FIELD OF THE INVENTION

The present invention pertains to the field of vaccines which are useful for stimulate a host- specific protection in human and animal populations against infections by novel coronaviruses including both current SARS-CoV-2 and potential future strains derived thereof

The present invention also relates to an in silico method of identifying immunogenic epitopes derived from novel coronaviruses by characterizing their physiochemical properties such as hydrophobicity and affinity for specific receptors and stability of such complexes to develop a vaccine composition capable of stimulating a specific subset of immune cells preferably but not limited to T cells with minimal unwanted immunologic reactions.

The present invention further relates to a composition capable of releasing on-demand specific amounts of the synthesized epitope carrier system to the specific tissues susceptible to coronaviruses for eliciting a protective immunologic reaction.

Finally, the present invention relates to a method for selecting T-cell specific immunogenic novel coronavirus epitopes for nanomaterial-based vaccine formulations.

BACKGROUND

The emergence ofthe Coronavirus Disease 2019 (COVID-19) outbreak from the end of2019 caused by a novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in several million infections in man with a fatality rate of around 4% based on early epidemiology [1], The causative agent SARS-CoV-2 is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae that has the ability to infect animals and humans. Coronaviruses generally cause mild respiratory infections with similar symptoms observed in the common cold [1-3]. However, in the case of the novel SARS-CoV-2, some patients are at risk of developing moderate to severe acute respiratory distress syndrome (ARDS), which requires expensive mechanical ventilation for several weeks, and the vast majority of deaths due to COVID-19 are within this patient group [1], Consequently, there is an urgent need to develop treatments, vaccines and countermeasures for hindering the spreading of this disease.

SARS-CoV-2, SARS-CoV and MERS-CoV all belongs to the betacoronavirus genus that has a genome size of approximate 30 kilobases encoding both structural and non- structural proteins. The structural proteins include the envelope (E) protein, spike (S) glycoprotein, the nucleocapsid (N) protein and the membrane (M) protein, whereas the non-structural proteins include, for example, the RNA-dependent RNA polymerase [1], The spike (S) glycoprotein decorating the coronavirus surface is a homotrimeric transmembrane protein, with each 180 kDa monomer comprising two functional subdomains, SI and S2, and where the SI subunit consists of two domains: a N-terminal domain (NTD) and a C-terminal domain (CTD) [1- 4], Depending on the coronavirus type, either the NTD or CTD of SI is used as the receptor binding domain (RBD) capable of binding to specific receptors at the host cell surface. SARS-CoV-2 and SARS-CoV both utilize the CTD as its RBD, recognizing the human angiotensin converting enzyme 2 (ACE2), allowing for specific internalization of the virus in the epithelial cells of the respiratory tract and possibly the intestine too where there is high expression of its target receptor [2], The S2 subunit from the S protein is necessary for viral fusion with the host cellular membrane, mediated by proteolytic cleavage by the human transmembrane serine protease 2 (TMPRSS2), leading to the internalization of SARS-CoV- 2 and enabling viral replication inside the host cell [1],

The human viral antigen presentation pathway of the immune system consists of the human leukocyte antigen (HLA) system, which is a group of proteins that are encoded by the major histocompatibility complex (MHC) genes. The immunological cascade leading to the elimination of infected cells relies on the MHC class I cell-surface molecules presenting viral epitopes - peptides -recognized by T-cell receptors (TCR) of the cytotoxic or killer T cells. These viral peptides are released from the intact viral proteins when they are processed in the cytosol of the host cells by the ubiquitin-proteasome system. Next, these viral fragments, i.e., the epitopes, are translocated to and processed within the lumen of the ER before being loaded onto the HLA receptors using a peptide loading complex consisting of antigen processing (TAP) proteins and other associated proteins [1,5].

Based on current reports related to SARS-CoV-2, there is a predicted increased risk of developing a severe COVID-19 disease among individuals with the HLA-B*46:01 allele [5], Whereas individuals with specific HLA-A*02 alleles have a better ability to present viral epitopes derived from SARS-CoV-2 to killer T cells that may give protection against the disease [1,5]. This has been further validated in COVID-19 convalescent patients that had reactive T cells against certain epitopes from five proteins: S, orf1a, N, ORF3a and M originating from SARS-CoV-2 in individuals with the allele HLA-A*02:01 [6], These activated CD88 cytotoxic T cells are vital for eliminating virus-infected cells, limiting the spreading of the disease to other cells and tissues [5-9], In children it is speculated that the untrained naive T cells are better to adapt and eliminate new invading viruses that might explain why young children represents only a fraction of COVID-19 infections [9], Hence, it is of extreme importance to understand this elimination process in order to understand COVID-19 disease progression and to identify immunogenic epitopes that can be used for stimulating specifically the T cell response as an efficient way of mounting a protective response against SARS-CoV-2.

A vaccine that seeks to protect against infections agents takes considerable time and resources to develop, since the vaccine must be effective and safe for patients, and consequently there are many clinical tests required before regulatory approval [8], A vaccine only works if the correct antigens from a specific pathogen are administered and produces a sufficient immunological reaction resulting in immunity against a specific disease with minimal unwanted side effects [1,5-8], For example, seasonal influenza strains vary during the years and the vaccines usually contain only a few epitopes from different influenza strains and the available vaccines are partly based on educated guess work. Furthermore, recent data on SARS-CoV-2 suggest that the virus has already mutated to a more contagious strain and that the mutated amino acids might be difficult to recognize by the immune system, especially if trained to recognize the original strain of the virus and not the mutated variant, thus rendering the development of CODIV-19 vaccines even more difficult [1-12],

One proposed immune system target is the spike protein of SARS-CoV-2, which is currently being developed for vaccination against COVID-19 by several organizations [12] Although an obvious target, the spike protein alters its shape during the infection process after binding to the host receptor that might allosterically hide certain parts i.e. antigens of the viral protein [13] and is also decorated with sugar moieties functioning as “camouflage”, potentially making it difficult for antibodies to bind and neutralize the intruder [11], Furthermore, many of the immunogenic epitopes from the spike protein are hydrophobic in nature and thus difficult to administer using traditional protein subunit vaccination approaches [1], and this is likely why mRNA-based vaccines have recently been reported to be effective [12], However, there are some potential risks when using whole attenuated virus or protein-based and nucleotide-based vaccination encoding whole proteins as the probability of administering viral epitopes that mimics human proteins increases that could in principle give rise to unwanted reactions. Indeed, 33 peptides from SARS-CoV-2 are identical to the human reference proteome that are significantly expressed in lungs and arteries that could at least partly explain some of the autoinflammation seen in severe COVID-19 patents [14], Furthermore, autoantibodies against cytokines are detected in patents with life-threatening COVID-19 disease [15]. Therefore, there is an unmet need of developing T-cell reactive, tailored vaccines that are aimed at protecting against the virus in the current pandemic, as well as protection from future novel coronaviruses [1,3,5].

Nanomedicine shows great potential in the field of targeted drug delivery where nanotechnology and medicine are combined for the development of personalized diagnostics, treatment and prevention of different diseases. Nanomaterials are man-made or naturally occurring objects with dimensions between 0.2 nm to 100 nm where the physical properties of these tiny materials can be drastically different compared to their bulk counterpart. For example, nanomaterials can be more reactive on both biological and chemical substances due to a higher surface-area-to-volume ratio. Functionalized nanoparticles have shown to be able to target specific cell types, opening the possibility for targeted drug delivery while simultaneously lowering the off-target effects [16,17], Combining these different fields, e.g., nanotechnology, biochemistry, in silico screening with immunology, it would be possible to develop a synthetic particle to be used in vaccinations that mimics SARS-CoV-2 by having epitopes derived from the virus or other novel coronaviruses that stimulate target T-cell populations - preferably but not limited to CD8+ cytotoxic T cells - and which would effectively eliminate coronavirus-infected cells and prevent coronavirus-based disease [1-20],

SUMMARY OF THE INVENTION

It is an aim of the present invention to provide means for controlling or hindering the spread of novel coronaviruses especially SARS-CoV-2 and future mutated strains and or variations derived thereof, which could otherwise give rise to allergic reactions, infections, diseases, or death of the host.

In particular, it is an aim of the invention to identify and utilize polypeptides originating from the amino-acid sequence from any of the proteins found encoded by the mRNA of the SARS-CoV-2 coronavirus.

In one aspect, the present invention relates to nano- and/or micromaterial-based carriers and/or adjuvants decorated and/or loaded with immunogenic novel coronavirus epitopes or nucleotides encoding for specific amino acids similar to that of the said virus to be used as a vaccination for targeted cell populations tailored to specific receptors responsible for presenting viral fragments of infected cells to be eliminated by the immune system.

Further, one aspect of the present invention provides a method of selecting one or more polypeptide epitope candidates to be used in the composition of a nanoparticle-based vaccine. The polypeptide, which includes both universal conserved epitopes and novel epitopes, is composed of a sub-sequence obtained from the complete amino-acid sequence of any given protein derived from SARS-COV-2 or from a novel coronaviruses derived thereof.

It is also an aim of the present invention to provide a method of selecting isolated nucleic acid molecules, i.e., RNA or DNA, having a nucleic acid sequence that encodes a polypeptide sequence for both universal conserved epitopes and novel epitopes that are composed of a sub-sequence obtained from the complete amino-acid sequence of any given protein derived from SARS-COV-2 or from a novel coronaviruses derived thereof.

Thus, in one aspect, the present invention relates to a nanoparticle-based vaccine composition comprising one or more nucleic acid molecules, i.e., RNA or DNA sequences having a nucleic acid sequence that encodes a polypeptide sequence for both universal conserved epitopes and novel epitopes that are sub-sequences obtained from the complete mRNA sequence and/or amino-acid sequence derived from SARS-COV-2 or from a novel coronavirus derived thereof. Thus, the present invention provides a method of protecting an animal and/or human from infection(s) by novel coronaviruses and especially SARS-CoV-2 by administering via a nanoparticle a given amount of one or more polypeptides and/or one or more nucleic acid molecules encoding for virus-derived polypeptide(s), where the objective is to elicit a protective immune response and especially a T-cell response.

One further aspect of the present invention provides for synthesized nano- or micro-sized materials, adjuvants or carriers, herein generally referred to as “nanoparticles”, which is composed as least partly of similar molecules as the coronavirus of interest (e.g. SARS-CoV- 2), by functionalizing the surface and/or loading the interior with small molecule compounds, polypeptides and/or nucleic-acid fragments that encode polypeptides capable of eliciting a protective immune response, especially a T-cell response.

More specifically, the present invention is characterized by what is stated in the characterizing parts of the independent claims.

Considerable advantages are obtainable with the present invention.

The present technology provides a method for selecting immunogenic epitopes from novel coronaviruses such as SARS-CoV-2 to be developed for nanotechnology based targeted vaccine compositions. The identified epitopes derived from SARS-CoV-2 have high affinity towards specific MHC molecules in humans that are known to elicit an effective T cell response in vitro.

The method gives a structural and physiochemical explanation for the binding strengths of both novel and conserved SARS-CoV-2-epitopes to MHC molecules. Detailed information are given of potential T cell specific immunogenic epitopes to human major histocompatibility complexes common in certain populations to develop efficient vaccines against SARS-CoV-2 and possible future outbreaks of coronaviruses derived thereof.

According to the invention, a synthetic carrier is provided for use in a method of preventing and/or reducing SARS-CoV-2 pathogen ability to infect and replicate in the host organism. In embodiments, the carrier comprises biocompatible particles having a maximum size in at least one dimension in the nanometer or micrometer range, forming a core, and further having a functionalized surface or loaded with the said immunogenic epitopes or nucleotides encoding for said polypeptides capable of eliciting a cell specific protection against the said virus.

The present nanoparticles can be synthesized using different materials and functionalized with virtually endless combinations. Thus nanoparticles as vaccine candidates may be decorated to the surface with polypeptides derived from novel coronaviruses, including making it possible to use polypeptides, including promising epitopes, i.e. hydrophobic epitopes that have poor water solubility under physiological conditions that otherwise limits their use [17].

The physiochemical properties of the nanoparticle with the selected epitopes provide for efficient delivery and stimulation of a T-cell specific immune response, increasing the immune system ^' s capability of clearing coronavirus infected cells.

Especially promising as nanocarriers are mesoporous silica nanoparticles (MSNs) and lipid micelles that have shown great potential for targeted drug delivery because they have tunable surface properties and can be loaded with different drugs; these particles can also be synthesized in various sizes and shapes. Furthermore, inorganic silica materials are generally recognized as safe (GRAS) by the FDA as silica degrades in aqueous solution to silicic acid and gets excreted via the urine and is therefore considered biocompatible [16].

By means of the present invention it is possible to synthesize nano- and/or micro-sized materials - nanoparticles - that mimic circulating epidemic and/or pandemic strains/types of a novel coronavirus such as SARS-CoV-2 in the sense of their size, morphology and presentation of polypeptides and/or nucleic acids to the host immune system. Thus, the surface adorned with the spike protein of the virus would ensure uptake into cells. Moreover, the surface properties of the nanoparticles, e.g., additional polypeptide epitopes of the said virus, become recognizable by the immune system, stimulate a T-cell response, making an effective vaccination strategy against viral infections, and the interior of the particle can function to carry e.g., nucleic acids or other desired small molecule compounds. In particular, a synthetic nanoparticle and/or microparticle can be used to reduce the spread of the SARS-CoV-2 virus or other derived novel coronaviruses viruses that cause symptoms such as general discomfort, respiratory infection, diarrhea, common cold, a cytokine storm, and death. To that end, the synthetic particles can be manufactured to match the characteristics for example of the SARS-CoV-2 virus.

In one embodiment, the particle fabricated to a size of around 100-120 nm and coated and/or loaded with amino acid sequences similar to that of any of the viral proteins, e.g., spike glycoprotein (S), membrane glycoprotein (M), small envelope glycoprotein (E) and nucleocapsid (N) protein, as well as other non- structural proteins found in the virus of interest, to be used for vaccination at target cell populations such as CD8+ cytotoxic T cells.

The nano- and/or micromaterial-based carriers decorated and/or loaded with immunogenic novel coronavirus epitopes or nucleotides can be formulated into compositions suitable for administration to mammals, such as humans, serving as hosts of the virus.

The compositions can be administered by e.g., intramuscular injection, inhalation device or nasal spray for the respiratory tract or tailored orally ingestible tablet for the gastrointestinal tract or a topically administrable cream or ointment for the skin of the subject in need for the vaccine.

Temporary or prolonged protection against novel coronaviruses is provided by stimulation of the immune system for efficiently identifying viral-infected cells. Next, embodiments will be described in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the distribution of predicted affinity (IC50) of all possible linear 8- to 11- mer peptides derived from the 26 proteins of the SARS-CoV-2 proteome to MHC-1 allotypes.

Figure 2 illustrates the predicted MHC class I binding epitopes classification: strong binders (IC50 ≤ 50 nM), weak binders (50 nM ≤ IC50 ≤ 500 nM) and non-binders (IC50 ≥ 500 nM).

Figure 3 illustrates the number of the predicted 9-mer peptides in SARS-CoV-2 proteins.

Figure 4 illustrates the three-dimensional structure models of MHC-I molecules with epitopes and T cell receptor (TCR). (a) and (b) Five epitopes were docked into the cleft of HLA-A*02:01 (PDB ID: 5TEZ, chain A) and HLA-A*02:06 (PDB ID: 30XR, chain A) alleles located between the al and a2 helices (shown in surface). The epitopes 1220FIAGLIAIV1228, 17VLLFLAFVV25 , 20FLAFVVFLL28, 204VLAWLYAAV212 and 184YLWAHGFEL 192 are colored cyan, yellow, blue, salmon and green, respectively; (c) Structure of the docked epitope 1220FIAGLIAIV1228 (cyan sticks) into the cleft of HLA-A*02:01 (PDB ID: 5TEZ, chain A; gray cartoon and sticks). Residues from G1223 to 11227 in the epitope and residues A69, K66, V76, T80, K146, V152 and Q155 in HLA- A*02:01 were observed to have solvent exposed side chains; (d) Conformations of the CDR loops (CDRlα, CDR2α and CDR3α in TCR-α chain; orange color) and (CDRiβ, CDR2β and CDR3β in TCR-β chain; blue color) in the ternary complex of HLA-A*02:01 (gray cartoon), epitope 1220FIAGLIAIV1228 (cyan loop and sticks) and TCR; (e) Side chains of residues in CDR3α (orange sticks) and CDRSβ (blue sticks) loops make hydrophobic interactions (dotted yellow line; distance in A) with both the epitope 1220FIAGLIAIV1228 (cyan sticks) and HLA-A*02:01 molecule (residues located in al and a2 helices; gray sticks).

Figure 5 illustrates the Ca atom Root-mean-square fluctuation of the HLA-A*02:01- 1220FIAGLIAIV1228 S protein epitope-TCR complex during a 100 ns simulation. Figure 6 illustrates the superimposed conformations of the HLA-A*02:01- 1220FIAGLIAIV1228 S protein epitope-TCR complex observed at 0 ns (blue), 50 ns (yellow) and 100 ns (pink) of the simulation. Figure 7 is a scanning electron microscopy image (Stereoscan 360; Cambridge Instruments, England) of mesoporous silica nanoparticles in a size range of around 300 nm according to some embodiments of the present technology; and the Tables show the predicted SARS-Co-2 epitopes according to some embodiments of the present technology.

Table 1 illustrates the most potent SARS-CoV-2-derived MHC class I binding epitopes identified with the combined in silico prediction methods. Table 2 illustrates SARS-CoV-2 derived HE A- A* 02 supertype binding epitopes that are identical to the experimentally known cytotoxic T-cell epitopes in SARS-CoV strains.

Table 3 illustrates the predicted half-life time for the SARS-CoV-2 derived epitopes and HLA-A*02 supertype complexes.

Table 4 illustrates the predicted half-lives of the novel SARS-CoV-2-derived and most immunogenic in-silico-identified epitopes.

EMBODIMENTS

In the present context, the term “around” means, when used in connection with numerical values, that a variation of ±25 %, in particular ±20 %, for example ±10 %, or ±5 %, of the exact value is included by a literal reading of that value.

Unless otherwise stated herein or clear from the context, any percentages referred to herein are expressed as percent by weight based on a total weight of the respective composition. Unless otherwise stated, properties that have been experimentally measured or determined herein have been measured or determined at room temperature. Unless otherwise indicated, room temperature is 25 °C.

Unless otherwise stated, properties that have been experimentally measured or determined herein have been measured or determined at atmospheric pressure.

The term "polymer" is used herein in a broad sense and refers to materials, compounds, nucleotides, amino acids and proteins characterized by repeating moieties or units.

The terms "functionalization" and “functionalizing” are used herein in a broad sense and refers to conjugating, coating, covalently or allosterically adding materials, compounds, drugs, amino acids, peptides, proteins, and nucleic acids to the synthetized particle or object. The term ’’biocompatible” refers herein to “the ability of a material to perform with an appropriate host response in a specific application” (William's definition) [18].

The term ’’polypeptide” refers herein to a peptide, polypeptide or protein molecule; a molecule including a peptide, polypeptide or protein molecule; or a molecule that contains amino acids that are linked by non-peptide bonds.

The term ’’immunogenic epitope” refers herein to a polypeptide(s) that include an incomplete protein fragment which is capable of eliciting a protective immune response against novel coronavirus in humans and animals susceptible to coronavirus infection. Immunogenic epitopes may comprise an amino acid sequence having preferably 8 to 11 or more amino acids from novel coronaviruses and may include additional or other amino acid sequences.

The term ’’immunogenic derivatives” refers herein to a to molecules, amino acid, peptide and nucleotide or any combination thereof containing or encoding for an amino acid substitutions that is capable of eliciting a protective immune response against novel coronavirus in humans and animals susceptible to coronavirus infection.

The term ’’conserved epitope” refers herein to an linear amino acid sequence that is evolutionarily stable amino acid sequence having identical amino acid sequences found in other viral strains derived thereof [1,19]. The term ’’novel epitope” refers herein to an linear amino acid sequence that is evolutionarily mutated and thus changing its amino acid sequence but having a similar but not identical amino acid sequence found in other viral strains derived thereof [1,19],

The term ’’nucleotides” refers herein to the basic building blocks of nucleic acids consisting an nitrogenous base, a pentose sugar and phosphate group.

The term “DNA” refers herein to deoxyribonucleic acid molecule consisting of a polynucleotide chains carrying genetic information.

The term “RNA” refers herein to ribonucleic acid molecule consisting of a single- or double- stranded chains of nucleotides carrying information regarding coding, decoding, regulation and expression of genes.

As used herein, the term “variant” as used herein means a sequence, in particular an amino acid or nucleic acid sequence, having a high sequence identity to a parent sequence. For example, the variant may have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the parent sequence. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLASTp and BLASTn 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402.

As used herein, the term “average particle size” refers to the number average particle size based on a largest linear dimension of the particles (also referred to as “diameter”) as determined using a technique known to those skilled in the art, such as Scanning Electron Microscopy, Transmission Electron Microscopy, and/or a Light Scattering technique.

As used herein, the term “average particle size” refers to the D ₅₀ value of the cumulative volume distribution curve at which 50 % by volume of the particles have a diameter less than that value.

In brief, the present technology provides a method for selecting immunogenic epitopes from novel coronaviruses such as SARS-CoV-2 to be developed for nanotechnology based targeted vaccine composition. Epitopes derived from SARS-CoV-2 have high affinity towards specific MHC molecules in humans and elicit an effective T cell response in vitro. Both novel and conserved SARS-CoV-2-epitopes are bonded to MHC molecules. Using information on T cell specific immunogenic epitopes to human major histocompatibility complexes common in certain populations efficient vaccines can be developed against SARS-CoV-2 and possible future outbreaks of coronaviruses derived thereof.

Thus, one embodiment provides a method for selecting T-cell specific immunogenic novel coronavirus epitopes for nanomaterial-based vaccine formulations.

According to an embodiment, a synthetic carrier is provided for use in a method of preventing and/or reducing SARS-CoV-2 pathogen ability to infect and replicate in the host organism. The carrier comprises biocompatible particles having a maximum size in at least one dimension in the nanometer or micrometer range, forming a core, and further having a functionalized surface or loaded with the said immunogenic epitopes or nucleotides encoding for said polypeptides capable of eliciting a cell specific protection against the said virus. A temporary or prolonged protection against novel coronaviruses is achieved by stimulation the immune system for efficiently identifying viral- infected cells.

Nanomaterials and nanomedicine can be classified with regard to targeting strategies used to either active or passive targeting. Passive targeting utilizes non- functionalized particles for accumulation in organs and tissues that are responsible for clearance of foreign objects such as macrophages, liver and spleen. Tumor microenvironments are typically showing an enhanced permeability and retention effect (EPR) which is a consequence of leaky and fenestrated blood vessels around tumors. Active targeting on the other hand uses a targeting ligand or functionalization that enhances the accumulation of the carrier at target site [16].

There are virtually endless functionalization possibilities by covalently attaching, adhering, saturating or allosterically binding molecules, polymers, proteins, amino acids, compounds and/or drugs onto the nanomaterial for achieving active targeting. One of the major advantages of functionalizing a smaller molecule to a larger entity, e.g. antibody to a nanomaterial, is to increase the combined objects stability and/or increase the desired immunologic effect and minimize the possible unwanted off-target effect [16, 17, 20], Embodiments disclosed herein comprise fabricated nano materials to be used for vaccines capable of stimulating a host-specific protection against infections by novel coronaviruses.

Embodiments disclosed herein have capabilities of carrying novel coronavirus epitopes of fragments thereof in the nano material delivering the molecule specifically to target tissues reducing the replication and growth of the infectious agent.

Embodiments allow for decreasing the risk of novel coronaviruses, especially SARS-CoV- 2, entering its host for a temporary or prolonged duration and to give a targeted immune stimulation capable of mounting a protection against the specific disease caused by the infectious agent.

In a first embodiment, a synthetic carrier is provided, which comprises biocompatible particles having a maximum size in at least one dimension in the nanometer or micrometer range, forming a core, and further having a functionalized surface with immunogenic epitopes capable of electing a targeted immunogenic response to eliminate, hinder, prevent or minimize novel coronaviruses viral replication inside host and, thus, reducing the risk of contracting the specific disease caused by the virus and lowering the risk of transmitting the virus to other organisms.

The term “host” stands for an individual animal, in particular mammal, such as human.

The synthetic carrier is typically a “nano” material which can be of nano- or micrometer or larger size, typically with at least a size in at least one dimension, preferably in two more more - or even all dimensions - which is/are in the nanometer scale. The nanomaterial can be formed as a particle, spheroid, cubical, cigar shaped, elongated, triangle, sharp and pointy or as a sheet and film. In one embodiment, the carrier is rotationally symmetric.

Typically, the material has a maximum size in at least one dimension which is smaller than 2500 μm, in particular smaller than 1000 μm, for example smaller than 500 μm, in particular smaller than 100 μm or smaller than 50 μm. In one embodiment, the material has a maximum size in at least one dimension which is smaller than 10 μm, in particular smaller than around 5 μm or around 1 μm. In one embodiment, the material in particular nanomaterial has a maximum size in at least one dimension which is smaller than 1000 nm, in particular smaller than around 500 nm or around 100 nm.

Embodiments comprise nanoparticles having a size, in particular an average particle size, in the range of 10 to 200 nm or 100 to 200 nm.

In one embodiment, the material (in particular nanomaterial) is biocompatible. Such a material causes no or only a minor unwanted reaction in the end-user, e.g. toxicity or off- target effects.

Generally, the carriers are synthetic which is used interchangeably with “synthesized” to denote that they are man-made or non-natural.

Embodiments comprise organic or inorganic materials, protein based, lipid droplets, micelles or any combination thereof and these materials can either be man-made or naturally occurring substances.

The synthetic material can be selected from inorganic and organic, monomeric and polymeric materials capable of forming biocompatible nano- or micro-sized particles as explained above.

Examples of materials include synthetic polymers, in particular thermoplastic or thermosetting materials, such as polyolefins, polyesters, including biodegradable polyesters, such as polylactides and polycaprolactones, polyamides, polyimides and polynitriles.

Further examples include silica, polysilo xanes and silicone materials which optionally may contain organic and metal residues. Silica and lipid particles are particularly preferred.

In one embodiment, the carriers are selected from mesoporous silica nanoparticles (MSNs) and lipid micelles. Such carriers are suitable for targeted drug delivery because they have tunable surface properties and can be loaded with different drugs. In one embodiment, the mesoporous silica nanoparticles have a particle size, in particular an average particle size, of about 200 to 250 nm, such as 230 nm a surface area in the range of about 800 to 1000 m ² /g, and pore size of generally about 2-5 nm.

In one embodiment, the lipid micelles have a particle size, in particular an average particle size, of about 10 to 100 nm, for example 20 to 80 nm.

In one embodiment, the lipid micelles comprise amphiphilic macromolecules with a hydrophobic core and hydrophilic shell.

In one embodiment, the material compromises a nanoparticle core with coated immunogenic epitopes with a possibility of loading the particle with additional molecules, proteins and amino acids, RNA or DNA and compounds of interest.

Thus, in one embodiment, the nanomaterial compromises a core particle or object functionalized with targeting moieties, drugs, amino acids, epitopes, protein or any combination thereof. The object is preferably loaded with an active substance proteins or nucleotides.

One embodiment provides a composition comprising a carrier formed by biocompatible particles having a maximum size in at least one dimension in the nanometer or micrometer range, forming a core, and further having a functionalized surface with epitopes or nucleotides encoding polypeptides from coronaviruses, such as SARS-CoV-2 and future viral strains thereof.

In embodiments of the present technology, two ways of synthetizing nanomaterials are in particular employed, viz. either the top-down or the bottom-up approach.

In the top-down approach the building materials have larger dimensions than the final product which means that the materials undergoes physical stresses e.g., grinding, milling etc. in order to be reduced in size which can lead to surface imperfections. The bottom-up method starts by using smaller building blocks usually in solution transforming gradually to the final product which in a more cost-efficient way of producing nano materials.

Another embodiment provides a composition, wherein the immunogenic epitopes are obtained by selection using in silico prediction tools including but not limited to IEDB web server, VaxiJen online tool, ToxinPred server and three-dimensional structures, molecular dynamics simulations and biophysical characterization.

In one embodiment, the nanoparticles comprise self-assembling recombinant protein-based nanoparticle constructs. In one embodiment, such nanoparticles have an average particle size of about 10 to 200 nm, or 10 to 100 nm.

Recombinant protein-based nanoparticle constructs are exemplified by the SpyTag/Spy Catcher system [21],

Thus, in one embodiment, carrier is assembled using the SpyTag/Spy Catcher system and then conjugated with the selected SARS-CoV-2 spike protein fragments or selected protein fragments from the membrane glycoprotein, small envelope glycoprotein, and/or nucleocapsid protein.

In one embodiment, for inhibiting the spread of the virus SARS-CoV-2 a mesoporous silica nanoparticle, lipid nanoparticle or protein-based nanoparticle or any combination thereof with similar size as the virus (around 10-120 nm) is fabricated using the bottom-up sol-gel method or top-down method.

By using the known novel coronaviral genetic information, it is possible to produce similar peptides, epitopes or fragments present for example in the viral glycoprotein spikes thus mimicking the viral surface properties that has the capability to stimulate a specific immunologic reaction protecting the host organism against the virus. The most potent amino acid sequence in terms of immunogenic epitopes found in the novel coronavirus can be determined based on the method described in the experimental procedure. Thus, allowing the synthetic particle to stimulate a cell specific immunologic response that protects the host from the disease by hindering viral replication by eliminating viral infected cells.

In one embodiment, the composition include a carrier which comprises nanoparticles, in particular synthetic nanoparticles, and the synthetic carrier exhibits a modified particle morphology, size or surface properties for increasing the vaccine efficacy, in particular for increasing the efficacy of stimulation a T cell specific immunologic reaction in said individual with variations in the receptors responsible for the said reaction.

The principle of predicting T cell specific coronavirus epitopes is illustrated in Figure 4. As will appear, by way of an example, the amino acid sequences of the known viral proteins are segmented to short 8-11 amino acids epitopes which affinities towards the known HLA allotypes in the MHC class 1 complexes is estimated by in silico analysis and classed to strong MHC binders, weak binder or non-binders.

Based on the fore-going, in an embodiment, in a carrier system the synthetic nanoparticles are selected such that they resemble or contains polypeptides from SARS-CoV-2 virus or mutated strains derived thereof. Preferably these epitopes are optimized for stimulated specific set of immune cells, i.e., T cells, in order for the host to gain a protection of said virus.

A carrier system as above, wherein the synthetic nanoparticle resembling the SARS-CoV-2 virus is, in one embodiment, optimized for personalized medicine as variations and mutations in individuals might give rise to slightly different target receptors, e.g., HLA allotypes. Thus, the surface properties and functionalization of the carrier can be changed to match the individual properties, e.g., mutations or variations in target receptors, for tailored vaccinations.

In one preferred embodiment, the synthetic particle is provided with a coating of polypeptides that has high affinity towards the MHC receptor favoring the binding of the synthetic epitope gaining a stronger immunologic reaction and thus better protection compared to naturally occurring epitope. The peptides especially but not limited to having experimentally determined or and/or predicted T-cell reactive or cross-reactive epitopes, may be highly conserved among coronaviruses and/or may be novel protein fragments that are proven to be capable or are predicted to be capable of eliciting a protective immune response in humans.

The polypeptides of the present invention refer to any sub-sequence derived from the complete amino acid sequence of an any of the novel coronaviruses’ proteins e.g., spike glycoprotein (S), membrane glycoprotein (M), small envelope glycoprotein (E), and nucleocapsid (N) protein, as well as including other accessory and non-structural proteins.

Both the polypeptide (subunit) based and nucleotide (recombinant) based vaccine construct may be decorated and/or loaded into or onto to the nanoparticle for enchased stability, solubility and efficacy and formulated by accepted conventions using known buffers, preservative, stabilizers, solubilizers and other compounds used to facilitate sustained release to target sites.

Stabilizers other than the nanoparticle itself include, in one embodiment, albumin and/or gelation.

Additional adjuvants, other than the nanoparticle itself, may be employed. Examples of adjuvants include mineral salts, such as aluminum hydroxide and magnesium hydroxide, cytokines and chemokines such as GM-CSF, Type I IFN-alpha and IL-12, modified microbial antigens, such as LT and CT derivatives, monophosphoryl lipid A (MPL), immunostimulatory oligonucleotides, such as CpG motifs, oil emulsions, such as MF59, AS02, AS03, Mantanide ISA-51 and ISA-720, and AF03, liposomes, such as AS01 and AS015, and particulate adjuvants such as virosomes, AS04 and ISCOMS, for enhancing the immunogenic reaction to said vaccine composition.

In one embodiment, the adjuvants are selected from mineral salts, such as aluminium hydroxide, aluminium gels and magnesium hydroxide, wherein the concentration ranges from 0.1 mg/mL to 2 mg/mL, cytokines and chemokines such as GM-CSF, Type I IFN-alpha and Il- 12, wherein the concentration ranges from 1 pg/ml to 100 μg/ml, modified microbial antigens, such as LT and CT derivatives, wherein the concentration ranges from 0.1 mg/mL to 10 mg/mF, immunostimulatory oligonucleotides, such as CpG motifs, wherein the concentration ranges from 1 μ g/mL to 1000 μg/mL, oil emulsions, such as MF59, AS02, AS03, Mantanide ISA-51 and ISA-720, and AF03, having a concentration in the range from 0.001 % to 5 % (w/v), liposomes, such as AS01 and AS015, having a concentration in the range from 0.001 % to 4 % (w/v), and particulate adjuvants such as virosomes, AS04 and ISCOMS, wherein the concentration ranges from 0.001 % to 4 % (w/v) and monophosphoryl lipid A (MPL) for which concentration ranges from 0.01 mg/mL to 2 mg/mL,

In one embodiment, the additives of the present compositions are selected from the group comprising preservatives, such as thimerosal and 2-phenoxyethanol, wherein the concentration ranges from 0.001 % to 2 % (w/v), stabilizers, such as sorbitol, sucrose, glycerol, trehalose, gelatin and urea, having a concentration in the range from 0.5 % to 40 % (w/v), and surfactants, such as polysorbate 80 and polysorbate 20, having a concentration in the range from 0.001 % to 4 % (w/v).

In one embodiment, the selected epitopes are maintained and/or stored in solution containing the designed nanoparticles for enhanced solubility and delivery of hydrophobic epitopes.

In some embodiment, the compositions are formulated in saline diluent with, e.g., 0.9 wt% sodium chloride.

In some embodiments, the compositions may include physiological buffer, antioxidants and chelators. The buffer can be selected from antacid agents, such as bicarbonate or bicarbonate-ascorbic acid, or phosphate-citrate buffers, to mention a few examples.

In one embodiment, the present compositions are capable of releasing on-demand specific amounts of the synthesized epitope carrier system to the specific tissues susceptible to coronaviruses for eliciting a protective immunologic reaction.

In one embodiment, the compositions are administered by intramuscular injection.

In one embodiment, the compositions are administered by inhalation device or nasal spray for the respiratory tract. In one embodiment, the compositions are formulated into orally ingestible tablets or oral for the gastrointestinal tract or formulated into topically administrable cream or ointment for the skin.

Nanoparticle based vaccines increases epitope stability may be stored conveniently in refrigerator temperatures in smaller containers such as vials, and directly used upon administration or then combined with sterile buffer solution for appropriate dosage.

The correct amount of polypeptide administered depends upon many factors, such as the size of the epitopes and the composition of the nanoparticle as well as the host organism being vaccinated e.g., species, age, weight, and physical traits.

In general polypeptide-based vaccines contains upon administration up to several thousand micrograms of specific polypeptide per milliliter of sterile solution. Recombinant vaccines containing the specific nucleotides encoding for the polypeptides and the vector are administered containing up to several thousands of infectious units per milliliter sterile buffer solution.

The vaccine is administered usually, but not limited, to using around 0.5 to 2 milliliters of solution containing the nanoparticle decorated or loaded with selected polypeptide or nucleotides.

The nanoparticle vaccine containing either polypeptides or nucleotides may be administered for example but not limited to inhaled, intranasal or orally ingested or administered thru intramuscular, intraperitoneal or subcutaneous injection.

Subsequent to initial vaccination, the host organism - animal or human - may be boosted by revaccination for prolonging the desired protection.

Based on the above the following represents embodiments of the present technology:

A synthetized carrier in the nano- or microscale decorated or loaded with epitopes or nucleotides capable of electing a immunologic reaction in target cells populations that prevents and minimize novel coronaviruses ability to infect and replicate in host organism and thus lowering the risk of contracting the specific disease.

A carrier as above, wherein the core structure of the carrier is obtained by 3D printing, microfluidics, sol-gel method or other bottom-up and/or top-down method of fabrication.

A carrier as above, where the core material is made of, however not limited to, organic or inorganic components, lipid droplets, micelles, amino acids, proteins, salts and minerals or other molecules.

A carrier as above, where the core material is made of, for example, mesoporous silica nanoparticles with ordered mesostructures of pores that can be loaded with different drugs and that these particles can be synthetized in various sizes and shapes.

A carrier as above, where the core material is functionalized or loaded with one or several of the following: polypeptides, peptide or amino acid sequence which includes both conserved epitopes and/or novel epitopes composed of a sub-sequence obtained from the complete amino-acid sequence of any given protein, e.g. spike glycoprotein (S), membrane glycoprotein (M), small envelope glycoprotein (E), and nucleocapsid (N) protein and several accessory and non-structural proteins derived from SARS-COV-2 or from a novel coronaviruses derived thereof.

In one embodiment, the core material is decorated with conserved epitopes selected from S protein (1220FIAGLIAIV1228) and the epitopes from the E protein (17VLLFLAFVV25, 20FFAFVVFFF28) and nsp3 (330FFSAGIFGA338) as well as 35EXO (184VFWAHGFEL192), and variants thererof. As explained above, the variants typically exhibit a sequence identify of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the parent sequence.

In one embodiment, the core material is decorated with epitopes preferably selected from but not limited to intravirion (738DTDFVNEFY746, 289SHFAIGLAL297, 217AMDEFIERY225), transmembrane (1505LVAEWFLAY15139,

1507AEWFFAYIF1515), and variants thereof. As explained above, the variants typically exhibit a sequence identify of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the parent sequence.

In a carrier, as dicussed above, the core material is functionalized or loaded with one or several of the following: nucleotides, RNA or DNA that codes for a sub-sequence obtained from the complete amino-acid sequence of any given proteins, e.g. (S) glycoprotein, membrane (M) glycoprotein, small envelope (E) glycoprotein, and nucleocapsid (N) protein, and several accessory and non-structural proteins derived from SARS-COV-2 or from a novel coronaviruses derived thereof

A carrier system which is loaded, as explained above, can be stored or dispersed in a device or vessel capable of on-demand release of the carrier to the end-user.

A carrier system which is loaded, as explained above, can be is loaded inside a dispenser such as an inhalation device, tablet, injectable substance, cream or ointment.

In further embodiments, the present invention is thus directed to a method for preparing a synthetic nanomaterial comprising a core object, particle, sheet, film or spheroid, tringle, star shaped, said object also compromising a coating or functionalization of organic polymers, amino acids, epitopes, proteins or molecules mimicking the surface proteins or any other proteins derived of the novel coronavirus of interest, i.e. SARS-CoV-2 and future variants thereof.

One embodiment comprises a) prediction of novel coronavirus epitopes using in silico methods; b) characterization of novel coronavirus epitopes using in silico methods; c) synthesis and/or expression of novel coronavirus epitopes and biophysical characterization; d) provision of a core material, e.g. a nano- and/or micro -material including nanoparticles, microparticles or any other object as disclosed herein; e) coating or functionalizing the core material with polypeptides, epitopes, amino acids, nucleotides, molecules, polymers, or other material as disclosed herein; f) loading the object with polypeptides, epitopes, molecules, DNA or RNA etc.; g) coating a second protective or functional layer on top of the object in particular for increasing its resistance that could be important in extreme environments such as the acidic environment in the stomach; and h) providing a small device, medical device, inhalation device or aerosol, sanitation product or consumer product that on-demand will release the containing synthetic material, particle or object for administration.

Further embodiments of the present technology are disclosed in the following:

1. A method of preventing or reducing pathogen binding to target areas of cell surfaces of a host selected from mammals, comprising administering to the mammal a carrier comprising biocompatible particles having a maximum size in at least one dimension in the nanometer or micrometer range, forming a core, and further having a functionalized surface capable of binding to said target areas of said cell surfaces to at least temporarily block said target areas to prevent or minimize pathogen binding and thus, reducing the risk of the host contracting a disease caused by said pathogen. The core material is, for example, functionalized with substance(s) selected from the group consisting of peptides, proteins such as antibodies, chemical agents, active pharmaceutical ingredients (API), organic or inorganic polymers or molecules and combinations thereof.

2. The method according to embodiment 1, wherein the synthetic nanoparticle and/or microparticle is used for reducing the spread of SARS-CoV-2 virus or other viruses that causes a respiratory infection, diarrhea, common cold, influenzas or generally discomfort or a combination thereof.

3. The method according to embodiments 1 or 2, wherein said synthetic nanoparticle has a 3D-configuration generally matching the characteristics of the SARS-CoV-2 virus, in particular the particle is fabricated to a size of around 100 nm and coated with similar amino acids as the glycoprotein spikes at the surface of the viral particle or similar molecules that mimic the surface of the viral envelope.

4 The method according to any of embodiments 1 to 3, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus is adapted for personalized medicine. 5. The method according to any of embodiments 1 to 4, wherein said the synthetic nanoparticle resembling the SARS-CoV-2 virus is loaded into or onto the nanoparticle for further enhancing the anti- viral properties.

6. The method according to any of embodiments 1 to 5, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus is loaded with vehicles or proteome inhibitors for efficiently delivering the compounds in the target tissues with minimal off-target effects.

7. The method according to any of embodiments 1 to 6, wherein the synthetic nanoparticle resembling the SARS-CoV-2 virus or any other pathogen is coated or decorated with epitopes to be used as a vaccination at target cell populations.

8. The method according to any of embodiments 1 to 7, wherein the carrier is loaded, stored or dispersed in a device or vessel capable of on-demand release of the carrier to the end-user.

9. The method according to any of embodiments 1 to 8, wherein the carrier system is loaded inside a dispenser such as an inhalation device, tablet, injectable substance, cream or ointment.

10. A synthetic carrier for use in a method of preventing or reducing pathogen binding to target areas of cell surfaces of a host, said carrier comprising biocompatible particles having a maximum size which, in at least one dimension, is in the nanometer or micrometer range, forming a core, and further having a functionalized surface capable of binding to said target areas of said cell surfaces so as to at least temporarily block said target areas to prevent or minimize pathogen binding and, thus, reducing the risk of the host contracting a disease caused by said pathogen, wherein said synthetic nanoparticle and/or microparticle is used for reducing the spread of SARS-CoV-2 virus or other viruses that causes a respiratory infection, diarrhea, common cold, influenzas or generally discomfort or a combination thereof.

11. The synthetic carrier for use in a method of preventing or reducing pathogen binding to target areas of cell structures of a host according to embodiment 13, wherein said synthetic nanoparticle has a 3D-configuration generally matching the characteristics of the SARS- CoV-2 virus, in particular the particle is fabricated to a size of around 100 nm and coated with similar amino acids as the glycoprotein spikes at the surface of the viral particle or similar molecules that mimic the surface of the viral envelope and thus binds to the same target receptor as the virus.

12. The synthetic carrier for use in a method of preventing or reducing pathogen binding to target areas of cell structures of a host according to any one of embodiments 1 to 9, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus or any other pathogen is coated or decorated with epitopes to be used as a vaccination at target cell populations making the administration potentially easier for the end user e.g. inhalation compared to intra muscular injection used in traditional vaccinations.

13. The synthetic carrier for use in a method of preventing or reducing pathogen binding to target areas of cell structures of a host according to any one of embodiments 10 to 12, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus is decorated with conserved epitopes preferably but not limited to the S protein (1220FIAGLIAIV1228) and the epitopes from the E protein (17VLLFLAFVV25, 20FFAFVVFFF28) and nsp3 (330FFSAGIFGA338) as well as 35EXO (184VLWAHGFEL192), and variants thererof.

14. The synthetic carrier for use in a method of preventing or reducing pathogen binding to target areas of cell structures of a host according to any one of embodiments 10 to 13, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus is decorated with novel epitopes preferably but not limited to intravirion (738DTDFVNEFY746, 289SHFAIGLAL297, 217AMDEFIERY225), transmembrane (1505LVAEWFLAY15139, 1507AEWFLAYIL1515), and variants thereof.

15. The synthetic carrier for use in a method of preventing or reducing novel coronaviruses ability to infect and replicate in a said host according to any one of the preceding embodiments, wherein said synthetic nanoparticle resembles the SARS-CoV-2 virus or is optimized for specific individuals, wherein preferably the synthetic carrier exhibits a modified particle morphology, size or surface properties for increasing the vaccine efficacy, in particular for increasing the efficacy of stimulation a T cell specific immunologic reaction in said individual with variations in the receptors responsible for the said reaction. 16. The synthetic carrier for use in a method of preventing or reducing novel coronaviruses ability to infect and replicate in a said host according to any one of the preceding embodiments, wherein said synthetic nanoparticle resembling the SARS-CoV-2 virus is adapted for personalized medicine such in the case of HLA and TCR receptor polymorphisms for achieving optimal receptor interaction.

17. The synthetic carrier for use in a method of preventing or reducing novel coronaviruses ability to infect and replicate in a said host according to any one of the preceding embodiments, wherein said carrier is loaded, stored or dispersed in a device or vessel capable of on-demand release of the carrier to the end-user, wherein said carrier system is preferably loaded inside a dispenser such as an inhalation device, tablet, injectable substance, cream or ointment.

18. A method according to any of embodiments 1 to 9 for preventing or reducing pathogen binding to target areas of cell structures of a host, comprising minimizing the spread of diverse pathogens by binding to the target molecule in the host body or binding to the infectious agent itself and potently inhibit the spread of the disease.

19. A method according to claim 18, wherein the synthetic nanoparticle resembles the SARS-CoV-2 virus or is optimized for competitive inhibition.

EXPERIMENTAL SECTION

Materials and Methods

In silico identification of the MHC-I specific immunogenic epitopes in the proteome of SARS-CoV-2

The IEDB web server was used to predict the binding affinities (IC ₅₀ ) of HLA class I specific top 1 percentile linear epitopes with lengths of 8 to 11 amino acids in the SARS-CoV-2 proteome (26 proteins). Based on the binding affinity scores, the predicted class I HLA- epitopes (eHLA-I) complexes were classified into three groups: strong binders (IC ₅₀ ≤ 500 nM), weak binders (50 nM < IC ₅₀ ≤ 500 nM) and non-binders (IC50 ≥ 500 nM). Both the 9- mer and 10-mer epitopes showed high binding affinities to MHC-I with least number of nonbinders in comparison with 8-mer and 11-mer epitopes. In the category of binders (IC ₅₀ ≤ 500 nM), the 9-mer epitopes were more than half in number as compare to the 10-mer epitopes. Thus, the 9-mer epitopes were selected for further analysis.

Predictive analysis tools come with their own specialties/limitations. Thus, a consensus approach is used to predict the HLA-I specific 9-mer epitopes in the SARS-CoV-2 proteome, where identical epitopes obtained from two independent predictions using the most reliable web servers: IEDB and NetCTL1.2 were considered for epitope immunogenicity prediction using the MHC-I immunogenicity server of IEDB. The in silico identified immunogenic (immunogenicity score > 0.25) 9-mer epitopes were searched against the experimentally known epitopes of SARS-CoV strains (experimental data available at IEDB database). Biophysical properties of the epitopes, such as grand average of hydropathicity index (GRAVY), secondary structure, potential transmembrane helices and half-lives (in hours) of predicted eHLA-I complexes were examined to understand the preference of predicted immunogenic epitopes to specific HLA-I allotypes.

In order to understand the possibility of relevant molecular interactions between the predicted immunogenic epitopes and HLA-I allotypes, three-dimensional structures of epitopes and HLA-I molecules were obtained from PDB database when available and modeled using molecular modeling approach. The modeled immunogenic epitopes were docked to the HLA-I receptor using the Rosetta FlexPepDock web server. Three- dimensional structural models of ternary complexes composed of T-cell receptor (TCR)- SARS-CoV-2 epitope-HLA-I allotype were modeled in PyMOL to understand the interaction pattern of amino acids at the interface of the TCR and eHLA-I complex.

Molecular dynamics simulations

The dynamics of the HLA-A*02:01- ¹²²⁰ FIAGLIAIV ¹²²⁸ S protein epitope-TCR complex structure was examined with a 100 ns molecular dynamics simulation. Prior to simulation the structure was prepared by adding hydrogen atoms, optimizing hydrogen bonds, determining protonation states of amino acids and energy minimizing the structure. The prepared structure was solvated with the TIP3P water model in an octahedral box leaving a distance of 12 Å between the surface of solute atoms and the box edge. The solvated system was neutralized by adding Na+ counterions. Additional Na+/Cl- ions were included to the system to achieve a 150 mM salt concentration. The simulation was carried out with the Amber program using the ffl4SB forcefield. The simulation protocol comprised four stages. (1) The system was subjected to 5000 cycles of energy minimization, with a solute atom restraint of 25 kcal mol ^{- 1} Å ^-2 , which was systematically lowered to 0 kcal mol ^-1 Å-. ² (2) The system was then heated to 300 K with a solute atom restraint of 10 kcal mol ^-1 Å ^-2 during 100 ps. (3) Thereafter, equilibration of the system was conducted for 6 ns gradually lowering the restraint force to 0 kcal mol ^-1 Å ^-2 . (4) Finally, the production simulation was carried out at constant pressure (1 bar) and temperature (300 K), saving coordinates every 20 ps. Three independent simulations were performed to enable better sampling of the conformational space. The resulting trajectories were analyzed using VMD, Cpptraj and Chimera programs. Root-mean-square fluctuation (RMSF) calculation was computed using the Cα atoms of the initial structure as a reference. Hydrogen bonds were defined with a bond length < 3.5 Å and a bond angle ≥ 135°.

Recombinant protein production.

Soluble recombinant proteins such as the epitopes derived from the S protein (1220FIAGLIAIV1228) and the epitopes from the E protein (17VLLFLAFVV25, 20FLAFVVFLL28) and nsp3 ( ³³⁰ LLSAGIFGA ³³⁸ ), and variants, may be expressed either in E. coli or in Bac-to-Bac baculovirus system using pFastBac-Dual vector (Invitrogen; or equivalent) potential fused proteins are generated in HEK293 cells. For analyzing thermal stability, folding and ligand binding; Mosquito (SPT Laptech) nano liter-scale pipetting robot may be sued to boost crystallization; Rock Imager (Formulatrix) for automatic imaging of crystallization plates; and PX Scanner (Agilent Technologies) for in situ X-ray analysis of protein crystals on 96-well plates to optimize results and prevent wasted experiments at remote synchrotron radiation facilities.

Nanoparticle synthesis and characterization

A particle system comprising core material(s) of e.g. polymer, protein, silica or lipids functionalized with either epitopes or nucleotides similar to that of SARS-CoV-2 capable of electing a specific immunologic reaction. Especially, mesoporous silica nanoparticles (MSNs) and lipid micelles have the capabilities of carrying large amount of different compounds inside the pores or conjugated onto the surface [16]. The nanoparticle may be synthetized using, top-down or bottoms-up methods such as supercritical solution or microfluidics allowing large quantities of the proposed nanoparticles to be produced. Then the particle would be functionalized or loaded with selected epitopes or nucleotides. The produced nanoparticles size, monodispersity, morphology and non-agglomerated state may be determined using electron microcopy and dynamic light scattering techniques amongst other [16].

For example, a mesoporous silica nanoparticle may be synthesized according to previously published protocols where 1.19 g of tetramethoxysilane (TMOS) mixed with 3- aminopropyltrimethoxysilane (APS) in an alkaline solution containing H20: MeOH with a ratio of 60:40, with the structure-directing agent cetyltrimethyl ammonium chloride (CTAC1) [16]. The alkaline solution with a molar ratio of 0.9 TMOS: 0.1 APS: 1.27 CTAC1: 0.26 NaOH: 1439 MeOH: 2560 H20 was kept overnight at room temperature under constant stir and the precipitate collected by centrifugation, washed with deionized water and ethanol. Nanoparticles were dried in vacuo and the structure-directing agent was removed by either repeated ultrasonic treatment in acidic ethanol and/or heat treatment to bum away the organic molecules from the MSNs [16]. The particles may be functionalized with the selected epitopes for example by carbamic acid and EDC/NHS molecules by resuspending the MSN pellet in a supernatant containing the epitopes at a weitght ratio of around 2 wt.% and mixed overnight at low speed at +4 °C [20], It will be obvious to a person skilled in the art that, as the technology advances and future novel coronaviruses emerges, the inventive concept can be implemented in various ways for example by finding novel immunogenic epitopes for the said virus. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

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