunogenic potential for eliciting neutralizing antibody responses, and or to exclude peptides that may elicit non-neutralizing antibodies. In one exemplary protocol, a sample comprising known or presumptive anti-SARS-CoV-2 neutralizing antibodies is contacted and incubated with Ii-Key-SARS-CoV-2 antigenic peptides (for example bound to a solid substrate, such as magnetic beads) then bound antibodies cognate for the Ii-K.ey-SARS-CoV-2 antigenic peptide are separated and tested for viral neutralization activity (e.g., using plaque reduction neutralization tests). These assays clarify that cognate Ii-Key-SARS-CoV-2 antigenic peptides bound by the neutralizing antibody are presumptively capable of eliciting a neutralizing antibody response in vivo. Related profiling methods and compositions are provided to screen and select li-Key-SARS- CoV-2 antigenic peptides that minimize incidence or severity of antibody dependent enhancement (ADE) in COVID-19 patients, likely to attend prior infection by endemic human coronaviruses, human SARS (hSARS) coronaviruses, or SARS-CoV-2.

Ii-Key-SARS-CoV-2 prophylactic peptide vaccines incorporate synthetic peptides engineered to exert multiple immune-regulatory effects, including to stimulate strong and specific helper (CD4+) T-cell responses. The minimal li-Key sequence (LRMK; SEQ ID NO: 37) works through its ability to deliver any desired peptide epitope directly to the MHC class II complex on the surface of antigen presenting cells. Helper T cells are essential for a robust antibody and cytotoxic T-cell response and long-term immunological memory (Crotty et al 2003: Lucas et al 2004). For example, dendritic cells require "licensing" by helper T cells before they can activate and expand cytotoxic T cells (Smith et al 2004), and influenza virosomes enhance class 1 restricted cytotoxic T cell induction by helper T-cell activation (Schumacher et al 2004). Helper T cells also activate and promote differentiation of antibody-producing plasma cells. The stimulation of helper T cells depends on the presentation of antigenic epitopes by MHC class II molecules. These are expressed by a subset of immune cells, including macrophages, dendritic cells, and B cells. Helper T cells recognize 9 amino acid epitopes within longer peptides that bind to the antigenic peptide binding sites of MHC class II molecules (with bound peptides extending 2 or more amino acids at either end beyond the open ends of the peptide binding trough). The binding affinity of epitopes to class 11 molecules is much lower than that of epitopes to MHC class 1 molecules that regulate immune activities of cytotoxic T cells. In ordinary MHC class II antigen processing by professional antigen presenting cells (APCs), allosteric antigenic peptide binding sites on MHC class II molecules are occupied and functionally blocked by a portion of the li protein during synthesis in the ER. Ml IC class II molecules with the site-blocking li protein traffic intracellularly to a post Golgi compartment, where cleavage of li protein occurs in a concerted process with MHC II binding of processed antigen fragments (Daibata et al 1994; Reyes et al 1994).

The portion of the li protein that interacts with the allosteric site, termed li-Key (core amino acid sequence LRMK), profoundly alters binding avidity of linked epitopes to MHC class II molecules. When li Key is covalently linked to an antigenic SARS-CoV-2 epitope, MHC class II molecules are readily charged with the linked epitope, even when cells have their MHC II epitope- binding trough already occupied by another peptide epitope (see, Figure 1 ). The li-Key addition profoundly enhances MHC class II loading and presentation of the linked antigen, directly charging MHC class II molecules on the surface of APCs (bypassing normal intracellular processing). li Key- antigen hybrid peptides of the invention dominantly appropriate MHC class 11 molecules on the surface of APCs. to potently activate helper T cells in an antigen-specific manner. This yields considerably stronger anti-SARS-CoV-2 cellular and humoral immune responses, through the interaction of helper T cells with B cells and other immune effector cells, including cytotoxic T lymphocytes (CTLs). li-Key modification enhances presentation of SARS-CoV-2 antigenic peptides by MHC class 11 molecules to induce potent stimulation of APC-mediated immune activation, for example stimulation of helper T-cell activation. Additional amino acids may be provided in the li-Key-SARS- CoV-2 hybrid peptide construct to interact with allosteric sites on MHC class II molecules adjacent to their epitope-binding troughs. Increased helper T-cell stimulation (for example as demonstrated by IFN-γ induction) has been demonstrated through a conclusive series of in vitro, ex vivo and in vivo assays for a large array of li Key-MHC class II epitope peptide vaccine constructs. Demonstration of the safety and multifunctional immunogenic activity of li-Key hybrids is also demonstrated herein through numerous preclinical and clinical studies.

Effective B-cell responses against infectious pathogens are critical to prevent and control infection. B cells mediate immunity through several pathways, cytokine signals and targets, including by producing virus-specific antibodies in both T-independent and a T-dependent pathways and in cognate and non-cognate cellular interactions to enhance activities of other cell types, most importantly helper T cells. In vivo experiments using B-cell-deficient mice demonstrate a major role for T-dependent versus T-independent antibody responses. These studies also indicate synergism between antibody and CD4+ T-cell activity. In addition to providing indirect "help" for B cells and cytotoxic T cells, additional effector functions have been described for CD4+ T cells in controlling viral infections, including potentiation of pathogen-specific cytolytic activity (Brown DM et al 2016). Additionally. CD4+ T cells also secrete antiviral cytokines such as IFN-γ and TNFa.

Extensive li-Key peptide vaccine studies have been completed to support the instant disclosure, including in vivo studies evaluating safety and efficacy of li-Key peptide vaccines directed against cancer and viral targets. Exemplary anti-cancer uses employed Ii-Key-HER2/neu antigenic hybrid peptides in Phase I and Phase 2 clinical studies, as described in the working examples below. Antiviral vaccine studies include a Phase 1 study conducted with Ii-Key-H5N 1 vaccine peptides against a potentially pandemic avian influenza.

The rationale for targeting viral proteins as prospective immunogens to generate protective immune responses against viral infection has been well established. Many noncellular vaccines are based on protein and peptide sequences derived from infectious disease agents, including recombinant peptide vaccines for influenza, hepatitis, and human papillomavirus. Common obstacles to rapid development and deployment of conventional vaccines for pandemic response, however, include lengthy production times and batch size restrictions. Using a traditional egg-based approach for influenza vaccine production, for example, the number of vaccine doses per egg is limited due to the viral growth characteristics. li-Key-SARS-CoV-2 prophylactic peptide vaccines of the invention are manufactured by an entirely synthetic process, enabling production of hundreds of millions of doses in a few months. Additional aspects of the invention are directed toward the selective design and construction of li-Key SARS-CoV-2 hybrid peptide immunogens that are "immune-regulatory" and "immune- selective" to simultaneously stimulate beneficial immune responses while averting or minimizing hyper-immune and hyper-inflammatory responses, as well as the potential for antibody-dependent enhancement (ADE) of infection and disease. ADE can occur, for example, when non-neutralizing antibodies bind to a newly infecting virus and promote enhanced virus uptake into host cells, presumptively via Fey receptors. In other instances, antibodies may directly enhance inflammation and thereby contribute directly to the severity of CSS/ARDS and other COVID-19 hyper-immune and hyper-inflammatory pathogenesis (de Alwis et al. 2020; Hotez et al. 2020). Data on previous vaccines for RSV and measles, and from nonclinical studies with coronavirus vaccine candidates, suggest that vaccines that are immune-selective towards a Th1 versus Th2 response are protective against infection while possibly minimizing pathogenesis (de Alwis et al. 2020), however the complexity of immune responses involved render these prospects speculative. The instant disclosure provides novel computational vaccinology methods and tools for mapping and selecting candidate SARS-CoV-2 antigenic peptides in combination with a novel peptide selection and characterization paradigm that includes optimizing candidate Ii-Key-SARS-Co-2 hybrid peptides for Th1 -biased antigenic potency, yielding surprisingly potent and selective immune-regulatory vaccine agents. Additionally, in certain embodiments the subject li-Key-SARS-Co-2 hybrid peptides are formulated for vaccine administration with a novel adjuvant partner, 3M 052, which also enhances Th1 T cell responses and other immune activities for liposomal antiviral vaccine formulations (Smirnov et al. 201 1).

Immune-regulatory Ii-Key-SARS-Co-2 hybrid peptide vaccine compositions are multi- functional and typically immuno-selective (for example, selective to elicit neutralizing antibodies, and discretely activate ThI -biased T-cell responses, while disaffecting or negating Th2-biased T-cell responses). Peptide selection and characterization methods and tools described herein provide for selection and optimization li-Key hybrid peptide antigens having multi-functional and selective immune-regulatory profiles, for example using ex vivo assays employing COVID-19 convalescent blood samples. In illustrative embodiments SARS-CoV-2 convalescent PBMCs are exposed to different candidate Ii-Key-SARS-CoV-2 peptide constructs, and ELISpot assays are used to select for peptides that are positive for eliciting Th1 -type T cell responses (for example as demonstrated by stimulation ofTh1 -positive markers, such as IFN-y. IL-2, and/or TNF-a), and null for eliciting Th2- type T cell responses (for example as demonstrated by non-detection of Th2-positive markers, such as IL-4. IL-5, and/or IL-13). The latter screening criteria can conversely be used to positively identify and characterize Ii-Key-SARS-CoV-2 peptides that do stimulate Th2-type T cell responses, for use in alternative embodiments of the invention.

Peripheral blood mononuclear cells (PBMCs) include a diverse array of peripheral, mononucleate blood cells, including T cells, B cells, NK cells and monocytes. This classification excludes enucleate erythrocytes and platelets, and granulocytes/polymorphonuclear cells (neutrophils, basophils, and eosinophils that have multi-lobed nuclei). In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, with a small percentage being dendritic cells. PBMCs are generally collected from whole blood using ficoll, a hydrophilic polysaccharide that separates blood during gradient centrifugation into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells. Monocytes represent a diverse immune/inflammatory cell type. They may be primed for phagocytosis, innate sensing/immune responses and migration, among other activation/effector states. Intermediate monocytes are capable of antigen presentation, cytokine secretion and apoptosis regulation, while other "non-classical" monocytes are involved in complement and Fc gamma-mediated phagocytosis and adhesion. Monocytes are heterogenous for expression of chemokine receptors, for ROS production and for chemotaxis activity, as well as their ability to secrete pro-inflammatory molecules, such as IL-6, IL-8, CCL2, CCL3, and CCL5. It is now' widely accepted that classical monocytes have the ability to differentiate into monocyte-derived macrophages (moMos) and DCs (moDCs) and play an integral part in shaping inflammation and its resolution in tissues. Intermediate monocytes express the highest levels of antigen presentation-related molecules, and are also known to secrete TNF-a, IL- 1β, IL-6, and CCL3. The precise roles of monocites in immunity and inflammation remain elusive.

An equally complex subset of immune/inflammatory cells are macrophages. Macrophages, are considered ancient cells in Metazoan phylogeny, found in all tissues and displaying great anatomical and functional diversity. In tissues, they are "territorial", forming a "tissue within a tissue". The simplest classification scheme for macrophages follows the "mononuclear phagocytic system" (MPS), which embraces all highly phagocytic cells and their bone marrow progenitors. In the MPS scheme, adult macrophages are defined as mature MPS cells, with tissue macrophages deriving from circulating monocytes in the bone marrow . However, macrophages have several distinct lineages classifiable based on their inflammatory state/activity. These include activated macrophages (AMs) and alternatively activated macrophages (AAMs). AMs are activated, for example, in Th1 mediated immune responses to viruses, while AAMs are activated during parasitic infections. Transcriptional profiling of resident macrophages in different tissues and compartments by the "Immunological Genome Project" indicate that these cells show great transcriptional diversity with minimal overlap, suggesting many discrete classes. Macrophages have roles in almost every aspect of an organism's biology, ranging from development, homeostasis, to immune and inflammatory responses to pathogens and injury. Macrophages of different phenotypes can be recruited from the monocyte reservoirs of blood, spleen and bone marrow, and possibly from resident tissue progenitors and through local proliferation.

Additional methods of the invention provide for development of Ii-Key-SARS-CoV-2 antigenic peptide hybrid vaccine compositions and methods that effectively elicit CD8+ "killer" T cell responses in immunized subjects. NaTve CD8 T cells have the potential to differentiate into both short-lived effectors and memory precursors following activation. Short-lived effector cells are commonly defined as CD127lo, KLRG-1 hi and express a panel of transcription factors which promote effector activities but limit their proliferative capacity and survival. By contrast, memory precursors are typically CD127hi, K.LRG- 1 Io. These cells also have certain effector properties including the ability to produce IFN-γ, but unlike their short-lived counterparts they are more likely to survive the downregulation of the response and transition into memory populations capable of persisting over time and confering long-lived immunological protection. Different priming strategies and infections can skew the developmental process in either direction, which is determined by various factors including duration of stimulation and the composition of the cytokine milieu. This likely results in a spectrum of CD8 T cell differentiated states, ranging from terminally differentiated effector cells to memory precursors.

The antigen-driven activation of naive CD8 T cells is a critical first step in a differentiation process that generates heterogeneous subsets of cells that vary in their phenotypes, functions/activities, anatomical location, and longevity. The developmental fate of individual naive CD8 T cells is not preset. Instead each naTve cell is immunologically pluripotent and possesses the capacity to give-rise to multiple distinct subsets. This permits the formation of short-lived, but highly functional, effector populations that operate to clear infections, as well as memory precursor effector cells that survive over longer periods, transition into memory T cells and contribute to long-lived immunological protection. Because the developmental pathway taken by a naive CD8 T cell is not predetermined, other factors such as the degree of antigenic activation, contact time with antigen-presenting cells, asymmetric division, and environmental cues, including cytokine availability and signaling, are vital forces that shape the overall outcome of CD8 T cell responses.

In various aspects of the invention, SARS-CoV-2 antigenic peptides and Ii-Key-SARS-CoV-2 antigenic peptide hybrid constructs arc selected for use in vaccine compositions and methods for their demonstrated ability to elicit CTL activation, that is, to induce a naive CD8+ T cell to differentiate into a short term or long term activated CTL. Various assays are useful for characterizing CD8+ T cell induction by SARS-CoV-2 antigenic peptides and Ii-Key-SARS-CoV-2 antigenic peptide hybrid constructs, including various cell-based, ex vivo and in vivo model systems known in the art. In one illustrative embodiment detailed in the examples below, PBMCs from healthy and COVID-19 convalescent subjects are studied to demonstrate the potential of a Ii-Key-SARS-CoV-2 antigenic peptide hybrid to induce CD8+ CTL maturation (e.g., as demonstrated by antigen-specific elevation in Granzyme B expression levels in treated/responsive cells relative to controls).

Further aspects of the invention employ design and construction methods and tools to identify immune-selective 1 i-Key SARS-CoV-2 hybrid peptide immunogens that eliminate or reduce risks of antibody dependent enhancement (ADE). This further selection eliminates li-Key SARS-CoV-2 hybrid peptide candidates that may elicit off-target antibody responses associated with ADE. In one embodiment. li-Key SARS-CoV-2 peptides formulated with an optimizing adjuvant yields an immune response profile compatible with protection against ADE. Alternatively, li-Key SARS-CoV-2 peptides are screened using an appropriate ADE assay to eliminate peptides that elicit off-target or non-neutralizing antibody responses. To address the potential for off-target and non-neutralizing antibody responses, exemplary screens employ COVID 19 convalescent blood samples that presumptively contain neutralizing antibodies and anti-SARS-CoV-2-competent immune effector cells. Presumptive neutralizing antibodies present in these samples are isolated based on cognate interaction with li Key SARS CoV-2 peptides, as described above, and the isolated antibodies are further screened to confirm neutralizing activity using a suitable viral neutralization assay (e.g., PRNT). Further screening processes may be used to exclude off-target and other ADE activities of immune-selective li-Key-SARS CoV-2 antigen peptides to confirm that they elicit discrete, neutralizing antibody responses without activating pro-inflammatory effector cells, signals and pathways identified to be associated with CSS and ARDS in severe COVID-19 patients.

In one such assay antibodies from a COVID-19 convalescent blood or serum sample, or pool of blood or serum samples, are isolated based on cognate binding to a li-Key-SARS CoV-2 antigen peptide (presumptively the sample(s) contain both neutralizing and non-neutralizing anti-SARS-CoV- 2 antibodies) is incubated with a suitable ADE assay subject or sample. Non-neutralizing antibody interactions and other ADE-contributory off-target responses are thereafter detected. In one exemplary embodiment, a comprehensive pool of antibodies from a COVID convalescent blood or serum sample, isolated through cognate binding to a candidate li-Key SARS CoV-2 peptide, is incubated with U937 cells. These cells model hyper-immune and hyper-inflammatory activation phenomena correlated with CSS. for example as detected by upregulation of pro-inflammatory cytokines and other indicia) (Liu et al. Virologica Sinica (2019) 34:648-661 ). These and comparable assays provide for exclusion of candidate li-Key SARS CoV-2 peptides that elicit non-neutralizing B-cell reactions and other potentially pathogenic responses, such as Th2-type responses and pathogenic hyper-inflammatory (e.g., CSS) responses

Design and Construction of Ii-Key-SARS-CoV-2 Hybrid Peptides

I lybrid li-Key SARS CoV-2 antigenic peptides are constructed to incorporate a functional li- Key sequence for mediating enhanced MHC II antigen binding and presentation. The hybrid peptides can incorporate a canonical mammalian li-key peptide sequence LRMKLPKPPKPVSKMR (SEQ ID NO: 36), a functional segment of this peptide sequence, or a natural or synthetic variant thereof. The mechanisms of MHC class II antigen processing, whereby li protein is cleaved to release fragments that regulate binding and locking-in of antigenic peptides to the antigenic peptide binding site of MHC class II molecules has been described (Humphreys U.S. Pat. No. 5,559,028, and Humphreys, et al. U.S. Pat. No. 5,919.639). One segment of the li protein. li (77-92) was found to act at an allosteric site outside the antigenic peptide binding site near the end of the site holding the N-terminus of the antigenic peptide. Humphreys and colleagues further disclosed three mechanisms of activity for li- Key peptides associated with MHC II peptide recognition and processing. In the first mechanism antigenic peptides are spilled from cell surface MHC class II molecules by the action of li-Key peptides. In the second, charging of the antigenic peptide binding sites on MHC class II molecules is promoted for antigenic peptides linked to li-Key peptides. The third mechanism modulates rates of association/dissociation of antigenic peptides linked to li-Key peptides from trimolecular MHC molecule/antigenic peptide-li-Key hybrid/T cell receptor complex, modulating cell-to-cell signal transduction interactions in a manner that regulates differentiation and function of interactive T lymphocytes.

Covalent coupling of functional li-Key peptides with SARS-CoV-2 antigenic peptides considerably increases potency of antigen presentation. By acting at the initial regulatory, antigenic peptide recognition stage of immune response, these hybrid peptides are particularly well-suited for prophylactic and early therapeutic use against SARS-CoV-2. Various modifications to Li-Key peptides for coupling to SARS-CoV-2 antigenic peptides are contemplated for use within the invention. Experimentation with modified li-key peptides reveal that a wide variety of modifications to the canonical Li-Key polypeptide sequence can be made without impairing MHC II antigen recognition/binding enhancement for coupled peptide antigens. Certain modifications actually improve antigen binding/presenting activity of the hybrid polypeptide. A diverse assemblage of modified li-key peptides that retain antigen presentation enhancing activity are contemplated and can be readily confirmed for operability within the compositions and methods of the invention. Modifications of the li-key peptides can include deletion, addition or substitution of one or more amino acids from the N-terminus, deletion, addition or substitution of one or more amino acids from the C -terminus, protection of the N-terminus, and other rational peptide modifications. Deletions of the li-key peptide to a minimal sequence that retains at least 4 contiguous amino acids of (LRMK; SEQ ID NO: 37), exhibit functional activity. Various natural or non-natural amino acids may be substituted at selected residue positions. Some examples of molecules that may be substituted are peptidomimetic structures, D-isomer amino acids, N-methyl amino acids, L-isomer amino acids, modified L-isomer amino acids, and cyclized derivatives. Modification of Li-Key peptides can be further guided by known principals of rational drug design and molecular modeling based on X-ray diffraction data, nuclear magnetic resonance data, and other computational methods. Examples of useful modified variants of Li-key peptides that have been demonstrated to retain activity for use within the methods and compositions of the invention are LRMKLPK (SEQ ID NO; 38), LRMKLPKS (SEQ ID NO: 39). LRMKLPKSAKP (SEQ ID NO: 40), and LRMKLPKSAKPVSK (SEQ ID NO: 41). Additional modified variants li-key peptides for use within the invention are described in Humphreys, et al., U.S. Pat. No. 5.919,639, and Humphreys U.S. Pat. No. 5,559.

Hybrid li-Key SARS CoV-2 antigenic peptides of the invention are typically terminally protected, for example by acetylation at the N-terminus and amidation at the C-terminus, to improve stability (e.g.. to inhibit activity of exopeptidases). Illustrative protected constructs for this purpose include Ac-LRMK (SEQ ID NO: 37)-5-aminopentanoyl; Ac-lAYLKQATAK (SEQ ID NO: 42)- NH ₂ ; AC-LRMK (SEQ ID NO: 37)-5-aminopentanoyl; Ac-LPKSIAYLKQATAK (SEQ ID NO: 43)- NH ₂ ; AC-LRMKLPKSIAYLKQATAK (SEQ ID NO: 44)-NH ₂ ; Ac-RMKLPKSAKPIAYLKQATAK (SEQ ID NO: 45)-NH ₂ ; and Ac-MKLPKSAKPVSKIAYLKQATAK (SEQ ID NO: 46)-NH ₂ .

Hie Ii-Key-SARS-COV-2 vaccine is based on the li-Key platform and uses a similar manufacturing approach as compounds currently in clinical trials targeting HER-2/neu. the li-Key component is shared across this platform. The I i-Key peptide moiety generally has a minimal effective amino acid sequence LRMK (SEQ ID NO: 37) and is usually bound by a linker to an MHC class 11 epitope or other antigenic determinant sequence. In general, the epitope or antigenic determinant is from about 6 to about 40 amino acids, more commonly from about 10 to 15 amino acids in length, and the linker is selected from a variety of useful linkers, for example a 5- aminopentanoic acid (ava) linker between the epitope and the li-Key (the ava linker is often chosen because of its inert character, comprised of amino acids lacking side chains). Thus, an illustrative Ii-Key-SARS-CoV-2 antigenic peptide hybrid of the invention may be constructed according to the following formula:

Ac-(li-Key)-ava-XXXXXX-NH2 wherein Ac denotes N-terminal blocking acetylation. li-Key refers to a functional li-Key peptide, -ava denotes a delta aminovaleric acid (5-aminopentanoic acid) linker, and XXXXXX designates a SARS-CoV-2 antigenic peptide of from about 6-40 residues comprising one or more MIIC class I and/or MHC II epitope(s) and/or other antigenic determinant(s).

Modified hybrid li-Key SARS CoV-2 antigenic peptides can be designed to be totally peptide in composition, or to have substantially non-peptide physiochemical properties. Certain variants are rendered substantially non-peptide in character to have more favorable in vivo properties such as, for example, to enhance penetration through cellular membranes, improve solubility, increase in vivo half life, improve resistance to proteolysis, reduce aggregation or conjugation, provide for oral bioavailability, and other improvements. In exemplary embodiments, hybrid peptides of the invention can be constructed wholly of peptide constituents, or include peptidomimetic or additional or substituent chemical groups that may synthesized and selected according to known rational peptide and chemical design methods and principals, as described for example in Geysen, et al., U.S. Pat. No. 4,708,871 ; Geysen, et al., U.S. Pat. No. 5, 194,392; Schatz, et al., U.S. Pat. No. 5,270, 170; Lam, et al., U.S. Pat. No. 5,382,513; Geysen. et al.. U.S. Pat. No. 5.539,084; Pinilla, et al., U.S. Pat. No.

5.556.762; Geysen, et al.. U.S. Pat. No. 5.595.915; Kay, et al., U.S. Pat. No. 5,747,334; and Nova, et al., U.S. Pat. No. 5,874,214.

Also contemplated within the scope of the invention are pharmaceutically acceptable salts of the hybrid peptide constructs, comprising an acidic or basic group incorporated into the hybrid peptide structure. Pharmaceutically acceptable salts include all biocompatible acceptable salts such as acetate, ammonium salt, benzenesulfonate, benzoate, borate, bromide, calcium ede- tate, camsylate, carbonate, chloride/dihydrochloride, citrate, clavulanate, edetate, edisylate, estolate, esylate, fumarate, hexy lresorcinate. hydrabamine. hydroxynaphthoate. iodide, isothionate, lactate, lactobionate, laurate, mesylate, methyl- bromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N- methylglucamide. oleaste. oxalate, pamoate, palmitate, panoate, pantothenate, phosphate/diphosphate, polygalacturonate, subacetate, sulfate, tartrate, tosylate, tri- ethiodide, valerate, and the like. Salt forms of hybrid peptides can improve solubility, bioavailability, hydrolysis characteristics, stability and other properties, and can be useful for sustained-release and pro-drug formulation. Salts of the hybrid peptides can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and from bases such as ammonia, arginine, choline, diethanolamine, diethylamine, ethylenediamine, lysine. N-methyl-glutamine. ornithine, piperazine, tris(hydroxymethyl)aminomethane. tetramethylenediamine hydroxide, and the like. Additional chemical variants of the hybrid peptides can be routinely constructed for improved properties as noted, according to conventional rational design methods and principals. For example, an acid (-COOR) or alcohol group may be targeted or introduced to form pharmaceutically acceptable esters of the hybrid peptides, acetate, maleate, pivaloyloxymethyl, and other esters, to yield improved solubility or hydrolysis characteristics and provide sustained release or prodrug formulations.

Among their many benefits. li-Key-SARS-CoV-2 hybrid peptide vaccines can be rapidly, efficiently and inexpensively manufactured, for example using solid phase peptide synthesis (Figure 2). This technique allows for construction of a desired Ii-Key-SARS-CoV-2 hybrid peptide chain through successive reactions of amino acid derivatives. Exemplary solid supports comprise resin beads that link to the peptide chain throughout the synthesis, allowing excess reagents and side products to be removed by washing and filtration between each step. The final peptide is purified using a simple chromatography step and tested to ensure purity. Applicants have qualified numerous agents for this purpose, including PolyPeptide Laboratories (Torrance, CA), CordenPharma (Boulder CO), and Bachem (Torrance, CA), all global industry leaders in clinical and commercial scale peptide synthesis. These and other agents ensure the necessary capacity and redundancy to provide bulk manufacturing in the United States for clinical and commercial Ii-Key-SARS-CoV-2 hybrid peptide pandemic response vaccination programs. Specifically, hundreds of kilograms of peptide product, equating to more than 200 million doses can be manufactured within a few months. Additionally, Applicants have identified agents, including Ajinomoto Bio-Pharma Services (San Diego, CA) and Thermo-Fisher (Waltham. MA) to provide fill/finish material for clinical and commercial vaccination requirements.

Ii-Key-SARS-CoV-2 hybrid peptides will typically comprise synthetic amino acid peptides optionally formulated in a liquid-in-vial formulation containing 250, 500, or 1000 μg of li-Key- peptides with or without adjuvant. The final product will be filled in 2-mL vials by the manufacturer. Stability will be determined by the manufacturer at -15±5°C (dedicated freezer) with sample analysis at 0, 3, 6. 9, 12, 18, and 24 months and at 4±2°C (refrigerated) and 25±2°C (stability chamber) with sample analysis at 0, 1. 3, and 6 months. Finished vaccine will typically be formulated as a refrigerated liquid formulation including adjuvant, in multidose vials. For study purposes, Ji-Key- SARS-CoV-2 hybrid peptide may be solubilized in either saline or in an adjuvant formulation designed to stabilize the multi-peptide vaccine mixtures and provide appropriate adjuvant properties.

Additional quality control studies are conducted after synthesis to ensure purity, stability, and biological compatibility of Ii-Key-SARS-CoV-2 hybrid peptides, including:

• MS for identity

• Sequencing by MS for identity

• AAA for identity

• RP-HPLC for purity (>95% for peptide)

• Quantification of counter ion • Quantitative AAA for peptide content

• Residual solvents

• Total fluorine

• Bacterial endotoxin (Limulus Amebocyte Lysate test)

• Karl Fisher for water content

• Bioburden

The resultant hybrid peptides will typically have greater than 95% purity and be substantially free of contaminants, such as residual organic volatiles, bacteria, fungi and the like.

Ii-Key-SARS-CoV-2 hybrid peptides will typically incorporate a functional "spacer" between the li-key and antigenic peptide members. The spacer is usually composed of a covalently joined group of atoms ranging from zero to a number of atoms which, when arranged in a linear fashion, would extend up to a length corresponding to a length of peptidyl backbone atoms of about 20 linearly-arrayed amino acids. Most often the pacer is less than a length of a peptidyl backbone of 9 amino acids linearly arranged. In certain embodiments, a spacer length corresponding to a peptidyl backbone length of between 4 and 6 linearly arranged amino acids is used. The spacer may be engineered to have limited or no ability to hydrogen bond in any spatially distinct manner to an MHC class II molecule. While the spacer may be constructed readily in whole or in part from amino acids, various chemical groups may be incorporated to form the spacer segment instead of amino acids (see, c.g.. Tournier, et al., U.S. Pat. No. 5,910,300).

In exemplary embodiments the spacer is comprised of an aliphatic chain interrupted by heteroatoms, for example a C ₂ -C ₆ alkylene, or =N-(CH ₂ ) ₂ -6-N= . Alternatively, a spacer may be composed of alternating units, for example of hydrophobic, lipophilic, aliphatic and aryl-aliphatic sequences, optionally interrupted by heteroatoms such as O, N, or S. These optional components of useful spacers may be chosen from various classes of compounds, including sterols, alkyl alcohols, polyglycerides with varying alkyl functions, alkyl-phenols, alkyl-amines, amides, hydroxyphobic poly-oxyalkylenes, and the like. Other examples include hydrophobic polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polycaprolactones, and polylactic, polyglycolic and polyhydroxybutyric acids. Exemplary spacers may also contain repeating short aliphatic chains, such as polypropylene, isopropylene. butylene, isobutylene, pentamethlyene, and the like, separated by oxygen atoms. Additional peptidyl sequences useful to construct spacers are described in Whitlow, et al., (U.S. Pat. "No. 5,856,456).

Spacers for constructing li-Key-SARS-CoV-2 hybrid peptides can also beneficially incorporate a chemical group that is subject to in vivo processing or modification, for example protease cleavage. Without limitation, such a chemical group may be designed for cleavage catalyzed by a protease, by a chemical group, or by a catalytic monoclonal antibody. Protease- sensitive chemical groups include, tryptic targets (two amino acids with cationic side chains), chymotryptic targets (with a hydrophobic side chain), and cathepsin sensitive targets (B, Dor S). In addition, chemical targets of catalytic mono- clonal antibodies, and other chemically cleavable groups are suitable, all of which are known to persons skilled in the art of peptide synthesis, enzymic catalysis, and organic chemistry.

The present invention provides a diverse array of anti-SARS-COV-2 antigenic peptides for use in li-Key peptide hybrids and other vaccine compositions and methods of the invention. li-H+Key hybrid peptides will contain an li-Key element fused or linked with a an antigenic peotide comprising one or more antigenic or immunogenic "epitopes" or "determinants". In certain aspects, the subject epitope(s) or dcterminant(s) will include one or more MHC Class II antigenic epitopes as described herein. Certain li-Key peptide hybrid constructs of the invention will contain multiple MHC Class 11 epitopes. The inclusion of multiple MHC Class II epitopes provides for efficacy in inducing an immunogenic response in a greater fraction of an immunized human population, because multiple epitopes are likely to be presented by different alleles. In addition to employing an optional plurality of multiple MHC Class II epitopes, the invention also contemplates inclusion of one or more MHC Class I epitope(s) within a single li-Key-SARS-CoV-2 antigenic peptide construct, or within multiple li-Key-SARS-CoV-2 antigenic peptide constructs used in a multi-peptide hybrid vaccine composition. Additionally or alternatively. Ii-Kcy-SARS-CoV-2 antigenic peptide constructs may include antigenic peptide residues that comprise one or more antibody recognized immunogenic determinants, or ARDs. As used herein, antigenic or immunogenic "epitopes" and "determinants" will be understood as broad synonyms for small peptide regions, domains or sequences that can be recognized by antibodies, receptors or other cognitive or receptor elements of the mammalian immune system, to elicit a cognate (epitope- or antigen-specific immune response). The terms antigenic "epitope" and "determinant" typically encompasses MHC Class II epitopes, MHC Class I epitopes and ARDs.

The Examples herein below illustrate a diverse array of "anti-SARS-CoV-2" MHC Class II and MHC Class I epitopes, and multi-epitope "cluster" peptides, computationally predicted and experimentally validated to elicit multi-functional and discrete anti-SARS-CoV-2 effective immune responses in immune helper, effector and memory cells, in vitro and in immunized mammalian subjects. The rationally and experimentally-determined epitopes and epitope clusters will often be effective to elicit or enhance an MHC Class Il-mediated immune response, and/or an MHC Class I-mediated immune response, and to. directly or indirectly, induce downstream effector immune activity, for example, T helper and/or B cell proliferation/activation, cytokine and/or antibody expression, and/or cytotoxic T lymphocyte (CTL) proliferation/activation. Interactions between li- Key-SARS-CoV-2 antigenic peptides of the invention with cognate cells of the immune system greatly amplify numbers of responsive immune cells, of diverse types. Large numbers of primed and responsive immune cell types are stimulated to proliferate, activate or alter gene expression patterns, activate or alter cellular receptor patterns and/or functions, and activate or alter cytokine, chemokine and/or growth factor expression patterns or functions.

In certain embodiments. Ii-Key-SARS-CoV-2 antigenic peptides of the invention enhance T helper cell stimulation mediated by Class II epitopes. This enhanced stimulation is extraordinarily potentiated by the li-Key component of the antigenic peptide hybrid, with T helper stimulation effects increasing up to about 250 times for li-Key fusions compared to the corresponding antigenic peptide alone. Immune stimulation by Ii-Key-SARS-CoV-2 antigenic peptides often leads to clonal expansion of immunoregulatory cells, such as activated T cells, coupled with a downstream "cascade" effect of immune activation throughout the immune system of vaccinees.

MHC Class Il-presented antigenic epitopes incorporated within Ii-Key-SARS-CoV-2 antigenic peptides exert immunogenic. effects through presentation by MHC Class I and MHC Class II molecules on surfaces of antigen presenting cells (APCs). Two principal APC types are dendritic cells and macrophages. These APCs have MHC Class 1 and MHC Class 11 molecules on their surfaces that function in antigen processing and presentation. Antigenic li-Key hybrid peptides of the invention interact with MHC Class 1 or MHC Class II molecules through noncovalent binding, to effect subsequent MHC-mediated presentation to antigen-specific receptors on T cells. This association between the antigen-primed APC and adjacent T cell typically triggers a cascade of immune effects, including induction of T cells and the subsequent expansion of this induced cell population. T helper cells stimulated in this manner respond in a variety of ways. For example, stimulated T helper cells may originally be in an "undetermined" state, designated as ThO, whereafter the antigen induction triggers their differentiation to one of two general pathways, designated as Th1 or Th2 (characterized by distinct differentiation pathways and cellular fates, including expression of different characteristic panels of cytokines and other immune and inflammatory effector molecules-as described elsewhere herein). In some cases, T helper cells are stimulated by Ii-Key-SARS-CoV-2 antigenic peptide hybrids to release cytokines and other factors that function as activation signals for B cells. In response, B cells can produce surface immunoglobulins that may recognize and specifically bind ARD elements within the SARS-CoV-2 antigenic peptide component of the hybrids. This can result in internalization of the antigenic peptide to be processed and presented by the B cell. In this case, when a li-Key- SARS-CoV-2 antigenic hybrid peptide is internalized by a B cell, MHC Class II epitopes present within the hybrid may be alternately processed for display on the B cell surface in association with MHC Class 11 molecules. Such presentation further stimulates helper T cells resulting in proliferation and maturation of B lymphocytes to plasma cells that produce antibodies cognate to the ARD sequence within the SARS-CoV-2 antigenic peptide hybrid.

MHC Class 1 and MHC Class II epitopes are typically comprised of from about 8 to about 12 amino acid residues. ARD elements usually have a size range of from about 6 to about 16 amino acid residues. ARDs are recognized based on their 3-dimensional structure, whereas MHC Class I and MHC Class II epitopes are recognized based on their linear, primary amino acid structure. MHC Class 1 epitopes, MHC Class 11 epitopes and ARD sequences may be arranged in an overlapping manner while retaining full functionality of all represented epitopes. The respective functions of each epitope within a multi-epitope hybrid can be independently expressed, not necessarily simultaneously (because the respective epitopes or antigenic determinants must be bound as a linear array of amino acid residues by a MHC Class 1 or MHC Class 11 molecule, or recognized as a folded peptide structure by an antibody), but certainly all can be active (even when present in a single peptide) within in multi- epitope peptide vaccine. In a population of injected peptides, with respective processing, binding to cell surface MHC molecules, and/or cognate antibody recognition and binding, all three classes of epitopes/determinants can be operative within one or a plurality of Ii-Key-SARS-CoV-2 antigenic peptide hybrids, each effecting an independent immunogenic activity within the immunized subject.

Length-minimized epitopes and antigenic determinants are preferred for several reasons, including simplicity and cost of synthesis, reduced susceptibility to proteolytic degradation, reduced likelihood of for metabolic change leading to clearance or adsorption, among other factors. In this regard, using a plurality of epitopes which overlap one another (i.e., wherein one or more amino acid residues may be integral to multiple epitopes) is often desired. Thus, while individual epitopes may typically comprise between about 8-12 amino acids, and ARDs may span between about 6-16 residues, a span of about 9-10, 10-12, 13, 14, 15, 16, 17-18, 20-22, 23, up to about 30 residues, will often contain a desired cluster of multiple epitopes, including a combination of one or more MHC I, MHC 11, and/or ARD determinants, as illustrated by the various cluster epitopes represented in the examples below. Within these examples, multiple epitopes, of the same or different class, are clustered in useful antigenic peptides of between about 13-22 amino acids, including many of intermediate lengths, such as 14, 15, 16, and 18 residues, comprising multiple epitopes of the same or distinct classes.

In exemplary embodiments of the invention, SARS-CoV-2 antigenic peptides are provided for constructing Ii-Key-SARS-CoV-2 peptide hybrids, wherein the unfused peptides comprise one of the following sequences, or at least 9 contiguous amino acid residues (that retain antigenic activity) therefrom: YSFVSEETGTLIVN (SEQ ID NO: 47); KKLLEQWNLVIGFL (SEQ ID NO: 48); LLQFAYANRNRFLYI (SEQ ID NO: 49); SYFIASFRLFARTR (SEQ ID NO: 50); ASFRLFARTRSM WSFN (SEQ ID NO; 51 ); GHLRIAGHHLGRSD (SEQ ID NO: 52); KEITVATSRTLSYY (SEQ ID NO: 53); SYYKLGASQRVAGD (SEQ ID NO: 54); GNYKLNTDHSSSSDNI (SEQ ID NO: 55): PAYTNSFTRGVYY; EGVYFASTEKSNI1R;

VYYHKNNKSWMESE (SEQ ID NO: 58): FRVYSSANNSTFEYV (SEQ ID NO: 59) DGYFKIYSKHTPINL (SEQ ID NO: 60): QTLLALHRSYLTPGDSS (SEQ ID NO: 61); TFLLKYNENGTITDA (SEQ ID NO: 62); SNFRVQPTESIVRF (SEQ ID NO: 63); GEVFNATRFASVYA (SEQ ID NO: 64): SVYAWNRKR1SNSVAD (SEQ ID NO: 65); GNYNYLYRLFRKSNLKPFERDI (SEQ ID NO: 66): EGFNSYFPLQSYGFQPT (SEQ ID NO: 67); QSYGFQPTNGVGYQPY (SEQ ID NO: 68); KSVNFNFNGLTGT (SEQ ID NO: 69); GFNFSQILPDPSKPSKRS (SEQ ID NO: 70); DEMIAQYTSALLA (SEQ ID NO: 71); IPFAMQMAYRFNGIGV (SEQ ID NO: 72); KLIANQFNSAIGKI (SEQ ID NO: 73); NQFNSAIGKIQDSL (SEQ ID NO: 74); GRLQSLQTYVTQQL (SEQ ID NO: 75); KYFKNHTSPDVDL (SEQ ID NO: 77); ISGINASVVNIQKEI (SEQ ID NO: 78).

In related embodiments, illustrative SARS-CoV-2 antigenic peptides for making li-Key- SARS-CoV-2 peptide hybrids comprise at least 10,11, 12, 13 or 14 or more contiguous amino acids from one of the following exemplary SARS-CoV-2 antigenic peptide sequences YSFVSEETGTLIVN (SEQ ID NO: 47); KKLLEQWNLV1GFL (SEQ ID NO: 48); LLQFAYANRNRFLYI (SEQ ID NO: 49); SYFIASFRLFARTR (SEQ ID NO: 50); ASFRLFARTRSMWSFN (SEQ ID NO: 51 );

Gl ILRIAGHHLGRSD (SEQ ID NO: 52): KEITVATSRTLSYY (SEQ ID NO: 53); SYYKEGASQRVAGD (SEQ ID NO: 54): GNYKLNTDHSSSSDNI (SEQ ID NO: 55); PAYTNSFTRGVYY: EGVYFASTEKSNIIR; VYYHKNNKSWMESE (SEQ ID NO: 58); FRVYSSANNSTFEYV (SEQ ID NO: 59) DGYFKIYSKHTPINL (SEQ ID NO: 60); QTLLALHRSYLTPGDSS (SEQ ID NO; 61 ); TFLLKYNENGTITDA (SEQ ID NO: 62); SNFRVQPTESIVRF (SEQ ID NO: 63); GEVFNATRFASVYA (SEQ ID NO: 64);

SVYAWNRKRISNSVAD (SEQ ID NO: 65); GNYNYLYRLFRKSNLKPFERDI (SEQ ID NO: 66); EGFNSYFPLQSYGFQPT (SEQ ID NO: 67); QSYGFQPTNGVGYQPY (SEQ ID NO: 68);

KSVNFNFNGLTGT (SEQ ID NO: 69); GFNFSQILPDPSKPSKRS (SEQ ID NO: 70); DEMIAQYTSALEA (SEQ ID NO: 71 ); IPFAMQMAYRFNGIGV (SEQ ID NO: 72); KLIANQFNSAIGKI (SEQ ID NO: 73); NQFNSAIGKIQDSL (SEQ ID NO: 74);

GRLQSLQTYVTQQL (SEQ ID NO: 75); KYFKNHTSPDVDL (SEQ ID NO: 77); ISGINASVVNIQKEI (SEQ ID NO: 78).

In other embodiments, exemplary SARS-CoV-2 antigenic peptides for making li-Key-SARS- CoV-2 peptide hybrids are modified variants or derivatives of a primary antigenic peptide, having at least 80%, 85%, 90%, 95% or greater amino acid identity with a full or partial SARS-CoV-2 antigenic peptide selected from YSFVSEETGTLIVN (SEQ ID NO: 47); KKLLEQWNLVIGFL (SEQ ID NO: 48); LLQFAYANRNRFLYI (SEQ ID NO: 49); SYFIASFRLFARTR (SEQ ID NO: 50);

ASFRLFARTRSMWSFN (SEQ ID NO: 51 ); GHLRIAGHHLGRSD (SEQ ID NO: 52); KEITVATSRTLSYY (SEQ ID NO: 53); SYYKEGASQRVAGD (SEQ ID NO: 54); GNYKLNTDHSSSSDNI (SEQ ID NO: 55): PAYTNSFTRGVYY; EGVYFASTEKSNIIR; VYYHKNNKSWMESE (SEQ ID NO: 58); FRVYSSANNSTFEYV (SEQ ID NO: 59) DGYFKIYSKHTPINE (SEQ ID NO: 60); QTLLALHRSYLTPGDSS (SEQ ID NO: 61); TFLLKYNENGTITDA (SEQ ID NO: 62); SNFRVQPTESIVRF (SEQ ID NO: 63);

GEVFNATRFASVYA (SEQ ID NO: 64); SVYAWNRKRISNSVAD (SEQ ID NO: 65); GNYNYLYRLFRKSNLKPFERDI (SEQ ID NO: 66); EGFNSYFPLQSYGFQPT (SEQ ID NO: 67); QSYGFQPTNGVGYQPY (SEQ ID NO; 68); KSVNFNFNGLTGT (SEQ ID NO: 69);

GFNFSQIEPDPSKPSKRS (SEQ ID NO: 70); DEMIAQYTSALLA (SEQ ID NO: 71 ); IPFAMQMAYRFNG1GV (SEQ ID NO: 72): KLIANQFNSAIGKI (SEQ ID NO: 73); NQFNSAIGKIQDSE (SEQ ID NO; 74); GRLQSLQTYVTQQL (SEQ ID NO: 75); KYFKNHTSPDVDL (SEQ ID NO: 77); ISGINASVVNIQKEI (SEQ ID NO: 78). In this context, one or more amino acid residues from a primary antigenic peptide, as exemplified above, can be routinely deleted, substituted, chemically modified or replaced, and/or added to make useful variants and derivatives having, for example, 1 -4 amino acid alterations made to the primary sequence without substantially reducing or abolishing antigenic activity of the subject antigenic peptide.

The MHC Class I epitopes. MHC Class II epitopes and ARDs within a particular li-Key- SARS-CoV-2 antigenic peptide hybrid construct can be structurally modified according to rational peptide design and optimization methods, with only routine experimentation needed to determine which peptide/epitope variants and derivatives are operable within the invention. Antigenic epitopes and determinants selected for use in li-Key peptide hybrids can modified, for example, by deletion, substitution or addition or one or more amino acid residues, as well as by incorporation of modified amino acids, peptidomimetic structures, and/or chemical structures which are not natural or modified amino acids but may nonetheless be incorporated within the epitope/determ inant elements of the li- Key peptide hybrids, without substantially impairing their immunogenic activity or vaccine efficacy. The addition, in whole or in part, of non-natural amino acids, or of other backbone or side chain moieties can be routinely made and tested for non-disrupt ion of binding specificities and antigenic efficacy. Certain modifications contemplated here may actually alter or increase recognition of the modified antigenic epitope(s), for example to increase recognition and activation of an original cognate T cell target, or to prompt recognition by a previously non-cognate subset of T cells.

Typical vaccine compositions of the invention will comprise one or more Ii-Key-SARS-CoV-2 antigenic peptide hybrids formulated for direct prophylactic administration to a subject, often with an adjuvant. It is, however, also possible to deliver Ii-Key-SARS-CoV-2 antigenic peptide hybrids indirectly, for example through administration of nucleic acid sequences encoding in vivo-expressible peptide hybrids. Recombinant DNA techniques for expressing Ii-Key-SARS-CoV-2 antigen fusion peptides in the form of a vaccine are well known, and are indeed the basic platform currently used for delivering SARS-CoV-2 spike protein-encoding DNA vaccines. A wide variety of delivery systems are available for delivering a DNA vaccine encoding a Ii-Key-SARS-CoV-2 fusion peptide, including viral and non-viral systems. Examples of suitable viral systems include, for example, adenoviral vectors, adeno-associated virus, retroviral vectors, vaccinia, herpes simplex virus, HIV, the minute virus of mice, hepatitis B virus and influenza virus. Non-viral delivery systems may also be used, for example, uncomplexed DNA, DNA-liposome complexes, DNA-protein complexes and DNA-coated gold particles, bacterial vectors such as salmonella, and other technologies such as those involving VP22 transport protein, Co-X-gene. and replicon vectors. Adenoviral vectors offer a variety of advantages for introducing expressible constructs into cells, including their tropism for a broad range of human tissues and ability to direct high level expression of vectored products. Adenoviral vectors have a relatively short duration of transgene expression, due to immune system clearance and dilutional loss during target cell division, but they are easily administered and widely regarded as safe, especially newer, replication incompetent forms that reduce a likelihood of inflammation and other side effects. Methods for constructing, optimizing, and administering nucleic acid-based delivery systems are well known in the art and routinely implemented to deliver and direct in vivo expression of vector encoded Ii-Key-SARS-CoV-2 fusion peptides of the invention.

The invention also provides methods and compositions employing Ii-Key-SARS-CoV-2 antigenic peptide hybrids of the invention in preparation of ex-vivo conditioned cellular therapeutics to treat COVID-19 patients or at-risk subjects. In exemplary embodiments, patients known or suspected to be infected with SARS-CoV-2. and particularly patients showing symptoms of COVID- 19 infection (e.g.. hospitalized COVID-19 patients, patients with hypoxemia, and patients presenting with respiratory distress), are administered autologous or heterologous immune cells conditioned ex- vivo to have an anti-SARS-CoV-2-primed or activated, differentiated immune-effector cell status. Such patient are administered a population of ex vivo-activated T cells produced by contacting the T cells with an antigen presenting cell (APC) presenting an li-Key SARS-CoV-2 antigenic peptide hybrid of the invention in a complex with an MHC class I or MHC class II molecule on the surface of the APC in an APC and T cell-sustaining medium, for a period of time sufficient to activate the T cell (e.g., to activate a T helper cell to differentiate to a Th1 cytokine secreting helper cell, or to activate a T cell to differentiate into an activated CD8+, Granzyme B-secreting "killer" T cell). Typically the T cells thus activated ex vivo will be autologous to the patient, harvested and conditioned/activated for return to the patient as a therapeutic, expanded and specifically anti-SARS-CoV-2 immunogenically- activated population of T effector cells, capable of mediating therapeutic, anti-SARS-CoV-2 immune therapeutic effects in the treated subject. In alternate embodiments, the subject T cells may be obtained from a compatible, healthy or successful COVID-19 convalescent donor. In more detailed embodiments, the T cells are obtained as peripheral blood mononuclear cells (PBMCs), and the ex vivo process of conditioning/activating the T cells includes expanding the T cells in vitro before administration in expanded numbers to the patient. The optional expansion step may be conducted in the presence of proliferation-enhancing nutrients, growth factors, conditioning agents and/or cytokines (for example anti-CD28 antibody and IL- 12). In other detailed embodiments, the APCs (e.g., macrophages and/or dendritic cells) may be infected with a recombinant virus expressing a vector- expressible form of the Ii-Key-SARS-CoV-2 peptide hybrid, wherein the antigen presenting cell is a dendritic cell or a macrophage. For administration of ex vivo conditioned/activated T cells, optional adjuvants may be used as described herein for the anti-SARS-CoV-2 li-Key peptide hybrid vaccines. Other useful adjuvants include anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib. bevacizumab, interferon-alpha, interferon-beta, CpG, oligonucleotides and derivatives, poly-(LC) and derivatives, RNA. sildenafil, particulate formulations with poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)- 1 , IL-2, IL-4, IL-7, IL- 12, IL- 13, IL- 15, IL-21 , and IL-23.

In related embodiments the invention provides conditioned antigen presenting cells, APCs, including conditioned dendritic cells (DCs) that can be used for directly immunizing subjects, or to condition T cells ex vivo for use as activated T cell vaccine agents. DC vaccination and DC immunization refer to a strategy using dendritic cells (usually autologous) to potentiate an immune against specific antigens. DCs are particularly useful APCs and can be derived from a hematopoietic stem cell or a monocyte. Dendritic cells and their precursors can be isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrow and peripheral blood. Typically, dendritic cells express high levels of MHC and costimulatory (e.g., B7-1 and B7-2) molecules. DCs can potently induce antigen specific differentiation of T cells in vitro, and are able to initiate primary T cell responses in vitro and in vivo. In the context of DC vaccine production, an activated DC is produced by as described abov e (either by incubating DC with an li-Key-SARS-CoV- 2 antigenic peptide hybrid of the invention to prov ide for MHC binding of the antigenic peptide hybrid on the DC cell, or transducing the DC to express the peptide hybrid). DCs thus activated can be used to condition T cells ex vivo for immunotherapy use, as described, or the activated DC's can be administered directly to patients to potentiate antigen-specific anti-SARS-CoV-2 T cell immune responses and dow nstream immunotherapeutic effects.

Immunogenic Potency, Immune-Regulatory Efficacy and Immuno-Selective Activitie s of Ii-Key-SARS-CoV-2 Hybrid Peptides li-Key -SARS-CoV-2 hybrid peptides of the invention are evaluated extensively to determine immunogenic potency , immune-regulatory efficacy and immuno-selective activity(ies). These ev aluations employ an extensive array of assay s, compositions and immune analytic tools that are widely know n and av ailable in the art. In general, these evaluations employ one or more immunological assay s designed to detect and/or measure recognition of an antigenic peptide sequence by a T cell in an in vitro, ex vivo or in vivo environment. For many of these studies, li-Key-SARS- CoV-2 hy brid peptides are contacted w ith a T cell-containing sample, for example a T cell hybridoma culture or a T cell-containing sample of blood from one or more COV1D-19 convalescent patient(s). Suitable procedures and observations are conducted to determine, for example, binding and recognition of the hy brid peptides by T cells, activation of T cells in response to hybrid peptides (e.g., a proliferativ e response, or a cytokine expression response), and/or effector functions mediated by T cells in response to hy brid peptides (for example downstream immune effects/responses, such as antibody secretion by B cells. CTL activation, or other downstream effects).

Preliminary studies for li-Key hybrid peptides compared the binding and activation potential of hybrid peptides to the binding and activation potential of the antigenic peptide member of the hybrid alone. These studies evince. that li-Key peptides exhibit much greater binding and activation potency than the corresponding peptide alone. For example, endpoints for half maximal stimulation of unincorporated antigenic epitopes averages about 20 nM. In contrast, endpoints for half maximal stimulation with corresponding hybrids are typically about 50 pM. These and related experiments demonstrate that efficacy of li-Key-peptide antigen hybrids in relation to MHC II binding and antigen presentation far exceeds corresponding performances by antigenic peptides alone. Additional assays are used to demonstrate effects of incorporating antigenic peptides into li- Key hybrid constructs, and for defining immunogenic activities of selected li-Key hybrid constructs. These assays are aimed at detecting and quantifying a diversity of anti-viral immune-stimulatory and immune-regulatory effects, such as, inducing activation or expansion of T helper cells, inducing Th1 - or Th2 selective immune differentiation/activation of CD4+ T cells, modulating inflammatory cytokine expression by immune effector cells, stimulating B cells to proliferate and/or regulating B cell immunoglobulin production, triggering activation of cytotoxic T cells, and many other immune- activating and immune-regulatory functions.

Ii-Key-SARS-CoV-2 hybrid peptides directly charge MHC class II molecules on the surface of APCs and are presented effectively to activate and program other immune effector cells. In certain embodiments of the invention, Ii-K.ey-SARS-CoV-2 hybrid peptides are identified and selected that induce differentiation of CD4+ T cells toward a ThI -biased differentiation. In alternate embodiments, Ii-Key-SARS-CoV-2 hybrid peptides are identified and selected that induce selective differentiation of CD4+ T cells toward a Th2-biased differentiation. In related vaccine compositions and methods, one or more Ii-Key-SARS-CoV-2 hybrid peptides may be administered that primarily or exclusively induce Th1 differentiation of CD4+ T. In alternative vaccine combinations one or a plurality of li- Key-SARS-CoV-2 hybrid peptide(s) may be administered that effectively induce both Th1 and Th2 differentiation of CD4+ T cells. In additional embodiments. Ii-Key-SARS-CoV-2 hybrid peptides screened for the ability to activate CD8+ CTLs can be included, or excluded, from vaccine compositions of the invention.

Figure 3 illustrates how APCs mediate activation of CD4+ T cells, and how different factors, including cytokines and transcription factors, determine or correlate with differentiation of CD4+ T cells along alternative, ThI or Th2 pathways. Th1 and Th2 cells are functionally distinct subclasses of helper T cells programed to become effector cells, distinguishable by the cytokines they secrete. Cells that differentiate to a Th1 effector phenotype secrete interferon-γ (IFN-γ) and tumor necrosis factor-a (TNF-a). If a naive T helper cell differentiates to a Th2 effector cell, it will secrete IL-4, IL-5, IL- 10, and IL- 13. Both Th1 and Th2 cells play critical roles in normal immune and inflammatory responses. Th1 cells induce cellular immune responses (e.g., by "licensing" dendritic cells to prime an effective MHC class 1 restricted CTL response), participate in macrophage regulation, and stimulate B cells to produce IgM and IgG antibodies. Th2 cells stimulate humoral immune responses, promote B cell proliferation and induce antibody production. Th2 cells are also associated with differentiation and proliferation of mast cells, and with differentiation and proliferation of eosinophilic leukocytes.

Under normal circumstances, the differentiation ofTh1/Th2 cells rests in a more or less balanced state. Substantial Th1/Th2 imbalance is broadly associated with disease. Hyper-activation of Th2 can lead to inappropriate immune responses, including inflammatory disease, allergies and asthma. Overexpression ofTh1 can lead to autoimmune disease, such as rheumatoid arthritis and multiple sclerosis.

The primary signal for T cell activation is binding of a helper T cell receptor (TCR) to a MHC II-antigen complex on the surface of an APC. When the antigen member of this complex is a li-Key- SARS-CoV-2 hybrid peptide, the identity and binding properties of the hybrid antigen will influence Th1/Th2 differentiation. T cell differentiation depends in part on the intensity ofT cell acquisition signal: weak TCR activation signal can induce IL-4 synthesis and promote T cell differentiation to Th2, whereas strong TCR activation signal can activate the MAPK pathway to induce IFN-y synthesis and promote ThI differentiation. The length of TCR triggering time is also known to affect differentiation of Th cells. Transient TCR triggering correlates with Th1 differentiation, while long- term TCR triggering correlates with Th2 differentiation.

Cytokines play a major downstream role in regulating Th1 T cell differentiation and fate. IFN- y promotes Th1 cell differentiation and inhibits Th2 cell differentiation. IFN-y production is in turn regulated by various transcription factors, including nuclear factor of activated T cells (NFAT), NF- κB, IRF- 1 family, ERM, T-bet, YY1 and Hix transcription factors. IL-12 is the most important cytokine that promotes Th1 cell differentiation, by activating STAT4 and upregulating IFN-y expression. IL-4 in turn inhibits IL- 12Rβ2 (only expressed in Th1 cells), rendering T cells refractory to IL- 12. In the absence of IL-12 recognition. Th1 cells differentiate into a Th2 phenotype (Glimcher et al., 2000). Therefore, IFN-y producing cells that fail to silence IL-4 production can destroy Th1 immunity by ablating their IL- 12 receptor functionality (Ansel et al., 2004). IL-18 acts indirectly but synergistically with IL- 12 to induce IFN-y production and promote IL-12-induced Th1 cell differentiation (O'Garra et al.. 1998). Cytokines that influence Th2 differentiation include IL-4, as noted, whose expression is in turn affected by C-maf, NF-KB, IL- 13 and IL-6, among other players. IL.- 13 and IL-4 share an IL-4R subunit known to induce inflammatory responses. The respective roles ofTh1 and Th2 differentiated helper cells in anti-SARS-CoV-2 protection and COVID-19 disease progression remain somewhat uncertain. From a broad biologic perspective, naive T-helper cells (ThO) have evolved to respond to a diverse array of novel pathogens. Depending on the nature of the infectious agent. ThO helper cells polarize the immune response into T-helper type 1 or 2. Th1 responses are biased toward pathogens vulnerable to cell-mediated responses, including intracellular or phagocytosable pathogens (viruses, bacteria, protozoa and fungi), and are mediated primarily by macrophages and CTLs. Th2 responses are classically directed against extracellular, non-phagocytosable pathogens, for example helminths, and are mainly effected ultimately by eosinophils, basophils and mastocytes. as well as B cells (humoral immunity). Eosinophils play a direct role in fighting RNA viruses, as demonstrated by the presence of RNases inside their granules. However, eosinophils are negatively associated with pathophysiology in respiratory viral infections, for triggering bronchoconstriction and dyspnea, virus-induced exacerbation of allergies and asthma, and releasing large amounts of inflammatory cytokines (Rosenberg, 2009) (including IL- 6, proposed as a key mediator of CSS in fatal COVID-19 cases (Zhang et al., 2020)).

Numerous studies point to a deleterious T helper differentiation biasing toward Th2 cell predominance in severe COVID-19 disease. Blood testing of hospitalized COVID-19 patients reveal cytological signals of Th2 dominance, for example eosinophilia plus basophilia, degranulated eosinophils, Turk cells or plasma cells, together with Th1 and CTL lymphopenia. For reasons that remain unclear, the immune system in severe COV1D-19 patients requiring IC is biased toward a predominately Th2 profile. This may be related to viral load, activation-induced apoptosis ofTh1 and CTL cells, phenotypic conversion ofThI cells to Th2. antigenic cross-reactivity, or the particular profile of APC stimulation of naive Th0 cells. This Th2 dominance is more likely to be observed in patients affected by secondary risk factors such as cancer, immunodeficiency, autoimmune disorders, congestive heart failure, chronic obstructive pulmonary disease, hepatic cirrhosis, major surgery or trauma, or total parenteral nutrition, all known conditions suppressive to Th1 immunity. For these and other reasons, at least for a subset of high-risk patients (e.g., patients with secondary risk factors, or patients previously exposed to SARS-CoV, SARS-CoV-2 or an endemic hCoV known to elicit cross- reactive immune potentiation, such as ADE, directed toward SARS-CoV-2), vaccine compositions and methods of the invention may be selected to comprise one or more Ii-Key-SARS-CoV-2 hybrid peptides that is/are immune-selective to primarily or exclusively induce Th1 differentiation of CD4+ T helper cells.

The skilled artisan will understand that the instant invention is not limited to the particular materials, process steps, or methods of design and use disclosed herein, as such are provided for illustrative purposes only. Following the discoveries and teachings of the invention as a whole, the invention can be adapted, optimized and expanded in equivalent form and purpose by the skilled artisan without undue experimentation. Likewise, the terminology employed herein is exemplary to describe illustrative embodiments, and is not intended to limit the scope of the present invention. The following examples are provided for the same, illustrative and non-limiting purpose.

EXAMPLE I

Identification and Characterization of Candidate Antigenic Peptides Comprising Putative Immunogenic Epitopes Having Antigenic Sequence Identity or Similarity to a Native SARS-CoV-2 Antigenic Epitope Using Computational Vaccinology Tools and Methods

The instant example applies computational vaccinology tools and methods to map and predict activity of presumptive antigenic epitopes within target protein sequences of the human coronavirus SARS-CoV-2 responsible for the 2019 Coronavirus Disease pandemic (COVID-19). The subject analyses were directed toward amino acid sequences of the envelope (E), membrane (M) and spike (S) proteins of SARS-CoV-2 Wuhan-Hu- 1 isolate (MN908947). These three proteins were selected because each comprises a substantial component of SARS-CoV-2 viral particles, making them likely targets for antigenic recognition by the human immune system. A transcript of the input sequences and GenBank accession numbers are presented below in Table 1.

Table 1. Overview of Input Sequences > Amino Acid Sequen

In brief summary, the foregoing amino acid sequences of SARS-CoV-2 proteins were analyzed for HLA class II epitope content from GenBank using the EpiMatrix® algorithm as well as the ClustiMer®, JanusMatrix®, and OptiMatrix® algorithms developed and exclusively licensed to Applicant company for use within the invention by EpiVax, Inc (Providence. RI). Using the ClustiMer® algorithm we screened results of EpiMatrix® analyses to identify T cell epitope clusters that include epitope rich segments, representing high probability candidate peptides include in Ii-key-SARS-CoV-2 hybrid peptides. The JanusMatrix® algorithm was applied to the data to determine whether any putative T cell epitopes clusters identified by EpiMatrix® and ClustiMer® contain predicted homologies with counterparts within the human proteome (e.g.. to exclude homologous peptide/epitope sequences that might elicit tolerance or autoimmune responses). Putative T cell epitopes clusters identified by EpiMatrix® and ClustiMer® were additionally screened against reference datasets comprising known B cell epitopes identified for the original SARS coronavirus, SARS-COV. Yet additional analyses of putative T cell epitope clusters identified by EpiMatrix® and ClustiMer® were conducted to identify Class 1 restricted T cell epitopes, useful in vaccine compositions of the invention to activate or direct differentiation of mature CD8+ T cells (to generate anti-SARS-CoV-2 cognate CD8+ memory T cells). Further studies examined whether predicted T cell epitope clusters contain epitopes of related pathogens previously encountered by humans, for which candidate sequences were screened against reference datasets for known isolates of SARS-COV-2, SARS-COV, and various endemic human coronaviruses (hCoVs).

Further detailing these studies, the proprietary computational vaccinology software system EpiMatrix® (EpiVax, Providence Rl) was used to analyze amino acid sequences of the SARS-COV-2 envelope, membrane and spike proteins collected from GenBank, to predict Class II HLA DR restricted HLA ligands and putative T cell epitopes. Activation of naive CD4+ T cells occurs through presentation of linear peptide ligands to HLA molecules on the surface of antigen presenting cells. Activated CD4+ T cells arc effective to drive adaptive immune responses. These CD4+-mediated downstream responses may include maturation of antigen-specific, antibody producing B cells CD8+ "killer" T cells. Successful vaccination employing li-Key-SARS-CoV-2 antigenic peptide fusions are thus contemplated to elicit both short and long-term memory responses in both T and B cell compartments.

Class II restricted T cell epitopes tend to occur in "clusters". T cell epitope clusters typically range from between 15 to 25 amino acids in length and each cluster may contain between four and 40 predicted T cell epitopes. T cell epitope clusters are very likely to be promiscuous HLA ligands (i.e. they typically bind to multiple HLA "alleles"). As such they have a very good chance of driving immune responses in large numbers of individuals even in an outbred population where a diversity of HLA haplotypes is present. Using a second computational vaccinology software tool ClustiMer® (EpiVax. Providence Rl). results of the EpiMatrix® analyses were further analyzed to identify T cell epitope clusters. These are presumptive epitope rich segments that will enhance (broaden and diversify) activity of anti-SARS-CoV-2 li-Key-peptide fusion constructs.

Yet another algorithm, JanusMatrix® (EpiVax, Providence Rl), was employed to determine whether putative T cell epitope clusters identified by EpiMatrix® and ClustiMer® may be homologous to predicted epitopes found within the human proteome. Human-like T cell epitopes may be tolerated by the human immune system or in some cases may be actively tolerogenic. Such epitopes are un-desired for inclusion in a vaccine formulation of the invention, because they can reduce vaccine-induced immune responses and block or attenuate memory formation. In other cases, vaccination-induced responses targeting these sequences may recognize healthy human tissues, resulting in unwanted auto-immune response. Consequently, these predicted homologous sequences are de-selected as candidates and excluded from use in li-Key-SARS-CoV-2 antigenic peptide fusions.

To further evaluate whether putative T cell epitope clusters identified by EpiMatrix® and ClustiMer® contain beneficial vaccine epitopes, each candidate peptide was additionally screened for the predicted presence of Class 1 restricted epitopes. Upon vaccination, companion Class I content can help to mature CD8+ T cells, thereby creating cognate CD8+ T cell memory.

To determine if optimized T cell epitope clusters contain epitopes previously encountered by human beings, these were further screened against reference datasets composed of known isolates of SARS-COV-2, SARS-COV1 , and other endemic human coronaviruses (hCoVs). Activation of pre-existing cognate T cells can help create a robust response to vaccination, leading to enhanced memory formation.

Yet additional screening of putative T cell epitopes clusters identified by the EpiMatrix® and ClustiMer® algorithms was conducted against reference datasets composed of known B cell epitopes found in SARS-COV-E Upon vaccination, companion B cell epitope content may help to mature B cells to provide cognate B cell memory.

Searching for T cell epitopes with EpiMatrix® focuses initially on targets likely to activate CD4+ "helper" T cells. Activation of CD4+ T ceils is necessary for induction of a robust anti-viral immune response, including CD8+ T cell-mediated cytotoxic response, and B cell-mediated antibody response. Antigen presenting cells such as macrophages and dendritic cells sample circulating proteins and present constituent T cell epitopes (short segments of amino acids in a linear conformation) on their surface in the context of Class II Human Leucocyte Antigens (HUA). When CD4+ T cells recognize a presented T cell epitope they activate and release pro-inflammatory cytokines and chemokines, initiating an immune signal- response cascade resulting in B cell class switching, affinity maturation, and antibody secretion. Without CD4+ mediated "help" B cell activation is limited resulting in typically weak and transitory antibody response. The immunogenic potential of a candidate vaccine peptide can thus be estimated based on the number and quality of Class II restricted T cell epitopes it contains. To refine this aspect of peptide selection, input sequences were parsed into overlapping 9-mer frames and evaluated each frame against a panel of nine common Class II HLA alleles. These alleles were selected because they are relatively common within the human population and relatively distinct from each other. These alleles as referred to as "super-types", each functionally equivalent to many additional family member alleles. Alleles within a super-type family share a set of common peptide binding preferences. Taken collectively, these nine super-type alleles, along with their respective family members, cover well over 95% of HL. A types present in most human population groups (Southwood et al., 1998).

Each frame-by-allele assessment refines the reliability of HLA binding affinity prediction. EpiMatrix® assessment scores range from approximately -3 to +3 and are normally distributed. EpiMatrix® assessment scores above 1 .64 are defined as "hits." Hit peptides have a significant chance of binding to HLA molecules with moderate to high affinity, and therefore, have a significant chance of being presented on the surface of antigen presenting cells (APCs), such as dendritic cells or macrophages, where they may be presented to T cells. On average, about 5% of these assessments score above 1 .64 as "hits".

I'he greater the number of predicted HI . A ligands (identified as EpiMatrix® hits) contained in a given peptide sequence, the more likely it is that the peptide will induce an immune response. An "EpiMatrix® Protein Score" is the difference between the number of predicted T cell epitopes expected in a given protein and the number of putative epitopes predicted by the EpiMatrix® System. EpiMatrix® Protein Scores are "normalized" and reported on a "per 1 ,000 assessments" scale. Normalizing for length makes EpiMatrix® Protein Scores directly comparable. Without normalization, longer proteins would always appear to be more immunogenic than shorter proteins and peptides, which naturally carry fewer HLA ligands and T cell epitopes. For proteins of average length, normalized EpiMatrix® Protein Scores correlate with observed immunogenicity. After normalization, the F.piMatrix® Protein Score of an "average" or randomly generated protein is zero. EpiMatrix® Protein Scores above zero indicate the presence of putative HLA ligands and denote a higher immunogenic potential, while scores below zero indicate fewer putative HLA ligands than expected and a low potential for immunogenicity. Proteins scoring above +20 are considered to have significant immunogenic potential.

Using the EpiMatrix® system to screen for putative Class II restricted T cell epitopes, a total of 603 frame-by-allele assessments were conducted on the SARS-COV-2 envelope protein sequence. The envelope protein sequence contained 128 EpiMatrix® hits. The E protein EpiMatrix® Protein Score was 332.60. In other words, the SARS-COV-2 envelope protein contains significantly more putative T cell epitopes than would be predicted for an "average" or randomly generated protein sequence of similar length. This is a high score indicating a high potential for T cell-dependent antigenicity.

For the SARS-CoV-2 membrane (M) protein 1 ,926 frame-by-allele assessments were conducted. The M protein sequence contains 186 EpiMatrix® hits, and its EpiMatrix® Protein Score is 89.43. In other words, the SARS-COV-2 M protein contains significantly more putative T cell epitopes than predicted for an average or randomly generated protein sequence of similar length. This is a high score indicating a high potential for T cell-dependent antigenicity.

For the SARS-CoV-2 spike (S) protein I 1 ,385 frame-by-allele assessments were made. The S protein sequence contains 753 EpiMatrix® hits, and its EpiMatrix® Protein Score is 22.19. Accordingly, the S protein contains more putative T cell epitopes than expected for average or randomly generated protein sequences, also indicating a high potential for T cell- dependent antigenicity.

A summary of these findings is presented below in Table 2. and the respective antigenicity scores for the SARS-CoV-2 E, M and S proteins are illustrated graphically in Figure 4.

Table 2. EpiMatrix® HLA Class II Analysis of SARS-COV-2 proteins

Further refinement of antigenic epitope predictions for SARS-CoV-2proteins employed the ClustiMer® tool to assess regional immunogenicity. Potential T cell epitopes are not randomly distributed throughout protein sequences, but tend to be clustered in specific regions. T cell epitope clusters range from about fifteen to twenty-live amino acids in length and, considering their affinity to bind multiple alleles and across multiple frames, can contain anywhere from four to forty binding motifs. Many of the peptides that induce strong T cell responses contain a cluster feature identified in the ClustiMer® system as an EpiBar®. An EpiBar® is a single 9-mer frame predicted to be reactive with at least four different HLA alleles. Sequences that contain EpiBars® include the well-known superantigens Influenza Hemagglutinin 306-318 (Cluster score of 23), Tetanus Toxin 825-850 (Cluster score of 46), and GAD65 557-567 (Cluster score of 23). An example of an EpiBar® is shown below in Figure 5.

Candidate peptide sequences that contain EpiBars® are predicted to bind very well to a range of HLA Class 11 molecules, and be highly immunogenic. The presence of one or more EpiBars® can drive effective anti-viral immune responses even in otherwise low-scoring peptides or proteins. Peptides containing promiscuously binding epitopes can be very powerful immunogens. For example, 100% of subjects exposed to either Tularemia or Vaccinia responded to T cell epitope pools containing between 20 and 50 promiscuous epitopes (McMurray et al., 2007; Moise et al. 2009). Thus, the presence of one or more dominant promiscuous binding motifs predicts significant antigenic potential.

In order to identify potential T cell epitope clusters, the EpiMatrix® analysis results for SAR.S- CoV-2 w ere further analyzed to identify regions with unusually high densities of putative T cell epitopes. The significant EpiMatrix® Scores contained within these regions were then aggregated to create an EpiMatrix® Cluster Immunogenicity Score. As for the EpiMatrix® Protein Immunogenicity Score, positive Cluster Immunogenicity scores indicate increased immunogenic potential relative to a randomly generated or "average'' sequence. T cell epitope clusters scoring above +10 are considered to have a significant immunogenic potential.

In general, exogenous vaccine peptides administered to mammalian subjects are expected to be captured by APCs and processed through the Class II presentation pathway. HLA ligands, such as those identified and analyzed here, may be presented on the surface of processing APCs, where they will be subject to immune surveillance. In general, promiscuous Class II HLA ligands are the most likely regions of the SARS-COV-2 envelope, membrane and spike proteins to induce a CD4+ T cell response. The ClustiMer® algorithm identified 48 putative T cell epitope clusters within the amino acid sequences of the SARS-COV-2 envelope, membrane and spike proteins. In addition, each of the detailed EpiMatrix® reports was reviewed by hand and additional pseudo-clusters (i.e. short regions which do not meet the ClustiMer® definition of an epitope cluster, but do include some Class Il- restricted epitope content along with additional interesting features such as significant Class I- restricted content, HLA DR9-restricted Class II epitope content, or significant human-like content) were selected. In total, 63 epitope clusters and pseudo-clusters were selected for further evaluation, five clusters were identified in the E protein, thirteen clusters in the M protein and forty-five clusters in the S protein, for every predicted epitope, in addition to having compatible residues in binding positions 1 , 4, 6, and 9, natural high-affinity EILA Class II ligands are also very likely to include two to three flanking residues on both the N- and C-terminal ends of the predicted 9-mer binding core (Nielsen et al.. 2010), These flanking residues are required for stabilization of the epitope in the HLA binding groove, but are not considered in calculating the EpiMatrix® Cluster Score. EpiMatrix® Cluster Scores of the 63 putative T cell epitope clusters and pseudo-clusters identified here ranged from 4.88 to 58.68.

T cell epitopes with sequence identity or similarity to endogenous human proteins are undesired, for their propensity to be tolerated, tolerogenic or even autoantigenic. The JanusMatrix® algorithm was employed to screen out such homologous peptide candidates. Within any given T cell epitope there are certain amino acids that contact and bind with the MHC molecule and certain amino acids that make contact with the T cell receptor (TCR) of responding T cells. For Class II restricted epitopes, relative positions 1 , 4, 6, and 9 are assumed to make contact with the MHC and positions 2, 3. 5, 7, and 8 are assumed to be available to the TCR. Peptide epitope pairs with compatible, but not exactly matched, MHC binding anchors and exactly matched TCR facing contours may be cross- reactive. In other words, CD4+ T cells engaged and activated by a given peptide epitope may also be engaged and activated by a TCR contour-matched homologue. The JanusMatrix® screening tool considers these principals to identify potentially cross-reactive candidate epitopes relative to a target reference sequence, in this case the human proteome.

As depicted in Figure 6, for a given 9-mer epitope, the JanusMatrix® algorithm searches a reference database to analyze the amino acid content of both the MHC-facing agretope and the TCR- facing epitope. Reference sequences with a compatible agretope (i.e. one that is predicted by EpiMatrix® to bind the same HLA as the input peptide) and exactly matching the TCR contacts of the input peptide are returned. The JanusMatrix® homology Score of a given peptide or protein indicates the average depth of coverage within the reference database for the HLA binding peptides contained within that sequence. When comparing peptide epitopes to the human genome, JanusMatrix® human homology Scores above two are considered significant; indicating an elevated level of conservation between the TCR-facing features of the input peptide or protein, and the TCR-facing features of the corresponding reference sequence in the human proteome. For a given EpiMatrix® Score, a high JanusMatrix® human homology Score suggests a bias towards immune tolerance, and high JanusMatrix® human homology Scores are considered to offset high EpiMatrix® Scores.

A review of 3,756 HLA Class Il-restricted T cell epitopes for which cytokine release data are catalogued by the I EDB indicates there is a statistically significant relationship between high JanusMatrix® human homology Scores and observed production of IL-10, a cytokine commonly associated with regulatory T cell response. Additionally, there is an inverse relationship between high JanusMatrix® human homology Scores and observed production of IL-4, a cytokine commonly associated with inflammatory T cell response (Moise et al., 2015) HLA Class II T cell epitopes derived from influenza A virus and hepatitis C virus with high JanusMatrix® Scores induce regulatory T cell responses (Liu et al., 2015; Moise et al., 2014). In addition, Tregitopes (actively tolerogenic HLA Class II T cell epitopes derived from human IgG and patented by EpiVax) are highly conserved not only amongst human antibodies, but also other human proteins (Moise et al., 2013). The prospect that natural regulatory T cells can be engaged and activated by peptide epitopes possessing TCR- facing motifs commonly found within human proteins is gaining acceptance (Moise et al., 2015; Daniel et al.. 2018).

Each of the T cell epitope clusters and pseudo-clusters discerned in the SARS-COV-2 E, M and S protein sequences was submitted to JanusMatrix® and screened against the human genome. Twelve of the selected epitope clusters scored above a determined threshold (JanusMatrix® Human I lomology Score of 2.00). All JanusMatrix® results were reviewed by hand. In some cases, matches to human proteins could be discounted based on weak predicted HLA binding and/or the presence of sub-optimal binding anchor residues in relative position Pl of either the SARS-COV-2 predicted ligand or the corresponding human sequence. Putative clusters and pseudo-clusters with significant human-like content were tagged. Optimizing Synthetic Peptide Sequences

In order to confirm the HEA binding affinity and T cell stimulating capacity of putative T cell epitope clusters and pseudo-clusters identified here, synthetic peptides are typically manufactured and characterized to confirm activity. Computer-identified sequences, such as ClustiMer®-identified T cell epitope clusters, do not always have optimal physiochemical properties for in vitro testing. It is often desired to synthesize modified peptides whose predicted binding motifs are well centered in the sequence, whose net charge is not zero, and whose amino acid composition avoid troublesome residues or combinations of residues. For example, cysteine residues can form sulfur bridges and induce aggregation in vitro, while methionine residues have a tendency to oxidize. Candidate epitope cluster identified by EpiMatrix® and ClustiMer® presenting these deficiencies are readily optimized, e.g., through amino acid substitution, addition or deletion, or simply excluded as problematic for synthesis or use.

Identifying Class I T Cell Epitopes

Sequences of all 63 of the identified Class II restricted T cell epitope clusters and pseudo- clusters were screened for the predicted presence of Class I restricted T cell epitopes. Using the EpiMatrix® system, input sequences were parsed into overlapping 9-mer frames, and overlapping 10- mer frames. Each of the parsed frames was then evaluated for binding potential with respect to a panel of six common Class I alleles: A*010 l , A*0201, A*0301 , A*2402, B*0702, and B*4403. These alleles are super-types. Each one is functionally equivalent to or nearly equivalent to many additional family member alleles. Taken collectively, these six super-type alleles, along with their respective family members, cover over 98% of the human population (Sette, 1999). As is true for Class II, Class I EpiMatrix® assessment scores range from approximately -3 to +3 and are normally distributed.

I piMatrix assessment scores above 1 .64 are defined as "hits’". In general, about 5% of all assessments are expected to score above 1 .64. These peptides have a significant probability of binding HLA molecules with moderate to high affinity, and thus are likely candidates for successful presentation and binding at the surfaces of both professional antigen presenting cells (APCs) (e.g., dendritic cells and macrophages) and non-professional APCs, where they can effectuate interactions with adjacent T cells. After parsing the 63 Class II restricted T cell epitope clusters and pseudo- clusters identified within the SARS-COV-2 E, M and S proteins into 9-mer and l Omer frames, and screening for the presence of putative Class I restricted T cell epitopes, the Class I "hits" for each cluster were identified. Observed Class 1 content ranges between I and 17 putative epitopes.

Identifying Significant Homologies

In order to understand how well conserved the putative T cell epitope clusters and pseudo- clusters identified by the EpiMatrix® and ClustiMer® algorithms are with respect to circulating stains of SARS-COV-2, a homology reference database was assembled by querying GenBank for known isolates of SARS-COV-2. The queries used are as follows:

For SARS-CoV-2 Envelope: TXID2697049[Organism] and 75[SLEN]

For SARS-CoV-2 Membrane: TXID2697049[Organism] and 222[SLEN]

For SARS-CoV-2 Spike: TXID2697049[Organism] and 1273[SLEN]

When these queries were executed each returned 53 protein isolates. Each of the putative epitope clusters identified by EpiMatrix® and ClustiMer® were BLASTed against this database. All of the selected epitope clusters are 100% conserved in at least 51 isolates and most are 100% conserved in all 53 isolates.

Activation of pre-existing cognate T cells can help create a robust response to vaccination leading to enhanced memory formation. In order to assess conservation between SARS-CoV-2 and SARS-COV. a SARS- COV reference database was assembled by querying GenBank for known isolates of SARS-COV. The queries used are as follows:

For SARS-CoV Envelope: ("SARS"[ALL] AND "Envelope"[ALL] AND 74:78[SLEN]

AND "Homo sapiens"[ALL]) NOT TXID2697049

For SARS-CoV Membrane: ("SARS"[ALL] AND "Membrane"[ALL] AND 220:223[SLEN] AND "Homo sapiens"[ALL]) NOT TX1D2697049

For SARS-CoV Spike: ("SARS"[ALL] AND "Spike"[ALL] AND 1245:1265[SLEN] AND "Homo sapiens"[ALL]) NOT TX1D2697049

When these queries were executed, those for Envelope and Membrane each returned 12 protein isolates. T'he query for Spike returned ll protein isolates. Each of the putative epitope clusters identified by EpiMatrix® and ClustiMer® were BLASTed against this database. About 60% of the selected epitope clusters could be matched to 1 1 (for Spike) or 12 SARS-COV- 1 isolates at 80% or greater sequence identity. In order to assess conservation with endemic human coronaviruses (hCoVs), a human coronavirus reference database was assembled by querying GenBank for known isolates of known human coronaviruses. The queries used are as follows:

For HCOV-229E and Envelope: ("Human coronavirus 229E"[ALL] AND 75:85[SLEN] AND

Envelope[ALL] AND "Homosapiens"[ALLJ) NOT X1D2697049

For HCOV-229E and Membrane: ("Human coronavirus 229E"[ALL] AND MembranefALL] AND 213:233[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-229E and Spike: ("Human coronavirus 229E"[ALL] AND SpikefALL] AND

1143:1173[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-HKU1 and Envelope: ("Human coronavirus HKU 1 "[ALL] AND 75:85[SLEN] AND

Envelope[ALL] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-HKU1 and Membrane: ("Human coronavirus HKU 1 "[ALL] AND Membrane[ALL] AND 213:233[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-HKU1 and Spike: ("Human coronavirus HKU 1 "[ALL] AND Spike[ALL] AND

1336:1356[SLEN] AND "Homo sapiens"[ALL|) NOT TXID2697049

For HCOV-NL63 and Envelope: ("Human coronavirus NL63"[ALL] AND 75:85[SLEN] AND

Envelope [ALL] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-NL63 and Membrane: ("Human coronavirus NL63"[ALL] AND Membrane[ALL]

AND 213:233[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-NL63 and Spike: ("Human coronavirus NL63"[ALL] AND Spike[ALL] AND 1336: 1356[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-OC43 and Envelope: ("Human coronavirus OC43"[ALL] AND 75:85[SLEN]

AND Envelope[ALL] AND "Homo sapiens"[ALLJ) NOT TXID2697049

For HCOV-OC43 and Membrane: ("Human coronavirus OC43"[ALL] AND Membrane[ALL]

AND 213:233[SLEN] AND "Homo sapiens"[ALL]) NOT TXID2697049

For HCOV-OC43 and Spike: ("Human coronavirus OC43"[ALL] AND Spike[ALL] AND

1344: 1364[SLEN] AND "Homo sapiens"[ALLJ) NOT TXID2697049

When these queries were executed, the queries for Envelope and Spike each returned 80 protein isolates. The query for Membrane returned 77 protein isolates. Each of the putative epitope clusters identified by EpiMatrix® and ClustiMer® were BLASTed against this database. No epitope clusters could be matched to any hCoV reference sequences evaluated at 80% identity or better.

To assess conservation with known B cell epitopes, a B cell epitope reference database was assembled from published sources [Cell Host Microbe. 2020 Apr 8;27(4):671 -680. e2. doi: 10.1016/j.chom.2020.03.002 and Viruses. 2020 Feb 25; 12(3). pii: E254. doi: 10.3390/v12030254], Seven of the selected epitope clusters could be related to known B cell epitopes. Activation of pre- existing cognate B cells can help create a robust response to vaccination leading to enhanced memory formation.

In the instant example, epitope selection included setting aside all putative epitope clusters identified as potential Tregitopcs. Putative epitope clusters were also set aside that were tagged as having potential synthesis "red flags." This yielded a total of 32 candidate peptides for further evaluation (six initial pool candidates were tagged as both potential Tregitopes and having synthesis red flags). Certain epitope clusters with conservation to either SARS- COV, SARS-CoV B cell epitopes, or hCoVs are tagged for independent development and evaluation.

Table 3 below provides a listing of 32 exemplary candidate T cell epitope clusters selected according to the staged computational vaccinology screening methods described above. Figure 7 illustrates immunogenicity projections for these identified clusters compared to a series of standard controls.

Table 3. Overview of Class II Clusters Derived from SARS-COV-2 Proteins

Based on the foregoing computational vaccinology analyses, findings and determinations, a panel of candidate li-Key-SARS-CoV-2 hybrid peptides was constructed using the general li-Key construction and peptide synthetic teachings above. For initial screening and characterization purposes, all of the li-Key fusions with SARS-CoV-2 antigenic peptides were constructed similarly, using a basic li-Key hybrid design. In particular, all Ii-Key-SARS-CoV-2 peptide constructs in this example use a minimal li-Key sequence LMRK (SEQ ID NO: 37), and have an ava linker between the li-Key and antigenic peptide components. Each is N-terminally acylated and C-terminally amidated for simplicity and uniformity. All of these exemplary Ii-Key-SARS-CoV-2 antigenic peptide hybrids presented in fable 4, below, are subject to alteration/optimization using the alternative construction options described herein, including by modifying the li-Key sequence, the antigenic peptide sequence, to vary linker length or properties, alter terminal modification design, etc.

TABLE 4 Exemplary Ii-Key-SARS-CoV-2 Peptide Hybrid Constructs

An additional epitope cluster peptide (designated herein "peptide 33") was identified as a B cell epitope EIDRLNEVAKNLNESLIDLQELGKYEQI) (SEQ ID NO: 79). This epitope, used to construct li-Key-SARS-CoV-2 peptide hybrid 33, exhibits 100% sequence conservation with SARS- CoV-1 and will be selected for certain li-Key vaccine formulations of the invention for its potent capacity in an li-Key hybrid construction for eliciting neutralizing antibodies. EXAMPLE II Evaluation and Selection of Anti-SARS-CoV-2 Effective li-Kcv-SARS-CoV-2 Hybrid Peptides For Vaccine Use To Elicit Selective, Multi-Functional Immune-Regulatory Responses

Further screening and characterization of Ii-Key-SARS-CoV-2 antigenic peptide hybrids include qualifying candidate constructs for their ability to bind known or presumptive neutralizing anti-SARS-CoV-2 antibodies, for example in a serum sample from one or more successfully recovered (convalescent) COVID-19 patient(s). In related studies, Ii-Key-SARS-CoV-2 peptide-cognate antibodies (i.e., that specifically recognize/bind a Ii-Key-SARS-CoV-2 antigenic peptide) are tested in a viral neutralization assay to confirm their virus-neutralizing activity, indicating the corresponding (cognate) li-Key hybrid peptide is presumptively able to elicit a neutralizing antibody response in human subjects in vivo. These same screening steps provide for rational exclusion of li-Key-SARS- CoV-2 antigenic peptide candidates recognized/bound by non-neutralizing antibodies, which candidates are predicted to elicit potentially adverse, non-neutralizing antibody responses in vivo.

Additional screening detects and characterizes patterns of immune activation and downstream activities of immune effector cells and their targets, induced by candidate SARS-CoV-2 li-Key antigenic peptide hybrids, for example using peripheral blood mononuclear cells (PBMCs) or other cellular assay subjects obtained from COVID-19 convalescent patients. These assays allow, for example, selection of SARS-CoV-2 li-Key antigenic peptides that elicit Th1 or Th2 T helper cell activation bias or exclusivity. Yet additional, or optional, assays to characterize and select SARS- CoV-2 li-Key antigenic peptide hybrids for use within vaccine compositions include various screening assays contemplated to screen for elimination of peptides having demonstrated CSS and ADE contributory impacts, such as induction of pro-inflammatory cytokines associated with hyper-immune or -inflammatory activation linked to CSS and ARDS. Based on activity in these and other screening assays, optimal Ii-Key-SARS-CoV-2 peptide vaccine candidates are selected and/or combined in a candidate vaccine composition comprising peptide(s) manufactured in a GMP facility for the Phase 1 human clinical trials.

Ii-Key-SARS-CoV-2 Epitope Binding to Antibodies from Convalescent COVID-19 Plasma

To determine if the Ii-Key-SARS-CoV-2 epitopes are directed toward neutralizing regions of the virus, we conducted a screening program to test the antibody binding from convalescent COV1D- 19 serum samples. The 32 Ii-Key-SARS-CoV-2 epitopes were screened against convalescent serum samples from 8 COVID-19 recovered patients. COVID-19 convalescent blood and serum is presumed to contain anti-SARS-CoV-2 competent neutralizing antibodies, and some of these should recognize and bind Ii-Key-SARS-CoV-2 antigenic peptides predicted using the above-described computational methods and tools. The following assays will detect "cognate" antibodies from COVID-19 convalescent patients that recognize and bind with specific affinity to Ii-Key-SARS-CoV-2 antigenic peptides.

Convalescent blood is collected according to standard operating procedures published by Cellular Technologies Limited (CTL) (Cleveland. OH ). Blood or serum samples may kept separate per patient, or be pooled for representation of general population demographics, including a discrete or broad spectrum of COVID-19 disease severity, and for specific or broad HLA population coverage. Donor subjects are determined COVID-19 positive by medical record review and documentation of positive rtPCR. Seroconversion is confirmed prior to sample collection by rapid qualitative ELISA IgG and confirmed again prior to laboratory analysis by ELISA IgG titer. Control assay samples will incorporate COVID-19 negative pooled serum collected prior to November 2019 and confirmed negative by ELISA IgG titer.

Standard peptide Elisa protocols were adapted as follows. Peptides were resuspended in DMSO at I mg/ml and directly coated onto high- binding 384-well plates (Corning, 3700) at 2 μg /mL overnight at 4oC. After washing, plates were blocked with 3% BSA in PBS for 1 h. After washing, serially diluted mAbs or sera were added into wells and incubated for 1 .5 h at RT. Detection was measured with alkaline phosphatase-conjugated goat anti-human IgG Fey (Jackson ImmunoResearch 109-005- 008) at 1 : 1500 dilution for Ih. After the final wash, phosphatase substrate (Sigma-Aldrich, S0942-200TAB) was added into wells. Absorption was measured at 405 nm.

In one exemplary series of studies, individual Ii-Key-SARS-CoV-2 hybrid peptides were screened for antibody binding in serum samples from 8 patients recently recovered from COVID-19. The Ii-Key-SARS-CoV-2 hybrid peptides were plated and incubated with the convalescent serum in a conventional ELISA format. Positive antibody binding to specific Ii-Key-SARS-CoV-2 hybrid peptides was measured with immunofluorescence, and these results are presented in heatmap tables below. Control wells indicate background fluorescence, and the intensity of green-shading in the cells correlates w ith the intensity of antibody binding detected. As Tables 5- 1 1 demonstrate, numerous li-Key-SARS-CoV-2 hybrid peptides were specifically recognized and bound by presumptive anti-SARS-CoV-2 antibodies in convalescent patient samples, and in general these cognate antibody-peptide interactions were consistently identified across multiple patient samples.

TABLE 5- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid Peptides 1-5 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19 Patients

TABLE 6- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid Peptides 6-10 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19 Patients

TABLE 7- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid Peptides 11-15 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19 Patients TABLE 8- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid

Peptides 16-20 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19

Patients

TABLE 9- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid

Peptides 21-25 in Triplicate Samples of Convalescent Serum From 8 Recovered CO VID-19

Patients TABLE 10- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid Peptides 26-30 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19 Patients

TABLE 11- Detection of Cognate Antibody Binding Against Ii-Key-SARS-CoV-2 Hybrid Peptides 31-33 in Triplicate Samples of Convalescent Serum From 8 Recovered COVID-19 Patients the foregoing data are used to guide selection of candidate vaccine peptides. These data, along with neutralization data, CD8+ data, CBA/CSS assay results, ADE results, all contribute to identification of specific lead candidates, and individually determine Ii-Key-SARS-CoV-2 hybrid peptides that are multi-functional (e.g., Th1 CD4+ T cell-activating, potent to elicit a neutralizing B cell antibody secretory response, and also CD8+ T cell-activating). Purification of Antibodies from Convalescent COVID-19 Plasma using Ii-Key-SARS-CoV-2 Antigenic Peptide Hybrids.

Ii-Key-SARS-CoV-2 peptide hybrids were manufactured with an N-terminal cysteine utilizing standard solid-state amino acid chemistry. The N-terminal cysteine enables conjugation of the epitopes with magnetic beads that can be used to purify antibodies that bind the li-Key epitopes. Bead Coupling employed I mL of NHS magnetic beads (ABC MBFB-0400) washed through magnetic separation with I mL of 1 XPBS twice. Beads were resuspended in 5mL of coupling buffer (150mM NaCI. 0.01 % Tween-20. 50mM MES, pH7.0). 5mL of protein solution was added to the bead suspension and incubated at 4°C overnight while rotating. 2mL of quenching buffer (150mM NaCI, 100mM Tris-HCl, pH7.0) was added and incubated at 4C o/n. The beads were collected through magnetic separation and the supernatant was discarded. The beads were then washed 2 times with I mL of quenching buffer for 15min at RT. The beads were then resuspended inn PBS and stored at 4°C. The conjugated Ii-Key-SARS-CoV-2 epitope beads were incubated with convalescent serum samples from COVID-19 recovered patients. Coupled beads were separated magnetically and the supernatant was discarded. The beads were then resuspended in 9mL of PBS and I mL of polyclonal sera/plasma from convalescent COVID-19 patients. The beads were incubated overnight at 4°C while rotating. The bead mixture was magnetically separated and the supernatant was stored. The beads w ere washed 3 times w ith 5mL of PBS. The beads were then incubated in 7.5mL of elution buffer (0.2M Citric acid pH 3.0) for 5 minutes at RT while rotating. The beads were magnetically separated and the supernatant w as saved. 2.25 mL of neutralization buffer (2M TRIS base pH 9.0) was then added to the supernatant. A 50K amicon tube (Millipore UFC905024) was used to exchange buffers to PBS. The sample was quantified and used in the neutralization (FRNT) assays below.

Neutralization Assays Using Antibodies Isolated From Convalescent COVID-19 Plasma Based On Their Cognate Binding to Ii-Key-SARS-CoV-2 Antigenic Peptide Hybrids

For the present example, in order to determine which of the "cognate" antibodies (e.g., antibodies from pooled COVID-19 convalescent serum that specifically bind Ii-Key-SARS-CoV-2 epitopes) can actually neutralize the SARS-CoV-2 virus, Applicants conducted focus reduction neutralization assays (FRNTs) in a BSL 3 laboratory at the University of California San Diego (UCSD). HeLa-ACE2 cells were seeded in 12 μL complete DMEM at a density of 2x10 ³ cells per well. In a dilution plate, plasma or mAb was diluted in series with a final volume of 12.5 μL. Then 12.5 μL of SARS-CoV-2 was added to the dilution plate at a concentration of 1.2x104 pfu/mL. After 1 h incubation, the media remaining on the 384-well cell plate was removed and 25 μL of the virus/plasma mixture was added to the 384-well cell plate. The plate was incubated for 20 h after which the plate was fixed for 1 h. The plate was then washed three times with 100 μL of IxPBS 0.05% tween. 12.5 μL of human polyclonal sera diluted 1 :500 in Perm/Wash buffer (BD Biosciences 554723) were added to the plate and incubated at RT for 2 h. The plate was washed three times and peroxidase goat anti-human Fab (Jackson Scientific) were diluted 1 :200 in Perm/Wash buffer then added to the plate and incubated at RT for 2 h. The plate was then washed three times and 12.5 μL of Perm/Wash buffer was added to the plate then incubated at RT for 5 min. The Perm/Wash buffer was removed and TrueBlue peroxidase substrate was immediately added (Sera Care 5510-0030). Plasma or mAbs were tested in triplicate wells. SZMAb-5, a Zika specific mAb, and normal human plasma were used as negative controls for mAb and plasma screening respectively. Infected cell non-linear regression curves were analyzed using Prism 8 software to calculate EC ₅₀ values.

PBMC Screening for CD4+ Th1 and Th2 Activation, and for CD8+ CTL Activation, by li-Key- SARS-CoV-2 Antigenic Peptide Hybrids

In order to minimize the potential for vaccine-associated hyper-immune or inflammatory activation, including CSS, vaccines will typically be constructed using Ii-Key-SARS-CoV-2 peptides selected for their immune-selective regulatory ability to induce Th1 -biased T helper cell differentiation and marker expression (e.g., Th1 - versus Th2- specific cytokine/chemokine/growth factor expression pattern). COVID-19 convalescent blood or other samples are utilized that contain competent immune cell samples, for example PBMC samples isolated from COVID-19 convalescent subjects, to evaluate induction ofTh1 -biased versus Th2-biased T cell differentiation and marker expression. In the instant example, cellular immune responses were evaluated using a 3-color T-cell ELISpot performed by CTL Laboratories. CD4+ Th1 responses were identified by measuring IFN-y; CD4+ Th2 responses were identified by measuring IL-5; and CD8+ responses were determined by measuring Granzyme B. This process limit risks of vaccinating a person with an off-target epitope that might generate a potentially risky Th2 responses that may put the vaccinee at for a future infection attended by hyper-immune or -inflammatory activation and/or CSS.

The enzyme-linked immune absorbent spot (ELISpot) is a highly sensitive and specific assay that quantitatively measures the frequency of cytokine or immunoglobulin secretion by a single cell. ELISpot has been widely applied to investigate specific immune responses in infections, cancer, allergies and autoimmune diseases. With detection levels as low as one cell in 100,000, ELISpot is among the most sensitive cellular assays currently available. The FluoroSpot Assay is a variation of the ELISpot assay, using fluorescence to analyze multiple cytokines in a single well. ELISpot assays are carried out in a 96-well plate, and an automated ELISpot reader is used for analysis. The assay is therefore robust, easy to perform and suitable for large-scale trials. T-cell ELISpot is widely applied in investigations of specific immune responses in infectious diseases, cancer, allergies, and autoimmune diseases. Within the instant invention, T-cell ELISpot assays are particularly useful to guide development and monitor the efficacy and safety of Ii-Key-SARS-CoV-2 antigenic peptide vaccines.

Cellular Technologies Limited (CTL) (Cleveland, OH) served as Applicants' agent to conduct a variation of the ELISpot assay utilizing CTL's proprietary ImmunoSpot® platform that permits optimized, scientifically validated single cell ELISpot analyses, for detecting and measuring effector molecule secretion by individual T cells in response to stimulation by Ii-Key-SARS-CoV-2 peptides ofthe invention. Within the instant example, ImmunoSpot® assays reveal specific numbers of antigen-specific T cells present in convalescent COVID-19 PBMC samples that respond to stimulation by specific Ii-Key-SARS-CoV-2 peptides using peripheral blood mononuclear cells (PBMCs). The frequency of the responsive T cells detected in a test PBMC sample reflects a magnitude of T cell immune response, while spot sizes indicate the quantity of cytokines secreted by activated cells.

The general protocol for ELISpot assays employed within the invention is illustrated in Figure 8. Cells are cultured on an antibody-coated surface in the presence or absence of stimuli. Proteins, for example cytokines, secreted by the cells are captured immediately after secretion and throughout the stimulation process by the specific antibodies. After an incubation period, the cells are removed by washing and the secreted cytokines are detected by biotinylated or enzyme-conjugated detection antibodies. By using a precipitating enzyme substrate, the end result is visible as a spot, where each spot corresponds to a single secreting cell.

ELISpot was used in the instant example to discriminate between subsets of li-Key-SARS- CoV-2 peptide-activated T cells. For example. ThI cells can be identified by their production of one or more "Th1 cytokines" selected from IFN-γ, IL-2, and/or TNF-a, while Th2 cells can be identified by their production of one or more "Th2 cytokines" selected from IL-4, IL-5, and/or IL-13. CD8+ cells are identified by detection of Granzyme B(GrB). We tested a total of 46 samples of frozen peripheral blood mononuclear cells (PBMC) in a IFN-γ/Granzyme B/lL-5-detecting fluorescent ELISpot assay. Eight (8) samples were provided by CTL (Cleveland, OH) and thirty-eight (38) were provided by the University of California San Diego. In addition, 30 healthy cryopreserved PBMC samples collected during or before 2018 were tested in the double color IFN-y/Granzyme B fluorescent ELISPOT assay. All samples were collected under IRB approved protocols. The aim of the experiments was to test recall responses to the Ii-Key-SARS-CoV-2 peptide hybrids provided in the ELISpot assays. Ii-Key-SARS-CoV-2 antigenic peptides were diluted and a stock solution was aliquoted and frozen at - 20°C. The Ii-Key-SARS-CoV-2 antigenic peptides were tested at a concentration of 100 ug/m; 25 ug/ml and 2.5 ug/ml. The ELISpot assays were performed according to the contractor. CTL's published, standard operating procedure (SOP). The PBMCs were pipetted into pre-coated wells at a concentration of 4x 10 ⁵ cells per well in duplicate or triplicate wells (depending on quantity of viable PBMCs) and cultured for 24 hours with each of the 33 Ii-Key-SARS-CoV-2 antigenic peptides (i.e.. 33 hybrid li-Key peptides using the 33 epitope-containing peptides identified from the computational epitope analyses). Medium alone served as the negative control. As positive control we used PHA (at 5ug/ml) to stimulate both CD4+ and CD8+ T cell subsets. The viability of the PBMCs was assessed using the Guava automated cell counter according to CTL's SOP. After the activation culture period, the cells were discarded, and labeled cytokine-specific detection antibodies were added to the plate. Subsequently, the plate-bound detection antibody was visualized via tri-color fluorometry, permitting detection and quantification of color precipitation "spot" (corresponding to a single cell's secretory activity).

Further details of the ImmunoSpot® assay protocols are as follows:

On day 0, capture solution was prepared by diluting the capture antibody according to cytokine specific protocols. PVDF membranes were pre-wet with 70% ethanol for 30sec and washed with 150μl of PBS three times before adding 80μl of the Capture Solution into each well. Plates were incubated overnight at 4°C in a humidified chamber.

On day I . CTL-Test™ Medium was prepared by adding 1 % fresh L-glutamine, and the antigen solutions at 2X final concentration in CTL-Test™ Medium. The plate was decanted with coating antibody from Day 0, washed one time with 150μl PBS, and plated the antigen solutions in

100μl/well After thawing, PBMCs were adjusted to the desired concentration in CTL-Test™ Medium and incubated at 37oC in a humidified incubator with 5-9% CO2. The cells were incubated for 24 hours.

On day 2 detection solution was prepared by diluting detection antibody according to the cytokine specific protocol. Plates were washed two times with PBS and then two times with 0.05% Tween-PBS, 200μl/well each time. 80pl/well of detection solution was added, then samples were incubated at RT for 2h. Tertiary solution was prepared by diluting the tertiary antibody according to specific protocol. Plates were washed three times with 0.05% Tween-PBS, 200pl/well. 80pl/well of Strep-AP Solution was added, and incubation continued at RT for 30min. Developer solution was prepared according to cytokine specific protocols. Plates were washed two times with 0.05% Tween- PBS. and then two times with distilled water. 200μl/well each time. Developer solution was added, 80pl/well. and incubation continued at RT for 10-20 min. The reaction was stopped by gently rinsing the membrane with water three times. T he plates were air-dried for a minimum of 2 hours and scanned for analysis.

Results of these assays with respect to Ii-Key-SARS-CoV-2 peptide-specific activation of CD4+ Th1 T cells (induced to express gamma IFN) are presented in Table 12, below.

Results of these assays with respect to Ii-Key-SARS-CoV-2 peptide-specific activation of CD8+ T cells (induced to express Granzyme B) are presented in Table 13, below. The results of T cell and antibody screening presented above demonstrate the potency and specificity of Ii-Key-SARS-CoV-2 hybrid peptides to regulate the immune system through Th1- specific activation of T helper cells potent induction of CD8+ lymphocytes, without activation of the Th2 response and without hyper-activation or dysregulation of immune cells to over-express detrimental cytokines associated with CSS and ARDS.

The CTL Primary T-Cell Screening data set forth in Table 12 and 13, above demonstrate strong, positive activation ofCD4+ Th1 responses (demonstrated by Gamma Interferon induction in PBMCs) by numerous Ii-Key-SARS-CoV-2 peptide hybrids. Similarly, positive CD8+ CTL activation is demonstrated for numerous Ii-K.ey-SARS-CoV-2 peptide hybrids by induction of Granzyme B (data presented in fable 13).

Despite exhaustive screening for Th2 T helper induction, none of the candidate li-Key-SARS- CoV-2 peptide hybrids activated Th2 pathway differentiation among PBMCs (no expression of IL-5 was observed in any of the samples tested).

An evaluation of patient blood samples from 2017 and 2018 prior to the COVID-19 pandemic yielded only three positive responses among the 32 Ii-Key-SARS-CoV-2 peptide hybrids tested (each the three positives having just I positive reaction out of 8 samples tested). These results demonstrate that Ii-Key-SARS-CoV-2 peptide hybrids designed and constructed according to the invention are highly specific for SARS-CoV-2, and will not be widely cross-reactive with immune cells having cognate immune memory for seasonal coronaviruses.

The B Cell epitope, li-Key-Epitope 33 (EIDRLNEVAKNLNESLIDLQELGKYEQU; SEQ ID NO: 79). which is 100% homologous with SARS-CoV-1 did show a positive response in 25% (2/8) samples tested, indicating potential homology with not only SARS-CoV-1 but also perhaps with seasonal coronaviruses.

The foregoing data, including the data for screening Ii-Key-SARS-CoV-2 peptide hybrids against healthy donor PMBCs, are both fundamentally surprising, and extraordinarily promising for SARS-CoV-2 vaccine development. There are four primary endemic human coronaviruses HCoV- 229E, -NL63. -OC43, and -HKU1, each causing substantial lower respiratory tract infections in adults and children (Corman. 2018). Researchers at the NIH have demonstrated serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal Betacoronaviruses (Hicks et. al., 2020). Since COVID-19 disease severity is closely associated with over-reactive (and likely non-specific, or non-neutralizing) immune responses, the instant invention provides novel solutions to avoid off-target responses and exacerbation of hyper-immune and hyper-inflammatory reactions associated with prior SARS-CoV-2 vaccination and/or prior substantial exposure(s) to other hCOVs. This is particularly critical in the current vaccine development context, because off target immune and inflammatory responses involving cross-reactivity with hCOVs are most often directed at the spike protein (Rogers, 2020, Wang. 2016). All of the SARS-CoV-2 vaccines in current deployment involve expression or administration of a complete or partial spike protein, and are therefore likely to trigger off-target or excessive immune responses in subjects presenting with a history of immune exposure to hCOVs.

In addition to the concern of latent serologic responses in subjects previously exposed to hCOVs, it is also likely that whole or partial spike protein vaccination will generate off-target and non-neutralizing immune responses that will contribute to ADE upon subsequent natural infection. The most worrisome aspect of vaccine related ADE was reported during Dengue vaccine development trials in Asia and Latin America for the first recombinant live-attenuated, tetravalent Dengue vaccine (who.int., 2020). As described in detail above, the risk of severe clinical outcome was actually increased by vaccination among seronegative persons not previously exposed to the virus (Sridhar et al., 2018). The proposed explanation is that Dengue vaccination mimics primary infection, whereafter waning immunity exposes vaccinated individuals to increased ADE risk.

To develop a truly safe and effective vaccine against SARS-CoV-2, it is therefore essential that the vaccine triggers neutralizing, long-term immune responses without activating off target responses that contribute to ADE or other hyper-immune responses, including CSS and multisystem inflammatory syndrome in children (MISC). The li-Key-SARS-CoV-2 vaccines of the invention have been target specific regions of the SARS-CoV-2 spike and membrane proteins that produce only desired, specific and discrete T cell and antibody responses for positive immunity, eliminating other regions with non-specific immunogenic character and with dangerous homology with endemic human coronaviruses.

The li-Key-SARS-CoV-2 antigenic peptide constructs of the invention have been evaluated against competent T cell populations from healthy (SARS-CoV-2-naiive) subjects. In particular, 11 representative li-Key epitopes were assayed w ith ELISpot against 30 blood samples from healthy donors collected in 2017-2018, before the SARS-CoV-2 pandemic. Out of a total of 660 tests (11 li- Key epitopes X 30 PBMC samples X 2 assays) there were only 8 positive responses (1.2%): five positive gamma interferon responses and 3 Granzyme-B responses. The results are both novel and highly unexpected, showing that only 2 out of 30 naiive T cell samples respond to stimulation with any tested li-Key epitopes in the IFN-Gamma-detecting assay, and two in the Granzyme B-detecting assay. Only one samples responded in both assays. In view of the data showing that the same li-Key- SARS-CoV-2 peptides elicit potent, specific T cell responses across multiple samples from convalescent COVID-19 patients, these negative results from naiive individuals demonstrate an unprecedented degree of specificity for Ii-Key-SARS-CoV-2 Ii-Key-SARS-CoV-2 hybrid peptides.

The most positive response in SARS-CoV-2-naiive subjects was elicited with li-Key construct 33, incorporating a B cell epitope within the sequence E1DRLNEVAKNLNESLIDLQELGKYEQU (SEQ ID NO: 79). This peptide was reported to elicit neutralizing antibodies in humans and primates against SARS-CoV-1 (Wang, 2016). Because this epitope has 100% with SARS-CoV-1 sequences, it will likely have greater cross-reactivity with endemic human coronaviruses. Since the epitope has been shown to neutralize SARS-CoV-1 , the cross-reactive immune responses are expected to be neutralizing for SARS-CoV-2, and perhaps can provide evidence of cross-reactive immune protection from specific regions of endemic coronaviruses. The theory of protective immunity due to previous infection with endemic human coronaviruses remains uncertain.

CSS/CBA Assays

Ii-Key-SARS-CoV-2 peptides of the invention are further screened and characterized for their safety and potential to elicit adverse hyper-immune and/or hyper-inflammatory responses, that could possibly contribute to CSS and/or ARDS in vaccinees, particularly upon later encounter with the wild- type SARS-CoV-2 virus. In view of the complex and uncertain immune and inflammatory interactions attending SARS-CoV-2 infection and COVID-19 disease, that have yet to be fully understood, the targets of these assays are fundamental — focusing on the potential for li-Key-SARS- CoV-2 peptides to induce expression of pro-inflammatory cytokines associated with CSS.

The subject assays employ an elegantly-modified cytometric bead array (CBA) screen, comprising a flow cytometry system adapted to quantify multiple cytokines simultaneously in cell culture supernatants (SN). or in biological fluids such as serum or plasma. The CBA system uses the broad dynamic range of fluorescence detection offered by flow cytometry, along with antibody-coated beads to efficiently capture analytes. Each bead in the array has a unique fluorescence intensity so that beads capturing different analytes can be mixed and run simultaneously in a single tube. This method significantly reduces sample volumes and time to results in comparison to traditional ELISA and Western blot techniques. Figure 9 provides a schematic diagram of an exemplary CBA assay system.

Briefly, target cytokines are captured from lysate, serum or supernatant by capture antibodies conjugated to beads. Detector antibody labeled with fluorochrome binds to various captured cytokines, and the fluorescent signals are recorded during sample acquisition by flow cytometer. The intensity of signal depends on the concentration of each cytokine and can be used to calculate the concentration of specific cytokines using a protein standard curve. Using different size and fluorescence of beads for different capture cytokines, the signals recorded for different cytokines can be distinguished by flow cytometric analysis for multiple analytes in one test sample.

Fluorescent signal provided by each cytokine captured by antibody-coated beads and labeled with detection antibody is defined as mean fluorescence intensity (MFI) that can be converted into concentration of each cytokine in a test sample using a standard curve generated by measuring MFI from the standards (samples with known concentrations of the given analyte). The increased concentration of cytokine is detected in the cell culture supernatant when the cells secrete cytokines in response to stimulation with a test Ii-Key-SARS-CoV-2 peptide hybrid. The baseline concentration of cytokine detected in cell culture supernatant from cells cultured with test medium alone serves as a negative control. rhe utility of these screening assays for rational design of Ii-Key-SARS-CoV-2 peptide hybrid vaccines is to eliminate risks of vaccine-induced hyper-immune or -inflammatory responses in vaccinees, particularly in elevated risk subjects, previously SARS-CoV-2-infected subjects, or subjects infected long after vaccination. It is well established that increased, unregulated expression of pro-inflammatory cytokines and chemokines (CSS) mediates excessive damage of organs and tissues. CSS is observed often during the acute phase of inflammation and infectious disease. Cytokines that are up-regulated and likely contribute to organ and tissue damage in severe COVID-19 disease patients have been reported to include:

IL-4, IL-6, IL-8, IL- 10, IL- 15 - Univ, of Modena, Italy, April 2020

IL-6, CCL2 (MCP- 1 ), CXCL9 (Mig) - Mt. Sinai (NY), Stanford (CA), USA, April-May 2020 IL-4. IL- 10, IL-12, IL-17, CXCL 10 (IP-10) - Beijing, Shenzhen, China, Feb-March 2020 Functional roles of pro-inflammatory cytokines in CSS and tissue/organ damage, for example, include the following: IL-6, IL-8, CCL2 - activation/recruitment of neutrophils and monocytes

IL- 17 tissue inflammation in autoimmune diseases (MS, IBD)

CXCL chemokines (Mig, IP- 10) - recruitment ofNK and T cells into organs/tissues

For calibrating and clarifying the results of CBA assays for use within the invention (to test li- Key-SARS-CoV-2 peptide hybrids for CSS-inducing potential), expression levels for a panel of pro- inflammatory cytokines known to be associated with CSS and ARDS in severe COVID-19 patients were tested and compared between severe COVID-19 patients and healthy controls. The panel of pro- inflammatory cytokines tested included human IL-6, IL-8, IL- 10, IL- 17, IFN-y, TNF, MCP-1 (CCL2) and Mig (CXCL9). Serum was collected and pooled from 5 healthy donors and 9 patients hospitalized for COVID-19. The assays were performed as described above. As shown in Figure 10, these CBA calibration assays establish a baseline for pro-inflammatory cytokines measured in healthy control samples, and exemplary profiles of elevated expression levels for each of the subject inflammatory cytokines measured in severe COVID-19 patients.

Comparable assays were conducted for each candidate Ii-Key-SARS-CoV-2 peptide hybrid using supernatants collected from ELlSpot assay plates of healthy PBMC samples and Covid-19 convalescent PBMC samples, after the Ii-Kcy-SARS-CoV-2 peptide stimulation and control treatments described above, as well as from a positive control Covid-19 convalescent PBMC sample un-stimulated by Ii-Key-SARS-CoV-2 peptide. Peptides that elicit excessive pro-inflammatory cytokine expression in comparison to levels detected in healthy control PBMC samples, and in positive control comparison to un-stimulated Covid- 19 convalescent PBMC samples, will be excluded from vaccine use.

ADE Assays

All cognate antibodies from pooled COVID-19 convalescent serum that specifically bound to Ii-Key-SARS-CoV-2 epitopes were screened in an antibody dependent disease enhancement (ADE) assay using U937 cells, which are an accepted cytological model for the immunological phenomenon known as cytokine storm syndrome (CSS). U937 cells were seeded in 12 μl . complete DMEM at a density of 2x103 cells per well. In a dilution plate, plasma or mAb was diluted in series with a final volume of 12.5 μL. Then 12.5 μL of SARS-CoV-2 was added to the dilution plate at a concentration of 1 ,2x 10 ⁴ pfu/mL. After 1 h incubation, the media remaining on the 384-well cell plate was removed and 25 μL of the virus/plasma mixture was added to the 384-well cell plate. The plate was incubated for 20 h after which supernantant is removed from the infected plates and added to the FRNT assay (see above) to determine the PFU/ml produced by U937 cells with and without plasma. The fold increase in PFU/ml is enumerated and used to define observed ADE.

Long-Term Memory B Cell Assays

In related examples Applicants have adapted ELlSpot assays for specific detection of long- term memory B-cells stimulated in ex vivo samples or in actual patients following exposure or vaccination with a li-Key-SARS-CoV-2 antigenic peptide hybrid. These B-cell ELlSpot assays provide a powerful tool used to analyze antibody immune responses. Principal applications include detection of B-cell responses to infections and responses elicited by vaccination. B-cell ELlSpot directly on antibody-secreting cells (ASCs), in contrast to assays designed to measure antibody reactivity in solution. This extremely sensitive method identification of ASCs in a sample, including determination of total positive cell numbers as well as determination of cells secreting antibodies directed against a specific, selected antigen. With B-cell ELlSpot, it is possible, for example, to demonstrate the presence and the frequencies of long-term memory B cells in the blood, which are difficult to assess by other methods.

Human Leukocyte Antigen (HLA) Testing of Convalescent COVID-19 Patient Samples

The li-Key is specific for human Ml IC complexes, and epitopes are specific to the HLA genetic makeup of individuals. It is important that the SARS-CoV-2 epitopes selected for the final vaccine formulation are recognized by HLA types across the population. Accordingly, Applicants have analyzed HLA profiles of convalescent COVID-19 blood samples used in the li-Key epitope T cell screening program. The final selection of li-Key-SARS-CoV-2 peptides for vaccine use will be guided by HLA screening, to select peptides that will activate the desired immune responses in greater than 95% of the population.

The most common HLA type is HLA-A2 allele. Caucasian and Native American populations appear to be the most homogeneous, exhibiting 95.7% and 94.3% of the A*02-01-l Allelle, respectively. Hispanic and Asian/Pacific Islander populations were the most allelicly diverse populations with 9 and 7 different HLA-A2 alleles present, respectively, but the majority of the populations were I IL. A- A *02-01 - I . African-Americans were also diverse, not in the number of alleles seen, but in the percentage of non-A*020l -l alleles in the population. HLA-A*02-02 (25.8%) and A*02-05 ( 12.9%) were present in a large percentage of African-Americans (Ellis. 2000). The EpiMatrix algorithms employed in the above examples predict HLA binding of the li-Key-SARS- CoV-2 epitope sequences. As indicated by the EpiMatrix Clustimer data, the epitopes are predicted to bind across DRB HEA subtypes, however, the predicted results do not always comport with the results of T cell screening as described above. For example, two of the li-Key-SARS-CoV-2 peptide hybrids predicted by the EpiMatrix algorithms (constructs 5 and 12) were shown to elicit strong positive Th! T cell responses as indicated by gamma interferon activation. However, two other li-Key-SARS-CoV- 2 peptide hybrids (constructs 22 and 27) that also elicited strong Th1 T cell responses scored low in the EpiMatrix system. It is important that the Ii-Key-SARS-CoV-2 T cell epitopes selected for vaccine use cover a broad range of HEA, so a further in-depth analysis of Ii-Key-SARS-CoV-2 peptide hybrid immune regulatory activity will compare and match T cell responses from blood screening data against HLA types of donors. Additional, related studies will employ DR4 transgenic mice for testing the vaccine immunogenicity of Ii-Key-SARS-CoV-2 peptide antigens, including to determine appropriate Th1 T cell responses and neutralizing antibody responses.

The foregoing detailed investigations and surprising results enable novel design and construction of multi-targeting, multi-functional, selective immune-regulatory Ii-Key-SARS-CoV-2 peptide vaccines, useful for both effective prophylaxis and long-term protection against SARS-CoV-2 infection and COVID-19 disease. The subject vaccines thus provided, typically comprised of multiple, distinctly active Ii-Key-SARS-CoV-2 hybrid peptides, minimize off-target immunogenic and inflammatory effects, including by minimizing or excluding potential for induction of Th2 T-cell response, cytokine storm responses, and antibody dependent enhancement (ADE) responses.

Prior efforts to develop SARS-CoV and SARS-CoV-2 vaccines have focused almost exclusively on generating prophylactic antibody responses. As noted here, however, overactive antibody responses, including production of high titers of non-neutralizing antibodies, are likely to exacerbate risks of ADE. clearly concerning in the case of SARS-CoVs. These concerns are particularly high with SARS-CoV-2 infection, which appears to be exacerbated by prior exposures to endemic coronaviruses (hCoVs), and may indeed be potentiated or exacerbated (at least in the long term, after neutralizing antibody titers wane) by the current leading, whole-spike protein RNA and DNA vaccines, and other candidate vaccines that immunize non-specifically with whole viral proteins or large protein subunits. Duration of immunity after vaccination is a related concern, especially if vaccines do not activate T helper cells and provide an effective memory immune response, which effects remain undetermined for all SARS-CoV-2 vaccines currently in use or development.

Lymphopenia is a hallmark feature ofCOVID-19 infection, and a signature correlate of severe disease (ARDS/pneumonia) (Jesenak, 2020). Absolute numbers of T lymphocytes, CD4+ T cells, and CD8+ T cells decrease in most patients, and are often critically exhausted in severe cases (Chen 2020). In severe cases, precipitous T cell declines may be due to a number of causes, including direct viral infection, reprogramming or dysregulation causing functional impairment, and activation-induced apoptosis.

Protective and enduring immune responses to viral infections or vaccines usually arise from the combined actions of lymphocytes: B cells (responsible for humoral antibody immunity) and T cells (responsible for cellular immunity and helping B cell responses). B cells produce detectable antibodies in classes IgM, IgG, and IgA along with lesser amounts of IgD and IgE. An effective immune response to SARS-CoV-2 or to a vaccine involves subsets of T cells including CD4+ T helper cells (Th1 ) that are responsible for cellular immunity and for helping B cells to produce neutralizing antibodies, cytotoxic T cells (CD8+) that directly kill infected cells with the aid of T helper cells, regulatory T cells (Tregs) that control and balance the immune response, and inflammatory T cells (Th2, Th17). Importantly, all B and T cell types have immunological memory after a first encounter with a pathogen or a vaccine, enabling a faster effective response after a subsequent infection. To provide an optimally and enduringly effective "Complete Vaccine™", the compositions and methods of the invention elicit produce both a robust cellular response and a targeted humoral response, effective to prevent acute SARS-CoV-2 infection and provide long-term immune memory against future infections. The instant vaccines are uniquely effective to promote T helper cell activation and clonal expansion, as well as IGg production and long-term memory T cell responses.

The lack of attention paid to cellular immune responses, and to the potential for T Cell vaccines to prevent and manage viral infections and control viral pandemics, points to a long, unmet need in the art— tracing back at least to the dire emergence of SARS-CoV in 2002. T cells are a critical component of naturally-acquired protective immunity against viruses, and the goal of inducing T cell responses through vaccination has persisted unsatisfied for decades. This need is now timely met to respond to the unprecedented SARS-CoV-2 pandemic, through the rapid and responsive deployment of novel vaccines and methods provided herein. As described above, the novel li-Key- SARS-CoV-2 hybrid peptide vaccines of the invention elicit specific, multi-functional and enduring T cell activation, attended by downstream B cell activation and antibody production. Even more surprising, the vaccines of the invention achieve these beneficial immune-regulatory effects in a manner that is uniquely specific, discrete and attenuated, to avoid hyper-immune and hyper- inflammatory responses in vaccinees, even those who have been previously exposed SARS-CoV-2 or another hCoV, or have been vaccinated against SARS-CoV-2 using a conventional vaccine. The novel vaccine compositions and methods of the invention will prove especially important in limiting and managing the ongoing COVID-19 pandemic, including as adjunctive vaccines to prevent adverse sequelae after first-line vaccines currently in development have been widely deployed.

EXAMPLE III

Safety and Efficacy of li-Key Hybrid Peptide Vaccines

Extensive studies presented herein below establish the safety and efficacy of li-Key antigenic peptide hybrids for anti-viral vaccine use generally, whereby the li-Key-SARS-CoV-2 peptides described and characterized above are qualified as save and effective for direct implementation into Phase I human clinical testing.

Over an extensive course of many years of research, Applicants and their agents have tested li- Key peptide vaccine constructs in thousands of mice, and over 300 human subjects. All of these subjects have been injected with li-Key hybrid peptide vaccines with no serious toxicity, no deaths, and no unexpected health conditions. Detailed safety and immune system data from these pre-clinical animal and human clinical studies have been collected and evaluated to support this determination. li- Key peptide vaccine development thus far has demonstrated clear safety of li-Key vaccines, as well as their effectiveness to activate CD-4+ and CD-8+ T-cells to induce peptide-specific antibody and cellular immune responses. Based on a wealth of non-clinical and clinical data demonstrating safety and efficacy, FDA authorized a Ii-Key-HER2/neu peptide cancer vaccine developed by Applicants and their agents to proceed directly to human clinical trials.

Nonclinical data are also established from Applicant's extensive research directed to li-Key influenza peptide vaccines. Using 3 li-Key H5 influenza peptide hybrids (H5 peptides 160, 551, and 239). priming with Ii-Key-H5 Influenza peptide hybrid vaccines in murine subjects augmented helper T-cell and antibody responses to a rHA booster dose. Mice primed with an H5 160, 551 , or 239 H5 li- Key-Influenza antigenic peptide hybrid vaccine exhibited strong, antigen-specific serum IgG responses.

Lethal challenge studies were also conducted for these vaccines, using BALB/c mice immunized with the subject li-Key H5 Influenza peptide hybrids for prime boost immunization, as well as using a heterologous prime boost immunization with the peptides for the first immunization followed by either recombinant H5 HA or a DNA vaccine (using a DNA coding sequence corresponding to the H5 HA protein) as a boost vaccination. Mice that received either a single dose of recombinant H5 HA or a heterologous prime boost survived lethal challenge with the H5N1 virus.

Related nonclinical studies showed that li-Key hybrids containing naturally processed MHC class II epitopes, as demonstrated by their recognition in splenocytes of recombinant H5 immunized mice, activated helper T cells and boosted immunological responsiveness to a subsequent dose of recombinant H5 protein.

To date more than 300 mice have been immunized Ii-Key-H5 Influenza peptide hybrid vaccines, and more than 2,000 mice have been immunized with li-Key hybrid peptide vaccines constructed for breast cancer treatment. All of these studies revealed favorable safety profiles, with no deaths or unexpected adverse health effects observed.

Two GLP-compliant toxicology studies have additionally been conducted to investigate potential toxicity of li-Key hybrid peptide avian influenza vaccine candidates. In one toxicology study, a li-Key avian influenza hybrid peptide vaccine was tested in BALB/c Mice. The objective of this study was to evaluate potential toxicity of 3 avian flu vaccine candidates following 3 SC injections (Days 1 , 22, and 43) and a 3-week recovery period in BALB/c mice. The study design is shown in Table 14.

Table 14 Study Design (A Repeat-Dose Subcutaneous Injection Toxicity Study of Three Avian

Flu Vaccine Candidates in BALB/c Mice)

Only for hematology and clinical biochemistry investigations, euthanized on Day -7.

For Groups 1 and 2, 8 animals/sex/group were euthanized on Day 44 and the remaining 8 animals/sex/group, euthanized on Day 63 (i.e. approximately 3 weeks following last immunization). For Groups 3 to 8, 10 animals/sex/group were euthanized on Day 44 and the remaining 10 animals/sex/group, euthanized on Day 63 (i.e. approximately 3 weeks following last immunization).

Only spleens collected on Day 44 and shipped to the Sponsor for pharmacodynamic evaluations (analysis of splenocyte activation).

Aluminum hydroxide gel adjuvant Prepared in a 1:1 ratio, Alum to saline, 50μL of each to give a total of 100μL.

1'he following were evaluated: clinical signs (daily signs of ill health or reaction to treatment and weekly detailed examinations), body weight (weekly), food consumption (weekly), local irritation (daily up to 7 days following each dosing occasion and once weekly thereafter), ophthalmology (predose and Day 44), hematology (at necropsy), clinical biochemistry (at necropsy), pharmacodynamic evaluations (at necropsy), macroscopic observations at necropsy, organ weights, and histopathology . Baseline animals were included in the study as a health screen and laboratory investigations conducted on 'these animals confirmed the suitability of the mice received for this study.

The repeated SC administration of avian flu vaccine (3 strains administered Days 1 , 22, and 43) with adjuvant (alurmsaline, 1 :1 ratio) was well tolerated. There was no treatment-related mortality. Deaths of 2 male mice during the treatment period were attributed to urinary tract obstruction, considered unrelated to treatment. Death of a single female mouse at the cessation of the treatment period, although undetermined, could not be directly attributed to administration of the test vaccine. Clinical signs of swelling and redness and/or scabbing and lesions of the skin, graded slight to moderate, were evident at the injection site of mice receiving adjuvant or adjuvant in combination with test vaccine (li-Key-avian influenza hybrid peptide constructs, designated MPS349, MPS350, MPS351) as a direct consequence of the SC injections. There was no compound-related effect on body weights, food consumption, ophthalmology, hematology, or serum chemistry.

There were no important organ weight changes at either interval and there was no evidence of systemic or delayed toxicity.

At the end of both the treatment and recovery period, treatment-related macroscopic findings were limited to the injection site(s) and/or adjacent SC tissue in mice from the adjuvant control group (Group 2) and all MPS treatment groups. At the lumbar and sacral injection sites and adjacent SC tissue, the observation of "thickening" was a common finding in males and females from the adjuvant control group (Group 2) and all MPS dose groups. Other macroscopic findings included foci dark, area dark, scab, area depressed, nodule, and/or mass.

Also at both intervals, treatment-related microscopic findings were present at the injection site(s) and/or adjacent SC tissue in mice from the adjuvant control group (Group 2) and all MPS treatment groups. Microscopic changes at these sites were mostly limited to the presence of inflammatory cell infiltrates (subacute and/or chronic) which were usually associated with and surrounded accumulations of amorphous material (adjuvant). Although inflammation and amorphous material were present in some animals from the adjuvant control and all MPS-treated groups, there was some indication that the reaction was very slightly more intense or persistent in mice receiving the MPS350 strain of the vaccine, although dramatic differences between mice receiving adjuvant only and those receiving adjuvant and the vaccine candidates were not apparent.

In view of the foregoing results, the administration of 3 avian flu vaccine candidates (MPS349, MPS350 and MPS351 ) to BALB/c mice by SC injections on Days 1, 22, and 43 resulted in no systemic toxicity. Subcutaneous masses/nodules (edema and/or erythema) were evident at the site of injection in mice from the adjuvant control group and all MPS treatment groups. Treatment- related macroscopic and microscopic findings were limited to the injection site(s) and/or adjacent SC tissue in mice. Remarkable differences between mice receiving adjuvant only and those receiving adjuvant and the vaccine candidates were not apparent, although there was some indication that the reaction was very slightly more intense or persistent in mice receiving the MPS-350 strain of the vaccine. Further toxicology studies validated safety of li-Key hybrid peptide avian influenza vaccine candidates in DR4 mice. The objective of these studies was to investigate the potential toxicity of 2 avian flu vaccine candidates following 3 SC injections (Days 1 , 22, and 43) and a 3-week recovery period in DR4 mice. The study design is shown in Table 15.

Table 15 Study Design (A Repeat-Dose Subcutaneous Injection Toxicity Study of Two Avian Flu Vaccine Candidates in DR4 Mice)

The following were evaluated: clinical signs (daily signs of ill health or reaction to treatment and weekly detailed examinations), body weight (weekly), food consumption (weekly), local irritation (daily up to 7 days following each dosing occasion and once weekly thereafter or between doses), ophthalmology (predose and Day 44), hematology (at necropsy), clinical biochemistry (at necropsy), pharmacodynamic evaluations (at necropsy), macroscopic observations at necropsy, organ weights and histopathology. Baseline animals were included in the study as a health screen and laboratory investigations conducted on these animals confirmed the suitability of the mice received for this study.

The repeated SC administration of Avian Flu vaccine (2 strains administered Days 1 , 22, and 43) with adjuvant (alum:saline. 1 : 1 ratio) appeared to be well tolerated. There were no deaths during the course of the study. Clinical signs of swelling, discoloration (primarily white, red), scabbing and/or lesions of the skin, graded slight to moderate, were evident at the injection site of mice receiving adjuvant or adjuvant in combination with test article (MPS-348, MPS-352). These observations were considered a direct consequence of the SC injections and/or related to the presence of adjuvant in the SC tissue. Edema and erythema (at a lower incidence), as part of the draize scoring, were observed in the majority of mice from the adjuvant control group and all MPS treatment groups (25 and 250 μg/mouse). which correlated with the clinical observations. The incidence and severity were comparable between dose levels and both vaccines.

There was no compound-related effect on body weights, food consumption, ophthalmology and no toxicologically significant changes in serum chemistry.

Increases in white blood cell mass seen for MPS-348 treated females at the end of the treatment period were considered likely related to the inflammatory response at the injection sites.

1'here were no important organ weight changes at either interval.

At both intervals, treatment-related macroscopic findings were limited to the injection site(s) in male and female mice from the adjuvant control group (Group 2) and all MPS-348 and MPS-352 treatment groups. At the injection sites, the observation of "thickening" was a common macroscopic observation in male and female from the adjuvant control group (Group 2) and all MPS dose groups from both intervals. Additionally, the macroscopic observation of "mass" was noted in a few female mice following the recovery period.

Also, at both intervals, treatment-related microscopic findings were present at (and limited to) the injection site(s) in mice from the adjuvant control group (Group 2) and all MPS treatment groups. Microscopic changes at these sites were mostly limited to the presence of inflammatory cell infiltrates (chronic and less frequently, subacute) that were usually associated with and surrounded accumulations of amorphous material (adjuvant). There did not appear to be significant Histopathologic differences between adjuvant control sites and those injected with either MPS-348 or MPS-352, at either dose level. Following the recovery, while the microscopic changes of amorphous material accumulation and inflammation persisted, they appeared somewhat resolved.

Summarizing these results, the administration of 2 avian flu vaccine candidates (MPS-348 and MPS-352) to DR4 mice by SC injections on Days 1. 22, and 43 resulted in no histopathological indications of sy stemic toxicity. Subcutaneous masses/nodules (edema and/or erythema) were evident at the site of injection in mice from the adjuvant control group and all MPS treatment groups. Changes in clinical pathology were occasionally noted for MPS-treated animals but were generally not associated with histopathological changes with the exception of a slight increase in white blood cell mass for MPS-348 treated females at the end of treatment. Treatment-related macroscopic and microscopic findings were limited to the injection site(s) and/or adjacent SC tissue in mice, which were somewhat resolved following the recovery period. Remarkable differences between mice receiving adjuvant only and those receiving adjuvant and the vaccine candidates were not apparent.

Consistent with WHO guidance on vaccine development (WHO 2005), dedicated safety pharmacology studies beyond the murine studies presented above are not planned for li-Key-SARS- CoV-2, unless future findings suggest potential effects on physiological functions (eg, central nervous system, respiratory, cardiovascular) other than those of the immune system, human clinical studies.

EXAMPLE IV

Clinical Safety and Efficacy of li-Key-SARS-CoV-2 Hybrid Peptide Vaccines Applicants have developed a clinical regulatory strategy comprising a master protocol with separate clinical trials that will include study subjects according to health status, age, and occupation:

1. Healthy adult (Phase la safety and immunogenicity trial)

2. Healthy older adults (Phase lb safety and immunogenicity trial)

3. Healthcare workers (Phase III efficacy trial to determine protection from COVID-19 infection and correlation with immune system biomarkers of efficacy)

4. Surveillance study (10,000 patients with option to expand)

5. Geriatric population (Phase III safety and biomarker trial)

6. Pediatric population (Phase III safety and biomarker trial)

7. At-Risk populations (Phase lll safety and biomarker trial)

A Complete Vaccine ^TM immune response is our clinical endpoint, which includes induction of antigen-specific CD4+ and CD8+ T cell and neutralizing antibody responses, without ADE, hyper- immune, hyper-inflammatory or cytokine storm syndrome (CSS)-related co-responses. Full protocols for each of these trials are in preparation for submission to the FDA. The proposed clinical studies will explore dose response and effect of 250 μg to 1000 μg of li- Key-SARS-CoV-2 hybrid peptide vaccine alone and in combination with 1 μg or 5 μg of adjuvant, administered Days 1 and 22 on neutralizing antibody titers, serum binding antibodies, and T-cell responses up to Day 50 after the first vaccination in healthy adult participants 18 to 55 years of age (Phase 1 ) and up to Day 365 in healthy adult participants >18 years of age (Phase 2). Evaluation of serum neutralizing antibodies specific for SARS-CoV-2 and serum immunoglobulin G (IgG), IgA, and IgM. and total (Pan Ig) binding antibody levels specific for SARS-CoV-2 and individual peptide epitopes in the study vaccine will be evaluated between arms, including placebo. In addition, T-cell activation will be evaluated through IFN-γ. IL-5, and Granzyme B enzyme-linked immunosorbent spot (ELlSpot) assays as generally described above and otherwise known in the clinical diagnostic and monitoring arts. The ratio of neutralizing to binding antibodies will also be characterized in each arm up to 50 days after first vaccination in Phase I and up to Day 365 in Phase 2. Solicited adverse events will be collected through 7 days post each vaccination. Clinical laboratory data will also be collected up to Day 50 after the first vaccination. Unsolicited adverse events and physical examination results will be collected in all arms through 1 year.

For demonstration of safety, Phase 1 , placebo controlled, blinded, dose response safety study of li-Key-SARS-CoV-2 vaccine with and without adjuvant in normal healthy adults (18-55 years) will be implemented, with a DSMB review when 15 patients are enrolled in each arm. The DSMB will have stopping rules for safety and futility. Only those arms that demonstrate safety and appropriate immune responses will continue in an extension protocol that will expand the healthy adult population to include healthy older adults (Age 56 - 75). The extension protocol will enroll an estimated 30 to 50 subjects per arm, with the goals to determine safety, whether or not to use an adjuvant, and selection of the optimal dose of li-Key-SARS-CoV-2 vaccine for the efficacy trials.

Different doses of li-key peptides with or without adjuvant will be (Oug, 250ug, 500ug,1000ug). Clinical analysis will be performed to evaluate the immunologic response to the li-Key- SARS- CoV-2 vaccine by B-Cells (antibody/humoral) and T-Cells (CD-4, CD-8), including the following diagnostic/monitoring studies using generally known methods and materials. • Plaque reduction neutralization test (PR.NT) by SOP

• ELISA titer for IgG, IgM. IgA by SOP

• IgG fractionation by SOP

• B cell ELISpot by SOP

• Antibody dependent enhancement (ADE) assay using U-937 cell lines by SOP

• CD4 Th1 response will be assessed by measurement of INFy

• CD4 Th2 response will be assessed by measurement of IL-5

• CD8 response will be assessed by measurement of Granzyme-B.

Cellular immune responses to the li-Key-SARS-CoV-2 hybrid peptide vaccines will be evaluated using a three-color T cell ELISpot performed by CTL in preclinical studies and PPD in clinical trials. CD4 Th1 response will be assessed by measurement of INFy. CD4 Th2 response will be assessed by measurement of IL-5. CD8 response will be assessed by measurement of Granzyme-B.

Humoral immune response will include evaluation of antibody response magnitude, longevity, and viral neutralization. Antibody response magnitude will be assessed using ELISA IgG, IgM, and IgA titers. Antibody response magnitude and longevity will be further assessed using B cell ELISpot for measurement of antibody secreting cell numbers. Titers and ELISpot will be performed by CTL. Antibody viral neutralization characteristics will be assessed using a plaque reduction neutralization test (PRINT)

T he Master Protocol will involve initial enrollment with DSMB review' after the first 15 subjects followed by expansion to full enrollment. The dosing schedule will be vaccination on Day 1 a booster on Day 21 and blood draw for final data analysis at a Day 31. Immunology laboratory testing will be conducted at Day 21 and 31. Safety follow-ups will occur at Day 60 w/labs and at 6 month safety review-.

Laboratory analysis will be conducted to evaluate the immunologic responses generated by the li-Key peptide vaccine. Clinical endpoints include the activation of a neutralizing antibody humoral response and Th1 cellular response without a negative non-neutralizing antibody production and Th2 responses.

To support the clinical program, assays will be developed, qualified and validated at PPD and subcontractor laboratories. Applicants will provide vaccine peptide antigen for all activities, and development and qualification will be completed within 4-5 months post contact award. Assays will be validated for use in Phase 3 clinical testing.

For the IgG, IgM and IgA ELISAs a cut-point format for Phase 1 clinical testing will be updated to incorporate a reference standard prior to assay validation and Phase 3 clinical testing. Applicants will provide serum samples from the Phase I study to support development of the reference standard. The qualification plan will include Precision/Ruggedness, Dilution Linearity, Relative Accuracy, Specificity and Selectivity, and setup and qualified.

For li-Key-SARS-COV-2 microneutralization assays, Applicants' agent, the San Diego Center for AIDs research, has a qualified viral neutralization assay that will be utilized for all required testing.

Safety and efficacy will be addressed in a pivotal study (NGIO-COV-002) of health normal subjects (18-55yrs) and Healthcare Workers (n= 1200). As above, this will be a multi-center, randomized placebo-controlled, double blinded trial design. Subject arms PLA, ii-Key SARS- COV2 Candidate # 1 with option for Ii-Key SARS-COV2 Candidate #2 (n=600/arm or n=400/arm)

Clinical laboratory analyses will be performed to evaluate the immunologic response to the li- Key-SARS-CoV-2 vaccine by B-Cells (antibody/humoral) and T-Cells (CD-4, CD-8) as described above for our Phase I study.

Biomarker analysis will be conducted to identify and validate the immunologic biomarkers of efficacy including the activation of a neutralizing antibody humoral response and Th1 cellular response without a negative non-neutralizing antibody production and Th2 responses.

The same master protocol as for the Phase 1 safety and immunogenicity trial will be used through Day 60, with dosing on Day 1, 21 with labs and a trial endpoint at Day 31 w/ labs.

DSMB review' triggers expansion of study arms and initiation of healthy older subjects cohort. We will have follow-up Day 60 with labs and followup monthly for 10 additional months.

Pooled Day 31 data by study arm will be analyzed to determine initial efficacy, and continued monthly safety analysis plus analysis of severity of illness for outcome analysis. Endpoint analysis will include the immunological biomarkers of efficacy, including the activation of a neutralizing antibody humoral response and TH1 cellular response.

The Master protocol includes longer-term follow-up of all subjects from phase 1/3 studies, with the ability to close out all phase 1/3 studies but continue to follow subjects to ascertain long term benefit and risk. Following these studies will be a safety surveillance trial of Ii-Key-SARS-CoV-2 peptide hybrid vaccine using a 10,000 patient approach with the potential for expansion to mass vaccination. This study will evaluate and track evolution of real world benefits and risks, collecting a 6 month survey of symptoms, COVID 19 diagnosis, COVID 19 exposure. PRN symptom-triggered chart review' w ith additional serology testing and SAE's and AE's of interest. This study will be a decentralized, technology-enabled approach with a follow-up period of three years. This approach will enable evaluation of long-term safety and efficacy of the vaccine in providing immunity from SARS-CoV-2 infection and disease. Additionally, an expanded surveillance study is proposed for eventual expansion up to I OOM people. Further advanced studies will evaluate pediatric, geriatric and co-morbidity vaccinee subgroups.

Adjuvant

As with all peptide vaccines, inoculation with Ii-Key-SARS-CoV-2 hybrid peptide vaccines w ill typically be accompanied by the use of an adjuvant to attract immune cells to the site of vaccination (although in higher doses, for example approaching the 1 mg range, li-Key peptides alone, without adjuvant, are sufficient to elicit specific and robust immune responses). A contemplated adjuvant for use w ith Ii-Key-SARS-CoV-2 peptide antigen vaccines of the invention is 3M-052, for which extensive nonclinical pharmacology testing has been completed in accepted animal models, adjuvant at doses of 250 μg or 125 μg . 3M-052-SE is a synthetic, small molecule imidazoquinoline Toll-like receptor agonist for human TLR7 and TLR8. 3M-052 directly activates innate immune cells including dendritic cells, plasmacytoid dendritic cells, monocyte/macrophages, and B cells. This activation results in innate and adaptive immune modulation such as activation of costimulatory molecules, production of antitumor and antiviral cytokines, and stimulation of adaptive immunity. 3M-052 and similar compounds have shown broad antiviral, antitumor, and adjuvant functions both preclinically and clinically.

A nanoparticulate squalene (SE) formulation of this adjuvant, 3M-052-SE, is contemplated for use within the invention. 3M-052-SE contains the component 3M-052 at 40 μg /mL, with 4% squalene and other excipients in an oil-in-water stable emulsion (SE). The 3M-052-SE formulation has been evaluated with HIV and influenza antigens in mice, rats, ferrets, and non-human primates, demonstrating potent immunogenicity and an excellent safety profile in a GLP toxicology study. Basic pharmacology studies of 3M-052 in mice demonstrate that SC injection of 3M-052 results in the preferential expression of cytokines, chemokines, and IFN-inducible genes in the draining lymph nodes rather than the spleen, indicating the pharmacological effects of 3M-052 are predominantly local (at or near the site of injection) rather than distant from the injection site. In addition, cytokine and chemokine gene expression is prolonged; a single SC dose of 3M-052 induces the expression of IFN-y, IL-6, and chemokine CXC ligand 10 at the injection site (skin) and in the draining lymph nodes for at least 33 days after injection. The prolonged immune activation in the area of drug administration is consistent with the PK and drug distribution findings. Initial studies with a liposomal formulation of 3M-052 and H1N1 HA showed enhanced Th1 antibody responses as well as enhanced Th1 CD4 responses (Smirnov et al., 201 1 ).

Studies evaluating 3M-052 as a vaccine adjuvant have shown that the compound acts as a potent adjuvant in mice and nonhuman primates when formulated into liposomes, oil-in-water emulsions or adsorbed to aluminum salts (Lynn et al.. 2015; Smirnov et al., 2011; Dowling et al., 2017: Van Hoeven et al.. 2017; Fox et al.. 2016). Initial studies w'ith a liposomal formulation of 3M- 052 and H1N1 HA showed enhanced Th1 antibody responses as well as enhanced Th1 CD4 responses (Lynn et al., 2015). These studies also demonstrated at least a 15-fold dose sparing potential and that 3M-052 was much more effective than resiquimod in this model. Studies in newborn and neonatal primates demonstrate that an oil-in-water emulsion formulation of 3M-052 enhanced opsonic antibody responses to pneumococcal polysaccharide antigen after a single immunization (Dowling et al., 2017).

Additional testing was performed under a contract from the Biomedical Advanced Research and Development Authority (BARDA; contract #HHSO100201000039). We found that a squalene emulsion formulation of 3M-052 (termed 3M-052/SE) improved the immunogenicity of H5N1 antigens, broadened the neutralizing antibody responses, provided antigen dose sparing and induced protection in multiple pre-clinical models (Van Hoeven et al., 2017).

In ferret challenge studies, immunization with an egg-derived split H5N1 vaccine (SP-H5, VN1203) in combination with 3M-052/SE protected animals from lethal challenge and from virus induced weight loss. The 3M-052/SE adjuvant resulted in significant increases in hemagglutinin inhibition (HAI) titer to the homologous strain (VN1203, Figure 2C). In addition, 3M-052/SE induced a broad, cross-clade antibody response, significantly increasing HAI titers against A/Whooper Swan/244/05 (WS05). Broadening of' the immune response to diverse H5N1 influenza strains also was demonstrated using an influenza HA chip-based assay (Van Hoeven et al., 2017).

Extensive preclinical pharmacology and toxicology testing in laboratory animals have established sufficient understanding of the pharmacology and safety of 3M-052 to support evaluating IM administration of 3M-052 with vaccines of the invention in humans at adjuvant doses of up to 5 μg . In both mice and rats. SC injection of 3M-052 results in very low levels of 3M- 052 in the systemic circulation, indicating mainly local exposure to the compound. After development of the 3M- 052 in a stable emulsion GMP manufacturing process, GLP toxicology study of a 3M-052/SE was combined with an influenza H5 antigen. The objectives of the preclinical study were to determine the potential toxicity of the vaccine with 3M-052/SE adjuvant in rats.

The toxicological response of the rat to H5 antigen with 10 μg 3M-052/SE was principally characterized by findings typically associated with administration of vaccines, including an acute phase response as well as a more extended immune response to the test article, including an inflammatory reaction to the test article at the injection site characterized by mononuclear cell infiltration. Additional test article-related findings included organ weight changes in the heart, spleen and thymus, as well as histopathology findings in bone marrow, liver, pancreas, thymus, spleen, and/or spinal cord. None of the findings were considered to be toxicologically significant. Similar results have been observed using 3M-052 along with HIV trimer in a toxicology study in guinea pigs.

3M-052 (MEDI9197) has been evaluated in a Phase I human clinical study using a sesame oil formulation to evaluate its effects following intratumoral injection (NCT02556463). A total of 52 cancer patients were dosed with 3M-052 alone, in combination with radiation, in combination with PD-I 1 antagonist (Durvalumab). or in combination with Durvalumab and radiation. 29 patients with solid tumors received 3M-052 monotherapy in the sesame oil formulation at doses of 55, 37, 12 and 5 micrograms. Q4W. The 37 microgram dose of 3M-052 was defined as the maximum tolerated dose for this study. Two dose-limiting-toxicities were observed: Grade 3 cytokine release syndrome (CRS) at 37 micrograms and Grade 4 CRS at 55 micrograms. The most common (> 15%) drug related adverse events (AEs) were pyrexia, fatigue, decreased lymphocyte count, chills, nausea, arthralgia, and injection site pain (Gupta S, et al, AACR 2017, Abstract CT091 , Cooper, Z, et al, S1TC 2017, Abstract P457). There was one Grade 5 serious adverse event (death) in the Phase 1 intratumoral study in a patient with liver metastasis that received 2 intratumoral doses of 3M-052 (12 micrograms) in combination with 2 doses of Durvalumab (1500 milligrams). Similar to other patients dosed with 3M- 052, this patient had Grade 1 fever, headache, myalgia, and fatigue. Unlike any other patient dosed with 3M-052, this patient reported upper right quadrant pain after intratumoral injection. The cause of death was not determined, because an autopsy was not performed (family decision) and post-mortem CT scans were not done due to local hospital rules.

An open-label Phase 1 dose escalation study in subjects with solid tumor cancers or cutaneous T-cell lymphoma was initiated to assess safety, pharmacokinetics (PK), and pharmacodynamics (Study D6410C00001 ). In this study, now terminated, 3M-052 was injected Q4W or Q8W into solid tumors (IT) as a single agent, in combination with durvalumab, or in combination with durvalumab and palliative radiation.

A Phase I placebo-controlled study is ongoing to evaluate the safety and immunogenicity of an HIV vaccine candidate with TLR agonist adjuvants, including 3M-052, with or without alum. In this study, healthy HIV-negative volunteers receive either a placebo intramuscular (IM) injection or an IM injection containing BG505 SOSIP.664 gp 140 admixed with a TLR agonist adjuvant (3M-052, CpG, or GLA-LSQ) and/or alum. This study establishes further precedent for investigations of 3M-052 adjuvant at 1 μg and 5 μg doses in healthy volunteers.

As noted. Applicants contemplate using the 3M-051 adjuvant in a squalene emulsion. There have been no clinical studies completed using the squalene emulsion formulation of 3M- 052 in humans with a vaccine. However, squalene emulsion-containing adjuvants have been tested in a number of clinical trials. Squalene emulsion is a squalene oil-in-water nanoemulsion that enhances Th2-based immune responses to a co-administered antigen. Alternatively, SE can be formulated with TLR ligands such as 3M-052 (TLR7/8; 3M Drug Delivery Systems) or the TLR4 ligand Glucopyranosyl Lipid A (GLA; IDRI) to modulate the immune response toward the Th1 paradigm. The GLA-SE has been tested in numerous clinical trials in combination with a wide variety of antigens for infectious diseases including influenza, leishmaniasis, tuberculosis, schistosomiasis, and malaria. Several trials evaluated GLA-SE given alone or in combination with an antigen as a cancer immunotherapy. These clinical trials evaluated SE containing GLA at dose levels ranging from 0.5 to 20 μg . In these studies, over 1 100 subjects received at least one study injection containing GLA-SE. Safety data have not revealed any significant safety issues at any dose level tested. These studies have revealed an acceptable safety profile: • Study injections are generally well tolerated

• There have been no serious adverse events related to study injection

• Injection site reactions are common and may include pain, tenderness, erythema, and induration

• Systemic reactions may include headache, fatigue, anorexia, fever, chills, myalgia, arthralgia, and anorexia

• Transient elevations in C-reactive protein (CRP) levels were noted in one study

• Hematologic changes may occur (including decreases in hemoglobin, WBC, and neutrophils). These reactions varied from study to study, were generally mild, resolved quickly, and are typical of vaccinations by the IM route. GLA-SE often increases the rate and severity of local and systemic reactogenicity compared to the antigen alone. This is in line with the nonclinical animal experience and is to be expected of a potent immunostimulant.

Immunogenicity analyses have shown that the adjuvant profoundly increases the immune response to the co-administered antigen, including:

• Antibody levels are increased as shown by enzyme-linked immunosorbent assay (ELISA) and functional assays

• Cellular immune responses are increased (intracellular cytokine staining [ICS]. Luminex, ELI Spot)

• Dose sparing potential is high (e.g., immunogenicity of 3.8 μg rHA + 1 μg GLA-SE exceeded that of 135 μg unadjuvanted rHA antigen).

3M-052 is currently being used in a US government clinical study trial IND 019275 for which a letter of authorization (LOA) has been obtained. "The Division of Acquired Immunodeficiency Syndrome (DAIDS), National Institute of Allergy and Infectious Diseases (N1A1D), authorizes the food and Drug Administration (EDA) to cross-reference IND 019275, Human Immunodeficiency Virus Type I (gp 140: subtype A env trimer; BG505 SOSIP.664; CHO cells; Ajinomoto Althea, Inc.) Vaccine with (CpG 1018 and Alum: 3M-052-AF and Alum; GLA-LSQ or Alum alone) Adjuvants, for nonclinical and clinical information for 3M-052 adjuvant that may be relevant to a new IND application containing Protocol NGIO-COV-001 (A Phase 1/11 Safety, Reactogenicity and Immunogenicity Study of Ii-Key-SARS-CoV-2 Peptide Vaccine [Ii-Key-SARS-CoV-2] Alone and in Combination with Adjuvant 3M-052 Against COVID-19 in Healthy Adult Subjects), sponsored by NuGenerex Immuno-Oncology, to be submitted to the FDA in the next few months."

3M will be conducting necessary steps for scale-up, formulation and manufacture of adjuvant to support Applicants' SARS-CoV-2 vaccine development program, including:

1 . Synthesis of GMP Material (3M-052 drug substance)

2. Production of cGMP batches of 3M-052-Alum and 3M-052-SE with 3 years stability monitoring

3. Scale-up of adjuvant formulation manufacture

4. Tech transfer of ad juvant formulation manufacture

5. Single vial vaccine development (antigen with selected adjuvant)

In order to minimize risk in the proposed protocols for li-Key-SARS-CoV-2 peptide vaccines, 1 μg and 5 μg 3M-052 will be administered subcutaneously with Ii-Key-SARS-CoV-2 peptide vaccine in the Phase 1 portion in 3 subjects per vaccine dose level in a sentinel cohort. Safety and reactogenicity data for all sentinel subjects through 7 days after first administration will be evaluated in a blinded data monitoring committee (DMC) review prior to expansion of the cohort and escalation of vaccine dose. Safety data will include solicited and unsolicited adverse events, clinical laboratory abnormalities, and physical examination results. In addition, if a participant experiences a grade 3 or 4 local or systemic adverse reaction to the study vaccine administered on Day 1, the participant will not receive any further doses of study vaccine on Day 22.

Contribution of the adjuvant will be evaluated by characterization of humoral response and cellular activation between adjuvanted and non-adjuvanted arms. Specifically, the ratio of neutralizing antibody titers at Day 50 between adjuvanted and non-adjuvanted arms within a dose level will be used to inform selection of adjuvant inclusion and adjuvant dose in the Phase 2 part of the study. This ratio with be considered along with other immunogenicity, cellular activation, and safety data for dose selection for vaccine and adjuvant.

Dosages selected for Ii-K.ey-SARS-CoV-2 peptide vaccines will be based in part on adjuvant selection, and on prev ious human dosing and efficacy experience. In prior human clinical trials of li- Key peptide vaccines, AE37(li-Key-HER2) was evaluated at doses of 100 μg and 500 μg , with and without GM-CSF adjuvant, and at a dose of 1000 μg of AE37 alone (Holmes et al 2008). All doses were shown to be safe with no grade 3 toxicities. The 1000 μg of AE37 alone showed equivalent CD4+ activation, as measured by interferon gamma, as 500 ug of AE37 in combination with GM- CSF.

With respect to adjuvant dosing, in vivo PK and drug distribution studies have been completed in either rats or mice, or in both species. Two formulations of 3M-052 were used. A primarily aqueous oil-in-water emulsion, consisting of soybean oil/citrate buffer/Span 85/Tween 80, was developed for the initial SC and IM administration studies and for the administration of an IV reference dose. The formulation was subsequently changed to a lipophilic sesame oil and dehydrated alcohol solution that matched the formulation used in toxicology and clinical studies. Pharmacokinetic results in rats indicate that 3M-052 slowly distributes into the blood after dosing, suggesting drug localization at or near the injection site. The serum drug levels after SC injection of a high dose of 3M-052 (10 mg/kg) were less than 1 % of the serum drug levels observed after IV dosing. Importantly, in vitro data indicate that 3M-052 concentrations at least 10 times higher than 0.0085 μM are required to induce immune activation from human or mouse immune cells; therefore, the serum drug levels of 3M-052 likely to be achieved at the proposed clinical doses are likely to be too low to activate immune cells in the blood or in non-injected tissues.

Related studies evaluated systemic toxicity of 3M-052 in repeat-dose studies in mice and monkeys by the SC route, as a surrogate for IT administration. Subcutaneous toxicology studies were conducted using 3M-052 in the steri le-fi Itered sesame oil-ethanol clinical formulation. An in vivo genotoxicity study was conducted using 3M-052 in an oil-in-water emulsion. 3M-052 did not exhibit genotoxic activity in bacterial reverse-mutation assays. Grossly enlarged lymph nodes were noted in 3M-052-treated mice groups upon necropsy, which were dose related and correlated with the desired pharmacological activity of 3M-052. In both mice and cynomolgus monkey, following SC dosing, changes to the skin at the site of injection (ie. swelling, redness, scabbing) were noted in all groups but occurred at a higher incidence and severity in the 3M-052-treated groups. Perivascular inflammation in the lung, liver, kidney, spleen, axillary lymph node, bone marrow, sciatic nerve, brain (choroid plexus, meninges, brain parenchyma) and spinal cord were observed in SC 3M-052-treated cynomolgus monkeys, attributed to cytokine induction caused by the expected pharmacological activity of 3M-052.

In order to understand the toxicity and adverse events (AEs) associated with the potential worst-case scenarios of accidental IV administration or leakage into vasculature of the entire or major portion of an 3M-052 dose, a repeat-dose IV study was conducted in rats at dose concentrations of up to 2.4 mg/mL (50 μL per dose). Test article-related mortality/moribundity was observed for rats in the 0.6 and 2.4 mg/mL IV groups with clinical observations of pale and/or cool body and/or extremities, thin body condition, unkempt appearance, and/or red material around the eye and/or nose. Effects on rat body weight and food consumption and microscopic findings of mixed-cell inflammation in the lung, bronchial and mediastinal node enlargement (due to macrophage infiltration in most instances), and lymphoid hyperplasia were noted at all dose levels. A no observed adverse effect level could not be determined; the severely toxic dose in 10% of the animals was 50 μL of 0.3 mg/mL, which is equivalent to 0.075 mg/kg.

Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications may be practiced within the scope of the appended claims which are presented by way of illustration not limitation. The invention will thus be understood not to be limited, except in accordance to the claims which follow or may later be presented for examination. Various publications and other references have been cited with the foregoing disclosure for economy of description. Each of these references is incorporated herein by reference in its entirety for all purposes.

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