INFECTION

FIELD OF THE INVENTION:

The invention is in the field of infection disorders. More particularly, the present invention relates to a method of preventing or treating a brain viral infection in a subject in need thereof.

BACKGROUND OF THE INVENTION:

SARS-CoV-2 infection is associated with a wide range of neurological symptoms (headaches, dizziness, nausea, loss of consciousness, seizures, encephalitis, cognitive deficits etc.) ((/-5), reviewed in (6-9), as well as anosmia or ageusia in more than two-thirds of patients (10, 11). Emerging reports also describe the post-mortem detection of the virus in the cerebrospinal fluid (CSF) (12, 13) or brain parenchyma of patients (14). Additionally, a large number of COVID-19 patients with severe disease do not respond well to artificial ventilation or display a phenomenon known as "silent hypoxia", where low blood oxygen levels fail to trigger the appropriate physiological response (75), suggesting an extra-pulmonary component to respiratory dysfunction, and cardiorespiratory function and fluid homeostasis are themselves subject to central nervous system (CNS) control.

Although the possibility of CNS infection was largely underestimated at the beginning of the pandemic due to the common view that angiotensin converting enzyme 2 (ACE2), the only confirmed cellular receptor for SARS-CoV-2 so far (16-19), was absent or expressed only at very low levels in the brain, in particular the cerebral cortex (20, 21)), other regions of the brain, notably the hypothalamus, are rich in ACE2 (22) (23) and more recent reports indicate that neuroinvasion can occur in a widespread manner (102, 103). Indeed, the highly related SARS-CoV has been observed in the post-mortem human brain tissue, including the hypothalamus (24-26), and could induce long-term neuroendocrine deficits in survivors (27, 28), supporting the notion of the hypothalamus as a route and/or target of viral infection. In animal models, SARS-CoV administered intranasally enters the brain through the olfactory route and is found at high concentrations in the hypothalamus and brainstem in a matter of days (29-32). This is of particular interest as the loss of the sense of smell or taste in the majority of COVID-19 patients suggests that the virus could invade the brain through sensory receptors. Indeed, the hypothalamus is also directly linked to regions implicated in the perception or integration of odor and taste such as the olfactory bulbs and presumptive vomeronasal neurons, the entorhinal and piriform cortices, the insula, amygdala, and thalamus (33-38), as well as other parts of the CNS involved in functions affected in COVID-19 patients, including several brainstem nuclei that control fluid homeostasis, cardiac function and respiration. In addition, some nuclei of the hypothalamus form part of the circumventricular organs, at which the classic blood-brain barrier is replaced by a fenestrated epithelium, and molecules or particles can cross from the blood into the brain parenchyma through specific mechanisms (39, 40). Intriguingly, most major risk factors for severe COVID-19 (male sex, age, obesity, hypertension, diabetes) ((41-44); reviewed by (45) (43, 46-48)) could be mediated by normal or dysfunctional hypothalamic neural networks that regulate a variety of physiological processes: sexual differentiation and gonadal hormone production, energy homeostasis, fluid homeostasis/osmoregulation and even ageing (33, 34, 49, 50).

Here, the inventors analyzed the expression of ACE2 and the transmembrane proteinase, serine 2 (TMPRSS2), which primes the SARS-CoV-2 spike (S) protein for internalization, in existing data from the Allen Human Brain Atlas (AHBA) (57) to determine the susceptibility of the hypothalamus and related brain regions to SARS-CoV-2 infection. The inventors also used network analysis and pathway enrichment tools to determine a number of genes and cellular, molecular or disease processes that were correlated with this susceptibility, of which some were differentially expressed in the respiratory epithelium of COVID-19 patients (52). The inventors thereby identified as a candidate molecule formyl peptide receptor 2 (FPR2), a gene involved in the immune response and known to bind to other viral surface proteins (53- 55), and which is correlated with ACE2 in the brain regions examined. Furthermore, the inventors performed the same gene expression, correlation and enrichment analysis, using FPR2 as the reference gene, and found a large list of 6554 genes and 60 functional pathways that were positively or negatively correlated with FPR2. In addition, the inventors identified among these genes as well as in the literature several molecules belong to the interactome for FPR2 and exerting an effect compatible with a modulation of viral infection and inflammation in the hypothalamus. Finally, the inventors showed by immunolabeling for ACE2, TMPRSS2 and FPR2 that the three proteins are present in the hypothalamus of mice and control human brains, indicating that the hypothalamus is a viable route for viral entry or propagation in the brain. The inventors also showed that in animal models of metabolic diseases (mice given a high-fat diet to induce obesity, and which also display type 2 diabetes and hypertension) and different gonadal steroid levels (ovariectomized mice used as a proof of concept for the lack of a protective effect of estrogen in men or decrease with age in both sexes), the expression of these molecules in the hypothalamus is different, providing a biological basis for the increased risk of severe COVID-19 in patients with obesity, diabetes or hypertension, male patients and aged patients. Specifically, ACE2 was localized in other cells and in different cellular components than in control animals, and FPR2 expression was greatly increased in microglial- like cells, consistent with the trigerring of inflammatory pathways under these conditions. The inventors show that infection with SARS-CoV-2 or a replication-deficient RNA pseudovirus greatly increases the expression of FPR2 in the brain of humans and animal models, and that treatment with Fpr2 antagonists could be of prophylactic or therapeutic value in limiting brain cell entry and propagation of SARS-CoV-2 and other viruses, and thus limiting the severe short- and long-term consequences of neuroinvasion by these viruses.

SUMMARY OF THE INVENTION:

The invention relates to a method of preventing or treating a brain viral infection or the resulting physiological consequences in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Formyl Peptide Receptor 2 (FPR2) modulator (herein defined as an inhibitor or as an activator of FPR2). In particular, the invention is claimed by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Most patients with COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), display neurological symptoms, and respiratory failure in certain cases could be of extra-pulmonary origin. With reports detecting SARS-CoV-2 in some post mortem patient brains, the routes, targets and consequences of brain viral infection merit investigation. Hypothalamic neural circuits play key roles in sex differences, diabetes, hypertension, obesity and aging, all risk factors for severe COVID-19, besides being connected to brainstem cardiorespiratory centers. Here, human brain gene-expression analyses reveal that the hypothalamus and associated regions express angiotensin-converting enzyme 2 (ACE) and transmembrane proteinase, serine 2 (TMPRSS2), which mediate SARS-CoV-2 cellular entry, in correlation with FPR2 and other molecules, which participate in several pathways involved in physiological functions, viral pathogenesis and inflammation. Immunolabeling in human and animal brains suggests that the hypothalamus could be central to SARS-CoV-2 brain invasion through multiple routes, and that metabolic diseases and the presence or absence of certain gonadal hormones increase its susceptibility. Accordingly, in a first aspect the invention relates to a method of preventing or treating a brain viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Formyl Peptide Receptor 2 (FPR2) modulator (herein defined as an inhibitor or an activator of FPR2).

In a particular embodiment, the invention relates to a method of preventing or treating a brain viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Formyl Peptide Receptor 2 (FPR2) inhibitor.

In a particular embodiment, the invention relates to a method of preventing or treating a brain viral infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a Formyl Peptide Receptor 2 (FPR2) activator.

In a particular embodiment, the invention relates to a Formyl Peptide Receptor 2 (FPR2) modulator (herein defined as an inhibitor or as an activator of FPR2) for use in the prevention or the treatment of a brain viral infection (e.g. coronavirus) in a subject in need thereof.

According to the invention, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, or a primate. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant. In some embodiments, the subject is a pregnant women. In some embodiments, the subject is a fetus. Particularly, the subject denotes a human with a pathogen brain infection. Particularly, the subject denotes a human with a brain viral infection. In a particular embodiment the subject is a human with co-morbidities and in the elderly (see for example Guan et al, 2020). As used herein, the term “subject” encompasses the term "patient”.

In a particular embodiment the subject of the present invention is obese.

An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m ² or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m ² . A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg/m ² to less than 30 kg/m ² or a subject with at least one co-morbidity with a BMI of 25 kg/m ² to less than 27 kg/m ² . The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m ² . An "obese subject" in these countries refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m ² . In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg/m2 to less than 25 kg/m ² .

In a particular embodiment the subject of the present invention suffers from diabetes.

As used herein the term “diabetes” has its general meaning in the art and refers to a chronic disease characterized by the presence of excess blood sugar called hyperglycemia. It is known if the fasting blood sugar level is equal to or greater than 1.26 g / 1 or 7 mmol / 1 of blood during two successive dosages.

As used herein the term “insulin” has its general meaning in the art and refers to a hormone made by the pancreas, which is permanently present in the blood. Its role is to maintain blood sugar around 1 g / 1 when sugar intake is high: insulin is a hypoglycemic hormone. Insulin allows the body's cells to take up the blood sugar when it needs it (such as muscle cells during exercise) and use it to turn it into energy. If necessary, it allows the storage of unused sugar, in the liver or fat cells. When the sugar level rises, for example after a meal, the pancreas produces more insulin to bring the blood sugar level back to normal. If insulin is insufficient or ineffective, sugar builds up in the blood and the blood sugar rises excessively: this is hyperglycemia. In the absence of treatment, this hyperglycemia is maintained at too high a level: it is chronic hyperglycemia which defines diabetes. There are 2 main types of diabetes:

"type 1" diabetes, is an autoimmune disease and is due to an absence of insulin secretion by the pancreas. In its absence, cells can no longer properly use the sugar that circulates in the blood. Hyperglycemia appears quickly, as soon as the insulin level becomes insufficient. The type 1 diabetes most commonly occurs in children, adolescents and young adults.

"type 2" diabetes, caused by the body's improper use of insulin. Its development takes place very gradually, insidiously over many years. First, the body’s cells become resistant to insulin. This resistance is normal with age, but it is aggravated by excess fatty tissue in the event of overweight and obesity. This stage is called: insulin resistance. Glucose builds up in the blood and hyperglycemia gradually sets in; the body is trying to adapt. The pancreas increases the production of insulin: this is called hyperinsulinism; after several years (10 to 20 years), the pancreas becomes exhausted and can no longer secrete enough insulin to regulate blood sugar: this is the stage of insulin deficiency.

In a particular embodiment the subject of the present invention suffers from hypertension. As used herein the term “hypertension”, also known as high blood pressure (HBP), has its general meaning in the art and refers to a long-term medical condition in which the blood pressure in the arteries is persistently elevated. High blood pressure is classified as primary (essential) hypertension or secondary hypertension. About 90-95% of cases are primary, defined as high blood pressure due to nonspecific lifestyle and genetic factor. Lifestyle factors that increase the risk include excess salt in the diet, excess body weight, smoking, and alcohol use. The remaining 5-10% of cases are categorized as secondary high blood pressure, defined as high blood pressure due to an identifiable cause, such as chronic kidney disease, narrowing of the kidney arteries, an endocrine disorder, or the use of birth control pills.

In a particular embodiment the subject of the present invention suffers from an ovariectomy. As used herein the term “ovariectomy” has its general meaning in the art and refers to the surgical removal of an ovary or ovaries.

In some embodiments the subject of the present invention includes but is not limited to menopausal women, pregnant women, women treated with GnRH inhibitors for endometriosis or dysmenorrhea, women taking hormonal contraceptives that modify hypothalamic control of gonadal steroid production (contraceptive pills, patches, injections, hormone-releasing IUDs...), women with PCOS/PCOD who have high androgen levels, men with prostate or testicular cancer or other cancer treated with androgen deprivation therapy, women with breast, uterine, cervical or ovarian cancer or any other cancer treated with selective estrogen receptor modulators or other anti-estrogens, men and women with abnormally high androgen levels for other reasons, men and women with abnormally low estrogen levels for other reasons (hypogonadotropic hypogonadisms, disorders of the pituitary).

The inventors show that Angiotensin-converting enzyme 2 (ACE2) and transmembrane proteinase, serine 2 (TMPRSS2) could form part of a pro-inflammatory pathway mediated by differential FPR2 expression, through which risk factors such as metabolic diseases and male sex hormones (or the absence of female sex hormones, or the modification of upstream hormones such as gonadotropins or gonadotropin-releasing hormones, or other hormones and neuroactive molecules that mediate the effect of sex, for example VIP, which is produced by the hypothalamus and is a ligand of FPR2 (54, 55)) could increase susceptibility to and outcome of SARS-CoV-2 infection by altering the pattern of ACE2 expression. The inventors show that FPR2 is expressed and dysregulated in the hypothalamus of the subject. FPR2 would have a potential role in coronavirus or other RNA virus (e.g. SARS-CoV-2) pathogenesis.

As used herein, the term “brain” has its general meaning in the art and refers to an organ that serves as the center of the nervous system (CNS) in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as vision. Neuroanatomists usually divide the vertebrate brain into six main regions: the telencephalon (cerebral hemispheres), diencephalon (thalamus and hypothalamus), mesencephalon (midbrain), cerebellum, pons, and medulla oblongata. Each of these areas has a complex internal structure. Some parts, such as the cerebral cortex and the cerebellar cortex, consist of layers that are folded or convoluted to fit within the available space. Other parts, such as the thalamus and hypothalamus, consist of clusters of many small nuclei.

As used herein, the term “hypothalamus” has its general meaning in the art and refers to a small region at the base of the forebrain, whose complexity and importance belies its size. It is composed of numerous small nuclei, each with distinct connections and neurochemistry. The hypothalamus is engaged in additional involuntary or partially voluntary acts such as sleep and wake cycles, eating and drinking, and the release of some hormones:

There are two types of hormones: 1) The hypothalamic neuro-hormones controlling the hormonal secretion of the anterior pituitary gland or adenohypophysis are synthesized by neurons in the neuroendocrine hypothalamus. These neuro-hormones act on the glandular cells of the adenohypophysis or anterior pituitary to stimulate them (also known as releasing hormones or factors/stimulating hormones or factors/liberins) or slow them down (also known as inhibiting hormones or factors/statins). The hypothalamic hypophysiotropic neuro-hormones currently identified are Thyrotropin-releasing hormone (TRH), Gonadotropin-releasing hormone (LHRH or GnRH), Corticotropin-releasing hormone (CRH), growth hormone releasing hormone (GHRH), vasoactive intestinal peptide (VIP), Prolactin-releasing hormone (PRH) as well as dopamine and somatostatin (SRIF) (PIF). 2) The so-called post-pituitary neuro-hormones (oxytocin and vasopressin - or antidiuretic hormone or ADH -) are secreted by the hypothalamic neurons of the supra-optic and paraventricular nuclei.

As used herein, the term “brain infection” refers to an infection that occurs in the brain of the subject.

As used herein, the term “a pathogen brain infection” denotes a brain infection induced by a biological pathogen or in other words an infectious agent.

According to the invention, the pathogen can be a virus.

In some embodiments, the brain infection is a brain viral infection. In some embodiments, the brain viral infection is caused by a virus selected from the group consisting a virus selected from the group consisting of, human immunodeficiency viruses (HIV), Zika virus, Cytomegalovirus (CMV), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie vims, echo vims, herpes simplex vims, coronavims (SARS-coronavims such as SARS-Covl or SARS-Cov 2), and smallpox. In some embodiments, the viral infection may be due to a member of the Pneumoviridae , Paramyxoviridae and/or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial vims (hRSV), type A and type B, human metapneumovims (hMPV) type A and type B; parainfluenza vims type 3 (PIV-3), measles vims, endemic human coronavimses (HCoV-229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle-East respiratory syndrome (MERS) coronavimses.

As used herein, the term “coronavims” has its general meaning in the art and refers to any member or members of the Coronaviridae family. Coronavims is a vims whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular vims. The virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronavimses the largest of the RNA vims genomes. In particular, coronavims RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non-stmctural proteins. In particular, the coronavims particle comprises at least the four canonical stmctural proteins E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein). The S protein is cleaved into 3 chains: Spike protein SI, Spike protein S2 and Spike protein S2'. Production of the replicase proteins is initiated by the translation of ORFla and ORFlab via a -1 ribosomal frame-shifting mechanism. This mechanism produces two large viral polyproteins, ppla and pplab, that are further processed by two virally encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro). Coronavimses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronavimses are transmitted by aerosols of respiratory secretions. Coronavimses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), Middle East respiratory syndrome-related coronavims (MERS-Cov) and Severe Acute Respiratory Syndrome (SARS)-coronavims (Center for Disease Control), in particular SARS- CoVl and SARS-CoV2.

According to the invention, the coronavims can be a MERS-CoV, SARS-CoV, SARS- CoV-2 or any new future family members. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS) and any neurological manifestations (headaches, dizziness, nausea, seizures, stroke, cognitive or sensory disturbances, etc.) or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances...) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances.

As used herein, the term “SARS-CoV-2” refers to severe acute respiratory syndrome coronavirus 2 known by the provisional name 2019 novel coronavirus (2019-nCoV) is the cause of the respiratory coronavirus disease 2019 (COVID-19). Taxonomically, it is a strain of the Severe acute respiratory syndrome-related coronavirus (SARSr-CoV), a positive-sense single- stranded RNA virus. It is contagious in humans, and the World Health Organization (WHO) has designated the ongoing pandemic of COVID-19 a Public Health Emergency of International Concern. SARS-CoV-2 virion is approximately 50-200 nanometres in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike protein, which has been imaged at the atomic level using cryogenic electron microscopy is the protein responsible for allowing the virus to attach to the membrane of a host cell.

In some embodiments, the subject of the present invention suffers from COVID-19.

As used herein, “SARS-CoV-2 infection” refers to the transmission of this virus from an animal and/or human to another animal and/or human primarily via respiratory droplets from coughs and sneezes within a range of about 2 meters. Indirect contact via contaminated surfaces is another possible cause of infection.

In some embodiments, the subject can be symptomatic or asymptomatic. As used herein, the term "asymptomatic" refers to a subject who experiences no detectable symptoms for the brain viral infection (e.g. coronavirus). As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of a pathogen brain viral infection and particularly a coronavirus infection. Symptoms of coronavirus infection include: neurological symptoms (headaches, dizziness, nausea, loss of consciousness, seizures, encephalitis stroke, cognitive or sensory disturbances...), as well as anosmia or ageusia; fatigue, cough, fever, difficulty to breathe or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances...) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances

As used herein, the term “formyl peptide receptor 2” (FPR2), also known as ALXR; HM63; FMLPX; FPR2A; FPRHl; FPRH2; FPRLl; LXA4R; FMLP-R-II, has its general meaning in the art and refers to a G-protein coupled receptor (GPCR) located on the surface of many cell types of various animal species. The human receptor protein is encoded by the FPR2. The term FPR2 includes naturally occurring FPR2 and variants and modified form thereof. The FPR2 can be from any source and is typically an animal FPR2, preferably a bird FPR2 or mammal FPR2, even more preferably a human FPR2. Nucleic acid sequence (Gene ID: 2358) and amino acid sequence of (UniProt: P25090) of human FPR2 in particular are described in the art.

As used herein, the term “modulator” refers to any molecule, agent or compound that increases or decreases FPR2 activity, including a molecule that changes FPR2 downstream signaling activities, said modulator being an activator or an inhibitor as defined herein below.

Another embodiment of the invention relates to an FPR2 inhibitor, as a modulator of FPR2, for preventing or treating a brain viral infection (e.g. coronavirus) in a subject in need thereof.

As used herein, the term “FPR2 inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of FPR2.

The term “inhibitor” as used herein, refers to an agent that is capable of specifically binding and inhibiting signaling through a receptor to fully block, as does an antagonist, or detectably inhibit a response mediated by the receptor. For example, as used herein the term “FPR2 inhibitor” is a natural or synthetic compound which binds and inactivates fully or partially FPR2 for initiating or participating to a pathway signaling (such as the ERK signaling pathway) and further biological processes. In the context of the invention the FPR2 inhibitor in particular prevents, decreases or suppresses the virus replication. The virus replication decrease observed can be by at least about 1%, 2%, 5%, 10%, e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to the replication observed in a referenced cell. The term inhibitor encompasses the term “antagonist”.

FPR2 inhibitory/antagonistic activity may be assessed by various known methods. A control FPR2 can be exposed to no antibody or antigen binding molecule, an antibody or antigen binding molecule that specifically binds to another antigen, or an anti-FPR2 antibody or antigen binding molecule known not to function as an inhibitor, for example as an antagonist.

In some embodiment, the FPR2 inhibitor inhibits the FPR2 actions that exacerbate the effects of viral invasion and inflammation of the hypothalamus and would be an effective therapeutic option for brain viral infection and its consequences.

In a particular embodiment, a FPR2 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a metabolic lipid, a pepducin, a polynucleotide and a compound comprising such a molecule or a combination thereof. A FPR2 inhibitor for use in the context of the present invention can be selected fromWRW4 (i.e. WRWWWW, in particular the WRWWWW-NH2 peptide); PBP10 (QRLFQVKGRRrhodamine-B as described in Forsman et al (2012) “Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending from a PIP2 -binding domain of gelsolin”. J. Immunol. 189-629.); BOC-2 (Boc-Phe- Leu-Phe-Leu-Phe-OH or also known as Boc-FLFLF and having the Molecular Formula: C44H59N508); Isopropylureido-FLFLF; the FPRL1 -inhibitory protein (as described in Prat C, Bestebroer J, de Haas CJ, van Strijp JA, van Kessel KP. (2006) “ A new staphylococcal anti inflammatory protein that antagonizes the formyl peptide receptor-like 1" . J. Immunol., Ill (11): 8017-26.), Pam-(Lys-PNSpe)r _> -NH2 (as described in Skovbakke SL, Heegaard PM, Larsen CJ, Franzyk H, Forsman H, Dahlgren C (2015) “The proteolytically stable peptidomimetic Pam-(l s-fiN pe) ₆ -NH 2 selectively inhibits human neutrophil activation via formyl peptide receptor 2” Biochem. Pharmacol. Jan 15;93(2): 182-95); and a synthetic molecule such as Quin- C7 (also known as 4-butoxy-N-[2-(4-hydroxyphenyl)-4-oxo-l,2-dihydroquinazolin- 3- yljbenzamide) and described in Zhou C, Zhang S, Nanamori M, Zhang Y, Liu Q, Li N, Sun M, Tian J, Ye PP, Cheng N, Ye RD, Wang MW. (2007) “ Pharmacological characterization of a novel non peptide antagonist for formyl peptide receptor-like 7”. Mol Pharmacol, 72: 976- 983.); BB-V-115 (Young SM, Bologa CM, Fara D, Bryant BK, Strouse JJ, Arterbum JB, Ye RD, Oprea TI, Prossnitz ER, Sklar LA, Edwards BS. (2009) “ Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes" . Cytometry A 75: 253-263); compound 796276 (Young SM, Bologa CM, Fara D, Bryant BK, Strouse JJ, Arterbum JB, Ye RD, Oprea TI, Prossnitz ER, Sklar LA, Edwards BS. (2009) “ Duplex high- throughput flow cytometry screen identifies two novel formylpeptide receptor family probes" . Cytometry A 75: 253-263); compound 1754-20 [((R)-4-(2-([l,19-biphenyl]-4-yl)ethyl)-5-(4- hy droxybenzyl)- 1 -((R)- 1 -(4-hy droxyphenyl)-3 -((S)-2-(((S)-6-isopropyl-2, 3 -dioxopiperazin- 1 - yl)methyl)pyrrolidin-l-yl)propan-2-yl)piperazine-2,3-dione] or compound 1754-31 [(R)-4- (cyclohexylmethyl)-5-(4-hydroxybenzyl)-l-((R)-l-((S)-2-(((S) -6-isopropyl-2,3- dioxopiperazin- 1 -yl)methyl)pyrrolidin- 1 -yl)-3 -(naphthalene-2-yl)propan-2-yl)piperazine-2, 3 - dione] both described in Pinilla et al. (“ Selective agonists and agonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries." Molecular Pharmacology 84:314-324, 2013). Other FPR2 inhibitors can be found in patent application WO201 1/073918. A preferred FPR2 inhibitor is selected from WRW4, PBP10 or BOC-2. WRW4 is a particularly preferred FPR2 inhibitor. As indicated previously the FPR2 inhibitor can be a peptide or peptide molecule comprising amino acid residues. As used herein the term “amino acid residue” refers to any natural/standard and non-natural/non- standard amino acid residue in (L) or (D) configuration, and includes alpha or alpha-di substituted amino acids. It refers to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, serine, tyrosine. It also includes beta-alanine, 3 -amino-propionic acid, 2,3-diamino propionic acid, alpha- aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, cyclopentylalanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, cyclopentylglycine, cyclobutylglycine, cyclopropylglycine, norleucine (Me), norvaline, 2-napthylalanine, pyridylalanine, 3- benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4- fluorophenylalanine, penicillamine, l,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2- thienylalanine, methionine sulfoxide, L-homoarginine (hArg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p-aminophenylalanine, N- methylvaline, selenocysteine, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry.

In a particular embodiment, the peptide used as a FPR2 inhibitor is WRW4.

In another particular embodiment, the peptide used as a FPR2 inhibitor is PBP10 (QRLF Q VKGRR-rhodamine-B) .

In a further particular embodiment, the peptide used as a FPR2 inhibitor is BOC-2 (Boc- FLFLF) or Isopropylureido-FLFLF.

Compounds of the present invention which include peptides may comprise replacement of at least one of the peptide bonds with an isosteric modification. Compounds of the present invention which include peptides may be peptidomimetics. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent, but wherein one or more of the peptide bonds/linkages have been replaced, often by proteolytically more stable linkages. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many or all of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, potential for hydrogen bonding, etc. Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, the peptide may have one or more peptide linkages replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis or trans), -CH(OH)CH ₂ -, or -COCH2-, -N-NH-, -CH2NHNH-, or peptoid linkages in which the side chain is connected to the nitrogen atom instead of the carbon atom. Such peptidomimetics may have greater chemical stability, enhanced biological/pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.) and/or reduced antigenicity relative its peptide equivalent.

The FPR2 inhibitor can also be a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Examples of small organic molecule are compound 1754-20, compound 1754-31, Quin-C7, BB-V-115 and compound 796276. In a particular embodiment, the FPR2 antagonist is a small organic molecule which can be selected from compound 1754-20, compound 1754-31, Quin-C7, BB-V-115 and compound 796276.

The FPR2 inhibitor can also be an antibody or an antigen-binding molecule. In an embodiment, the antibody specifically recognize/bind FPR2 (e.g. FPR2 of SEQ ID NO:2) or an epitope thereof involved in the activation/stimulation of the ERK-pathway. In another preferred embodiment, the antibody is a monoclonal antibody. In an even more preferred embodiment the antibody is the antibody obtained with the clone FN-1D6-AI described in De Santo et ak, Nat. Immunol. ²⁷ (available for sale from Genovac AG, Freiburg, Germany).

The term “antibody” is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies and humanized antibodies, so long as they exhibit the desired biological activity (e.g., as indicated previously, inhibiting the binding of/activation by the virus of FPR2, typically the binding of ANXA1 to FPR2, and/or of any other ligands/agonists to FPR2 such as LPXA4 (or LXA4) or formylated proteins/peptides). Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ⁷ , F(ab ⁷ )2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H, V H-V H), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Antibodies according to the present invention can be of any class, such as IgG, IgA, IgDl IgEl IgMl or IgY 1 although IgG antibodies are typically preferred. Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken. The antibodies can be modified by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.

In general, techniques for preparing antibodies (including polyclonal antibodies, monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art, see .g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Kohler & Milstein, Nature 256:495- 497 (1975); Kozbor et al, Immunology Today 4:72 (1983); and Cole et ah, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 1985. Additionally, antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag. The antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art. Other methods of preparing these monoclonal antibodies, as well as chimeric antibodies, humanized antibodies, and single-chain antibodies, are known in the art.

The FPR2 inhibitor can also be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Also within the scope of the invention is a metabolic lipid as a FPR2 inhibitor. Disorders in endogenous lipid metabolism have been associated with infectious disease. Lipid metabolites (such as Lipoxin A4) can act on FPR2 and modulate inflammation. Targeting metabolism of lipids (lipoxygenase 12/154, lipoxygenase 5) or use of specific active lipid metabolites such as lipoxin A4 have the following advantages: it overcomes the traditional challenge of resistance and should have a broad antiviral activity against circulating and future virus strains by targeting the host instead of the virus. An example of metabolic lipid usable in the context of the invention is lipoxin A4.

The FPR2 inhibitor can also be a pepducin. Pepducins are cell-penetrating that act as intracellular modulators of signal transference from receptors to G proteins (cf. Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A (January 2002). " Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides" . Proc. Natl. Acad. Sci. U.S.A. 99 (2): 643-8). Pepducins utilize lipidated fragments of intracellular G protein- coupled receptor loops to modulate GPCR action in targeted cell-signaling pathways. A pepducin molecule comprises a short peptide derived from a GPCR intracellular loop tethered to a hydrophobic moiety. This structure allows pepducin lipopeptides to anchor in the cell membrane lipid bilayer and target the GPCR/G protein interface via a unique intracellular allosteric mechanism.

An example of pepducin usable in the context of the invention is for example the FlPali ₆ pepducin described in Winther et al. (“ A neutrophil inhibitory pepducin derived from FPR1 expected to target FPR1 signaling hijacks the closely related FPR2 instead ” FEBS Letters 589 (2015) 1832-1839).

The FPR2 inhibitor can also be a polynucleotide, typically an inhibitory nucleotide. These include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, complementary DNA (cDNA) or guide RNA (gRNA usable in the context of a CRISPR/Cas system). In some preferred embodiments, a siRNA targeting FPR2 expression is used. Interference with the function and expression of endogenous genes by double- stranded RNA such as siRNA has been shown in various organisms. See, e.g., A. Fire et al., “Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans” Nature 391 :806-811 (1998); J. R. Kennerdell & R. W. Carthew, “Use of dsDNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway,” CeJ 95:1017-1026 (1998); F. Wianni & M. Zemicka- Goetz, “Specific Interference with Gene Function by Double- Stranded RNA in Early Mouse Development,” Nat. Cell Biol. 2:70-75 (2000). siRNAs can include hairpin loops comprising self-complementary sequences or double stranded sequences. siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. Such double-stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double-stranded RNA targeting FPR2 can also be synthesized from a cDNA vector construct in which a FPR2 gene (e.g., human FPR2 gene) is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal. Double-stranded RNA targeting the FPR2 gene can be introduced into a cell (e.g., a tumor cell) by transfection of an appropriate construct.

Typically, RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules.

The structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R. F. Gesteland et ak, eds, “The RNA World” (3 ^rd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2006), pp. 535-565, incorporated herein by this reference. For these approaches, cloning into vectors and transfection methods are also well known in the art and are described, for example, in J. Sambrook & D. R. Russell, “Molecular Cloning: A Laboratory Manual” (3 ^rd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001), incorporated herein by this reference.

In addition to double stranded RNAs, other nucleic acid agents targeting FPR2 can also be employed in the practice of the present invention, e.g., antisense nucleic acids. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA. Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., C J. Marcus- Sekura, “Techniques for Using Antisense Oligodeoxy ribonucleotides to Study Gene Expression,” Anal. Biochem. 172:289-295 (1988); J. E. Hambor et al, “Use of an Epstein-Ban Virus Episomal Replicon for Anti-Sense RNA-Mediated Gene Inhibition in a Human Cytotoxic T-Cell Clone,” Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014 (1988); H Arima et al., “Specific inhibition of Interleukin- 10 Production in Murine Macrophage-Like Cells by Phosphorothioate Antisense Oligonucleotides,” Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); and W.-F. Hou et al., “Effect of Antisense Oligodeoxynucleotides Directed to Individual Calmodulin Gene Transcripts on the Proliferation and Differentiation of PC 12 Cells,” Antisense Nucl. Acid Drug Dev. 8:295-308 (1998), all incorporated herein by this reference. Antisense technology is described further in C. Lichtenstein & W. Nellen, eds., “Antisense Technology: A Practical Approach” (IRL Press, Oxford, 1997), incorporated herein by this reference. FPR2 polynucleotide sequences from human and many other animals in particular mammals have all been delineated in the art. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting FPR2 can be readily synthesized using methods well known in the art.

Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex).

The FPR2 inhibitor can also include the inhibitors described in Yingying Le, Philip M. Murphy and Ji Ming Wang, (2002) “Formyl-peptide receptors revisited”, TRENDS in Immunology adnd in Hui-Qiong He, Richard D. Ye (2017), “The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition”, Molecules.

Another embodiment of the invention relates to a FPR2 activator, as a modulator of FPR2, for preventing or treating a brain viral infection (e.g. coronavirus) in a subject in need thereof.

The term “activator” as used herein, refers to an agent that is capable of specifically binding and activating FPR2 for initiating a pathway signaling and further biological processes through a receptor to fully activate, as does an agonist, or detectably induce or stimulate a response mediated by the receptor. For example, as used herein the term “FPR2 activator” is a natural or synthetic compound which binds and activates fully or partially FPR2 for initiating or participating to a pathway signaling (such as the ERK signaling pathway) and further biological processes. In the context of the invention the FPR2 activator in particular induces or stimulates influenza virus replication. FPR2 agonistic activity may be assessed by various methods known by the skilled person.

In some embodiment, FPR2 activator promotes the FPR2 actions that limit viral proliferation and resolve inflammation and would be an effective therapeutic option for brain viral infection and its consequences. In a particular embodiment, a FPR2 activator according to the invention can be selected from a peptide, typically a formylated peptide, a peptide, a peptide mimetic (or peptidomimetic), a protein, a small organic molecule, a metabolic lipid, and a pepducin as herein defined.

Preferred formylated peptides have a mitochondrial or bacterial origin.

In a particular embodiment, the peptide used as a FPR2 activator is the WKYMVM peptide, in particular the WKYMVm-NH2 peptide; fMLP (formyl-Met-Leu-Phe); TIPMFVPESTSKLQKFTSWFM-amide (also known as CGEN-855A, described in Hecht I, Rong J, Sampaio AL, Hermesh C, Rutledge C, Shemesh R, Toporik A, Beiman M, Dassa L, Niv H, Cojocaru G, Zauberman A, Rotman G, Perretti M, Vinten-Johansen J, Cohen Y “A novel peptide agonist of formyl-peptide receptor-like 1 (ALX) displays anti-inflammatory and cardioprotective effects” J Pharmacol Exp Ther. 2009 Feb;328(2):426-34); sCKbeta8-l (as described in Elagoz Al, Henderson D, Babu PS, Salter S, Grahames C, Bowers L, Roy MO, Laplante P, Grazzini E, Ahmad S, Lembo PM, “A truncated form of CKbeta8-l is a potent agonist for human formyl peptide-receptor-like 1 receptor”, Br J Pharmacol. 2004 Jan;141(l):37-46); PSMa3 (i.e. MEFVAKLFKFFKDLLGKFLGNN described in Kretschmer D, Gleske AK, Rautenberg M, Wang R, Koberle M, Bohn E, Schoneberg T, Rabiet MJ, Boulay F, Klebanoff SJ et al. (2010) “Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus”, Cell Host Microbe, 7 (6): 463-73); PSMa2 described in Forsman, H. et al. (2015 “Structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group.”, Biochim. Biophys. Acta 1853, 192-200); MMKl (for example the LESIFRSLLFRVM-NH2 peptide described in Hu JY, Le Y, Gong W, Dunlop NM, Gao JL, Murphy PM, Wang JM. (2001) Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRLl. J. Leukoc. Biol., 70 (1): 155-61.).

In a particular embodiment, the metabolic lipid used a FPR2 activator is lipoxin A4 (LXA4).

In a particular embodiment, the small organic molecule used as FPR2 activator is Quin- C1 (i.e. 4-butoxy-N-[2-(4-methoxyphenyl)-4-oxo-l,2-dihydroquinazolin- 3-yl]benzamide, described in Nanamori M, Cheng X, Mei J, Sang H, Xuan Y, Zhou C, Wang MW, Ye RD. (2004), “A novel nonpeptide ligand for formyl peptide receptor-like 1”. Mol. Pharmacol., 66 (5): 1213-22).

In another particular embodiment, the protein used as a FPR2 activator is Annexin-1 (ANXA1). In a particular embodiment, the small organic molecule used as FPR2 activator is BMS- 986235/LAR-1219, described in Yoshikazu Asahina, Nicholas R. Wurtz, Kazuto Arakawa, Nancy Carson, Kiyoshi Fujii, Kazunori Fukuchi, Ricardo Garcia, Mei-Yin Hsu, Junichi Ishiyama, Bruce Ito, Ellen Kick, John Lupisella, Shingo Matsushima, Kohei Ohata, Jacek Ostrowski, Yoshifumi Saito, Kosuke Tsuda, Francisco Villarreal, Hitomi Yamada, Toshikazu Yamaoka, Ruth Wexler, David Gordon, and Yasushi Kohno, (2020), “A Potent Formyl Peptide Receptor 2 (FPR2) Selective Agonist for the Prevention of Heart Failure”. J. Med. Chem.

Other FPR2 activators can be found in WO2011073918.

The FPR2 activator can also include the inhibitors described in Yingying Le, Philip M. Murphy and Ji Ming Wang, (2002) “Formyl-peptide receptors revisited”, TRENDS in Immunology, in Fabio Cattaneo, Melania Parisi and Rosario Ammendola (2013), Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2) Agonists, Int. J. Mol. Sci. and in Hui-Qiong He, Richard D. Ye (2017), “The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition”, Molecules.

Another aspect of the invention relates to the use of a modulator of FPR2 (herein defined as an activator or as an inhibitor of FPR2) for preparing a composition, typically a pharmaceutical composition, for preventing or treating a brain viral infection (e.g. coronavirus) or brain inflammation resulting from a peripheral infection in a subject in need thereof.

As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

The terms “prevention”, “preventive treatment”, “prophylactic treatment” and “preventing” refer to prophylactic or preventive measures comprising the administration of a FPR2 antagonist or composition comprising said antagonist that prevents the symptoms of a brain viral infection (e.g. coronavirus), enhances the subject’s resistance or retard the progression of a brain viral infection (e.g. coronavirus).

Such treatments are intended for a animal subject, typically a mammal subject as herein identified below, preferably a human subject in need thereof. Are considered as such, any subject that will benefit or that is likely to benefit from the modulator (herein defined as an activator or as an inhibitor of FPR2) of the present invention, typically the subjects suffering from a brain viral infection (e.g. coronavirus), or those who are not already suffering of a brain viral infection (e.g. coronavirus) but are considered “at risk of’, or as having a predisposition to, developing such an infection or associated complications (in whom this has to be prevented).

A particular pharmaceutical composition for use for preventing or treating brain viral infection (e.g. coronavirus) in a subject in need thereof according to the invention comprises a modulator of FPR2 (herein defined as an activator or as an inhibitor of FPR2) presents in a therapeutically effective amount, at least one distinct compound selected from a therapeutic agent, an adjuvant, and a combination thereof, and a pharmaceutically acceptable support or excipient. In a preferred embodiment, a composition for use for preventing or treating brain viral infection (e.g. coronavirus) in a subject in need thereof according to the invention comprises a modulator of FPR2 and an antiviral both present in a therapeutically effective amount, more particularly a FPR2 inhibitor selected from WRW4, BOC-2 and PBP10 in combination with an antiviral.

Examples of antiviral include but are not limited to amantadine, rimantadine or pleconaril. The FPR2 modulator or composition comprising said modulator can be combined with a pharmaceutically acceptable support or excipient, and optionally sustained-release matrice, such as biodegradable polymer, to form therapeutic composition. “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. The terms “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. The term can refer to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. The pharmaceutical compositions of the present invention may therefore contain non-limiting pharmaceutically acceptable excipients/carriers such as solubilizing/diluting agents, antioxidants, enteric coatings, absorption enhancers, pH adjusting agents and buffers, dispersing agents, coatings, antibacterial, antifungal agents, absorption delaying agents (controlled time-release), osmolarity adjusters, isotonic agents, preservative agents, stabilizers, surfactants, emulsifiers, sweeteners, thickening agents, solvents, emollients, coloring agents, wetting agents, as well as colors and flavors and salts for the variation of osmotic pressure. The carrier/excipient is selected for administration by the selected route of administration.

Another object of the invention relates to a method for preventing or treating a brain viral infection or brain inflammation from a peripheral infection (e.g. coronavirus) in a subject in need thereof with a FPR2 modulator as herein described. The method comprises a step of administering a modulator of FPR2 in a therapeutically effective amount, or a composition as herein described comprising said modulator, to the subject.

By a “therapeutically effective amount” is meant a sufficient amount the FPR2 antagonist according to the invention to treat or prevent a brain viral infection (e.g. coronavirus) at a reasonable benefit/risk ratio applicable to any medical treatment. An effective amount can be administered in one or more doses. It will be understood that the total daily usage and frequency of administration of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the deficit being treated and the severity of the deficit; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The daily dosage of the modulator of FPR2 may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15,0, 25.0, 50.0, 100, 250 and 500 mg ofthe active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the modulator of FPR2 is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The effective amount may be given daily in a single or several doses (e.g., twice daily, three times per day or 4 times per day). It may also be given every 2 days, every 3 days or once a week, as prescribed. Preferably, the effective amount is given once daily.

When the composition comprises several therapeutic agents, those may be administered simultaneously, separately or sequentially.

FPR2 modulators may be administered in a pharmaceutical composition. Pharmaceutical compositions may be administered in unit dosage form. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals, and particulars about the subject. Any appropriate route of administration may be employed, for example, nasal, transdermal (topical), parenteral, subcutaneous, intramuscular, intramammary, intracranial, intraorbital, ophthalmic, intraventricular, intracap sular, intraarticular, intraspinal, intraci sternal, intraperitoneal, or oral administration. Examples of specific routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramammary; oral; transdermal (topical); transmucosal, and rectal administration

Therapeutic formulations may be for example in the form of tablets, capsules, troches, dragees, hard or soft gelatin capsules, solutions (e.g; syrops), aerosols, emulsions or suspensions, for oral administration; in the form of ointments, powders, nasal drops, sprays/aerosols or suppositories for transmucosal (e.g., rectal, oral, buccal, sub-lingual, intranasal) or transdermal/percutaneous administration; in the form of ointments, creams, gels or solutions for topical administration; or in the form of an injectable solution for parenteral administration (e.g., intraperitoneally, intrathecally, intravenously, intramuscularly, intradermally, transdermally, or subcutaneously). Intranasal or oral administration is a preferred form of use. The modulator of FPR2 or composition comprising said modulator is preferably administered in an intracranial form or oral or intranasal administration form with a nebulizer, inhaler, spray or aerosol. Inhaler and nebulizer allow a direct administration to the lung of the subject to be treated.

Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent useful to treat brain viral infection (e.g. coronavirus) or the symptoms induced by the pathogen brain viral infection. For example, further agent may be selected in the group consisting bronchodilators like b2 agonists and anticholinergics, corticosteroids, beta2- adrenoceptor agonists like salbutamol, anticholinergic like ipratropium bromide or adrenergic agonists like epinephrine.

Further agent may be also an antidiabetic agent like biguanide, sulfonylureas, meglitinides, DPP-4 inhibitors, GLP-1 agonists, SGLT-2 inhibitors, alpha-glucosidase inhibitors, thiazolidinediones, amylin analogs.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Expression of ACE2 and FPR2 in the adult human and mouse hypothalamus. (A) In the adult human hypothalamus, the receptor of SARS-Cov-19 ACE2 is expressed in neurons of the paraventricular nucleus of the hypothalamus (PVH) that also express FPR2. (B) Cells bordering the wall of the third ventricle (3 V) in the tuberal region of the hypothalamus at the level of the median eminence (ME) express FPR2. FPR2 may be expressed in tancycytes as some of the FPR2-expressing cells also express vimentin. (C) In the mouse hypothalamus, Fpr2 immunolabelling is found to be more intense in the PVH, the ME and the arcuate nucleus of the hypothalamus (ARH), in mice fed on high fat diet (HFD; right panels) for 9 weeks than in mice on chow (left panels). (D) However, Fpr2 labeling appears to be less intense in female mice after ovariectomy (right panel) than in intact females (left panel) in the ME. (E) FACS-based sorting show that in the median eminence, both tanycytes (tdTomato positives cells) and non-tanycytes (tdTomato negative cells) express Fpr2 in the ME/ARH region. Figure 2. Immunolabeling for FPR2 and viral susceptibility markers (ACE2 and TMPRSS2) in the post mortem brains of COVID-19 patients, showing: (A) SARS-CoV-2 spike protein in the olfactory nerve fibers surrounding and entering the olfactory bulb and colocalizing with viral susceptibility markers; (B) increased expression of FPR2 colocalizing with the same fibers in a COVID-19 patient olfactory bulb; (C and D) FPR2 expression in the hypothalamus of COVID-19 patients in two types of cells.

Figure 3. Immunohistochemistry for the highly conserved SARS-CoV N protein showing widespread infection in the brain of K18-hACE2 mice 7 days after infection with SARS-CoV-2.

Figure 4. Immunohistochemical labeling for SARS-CoV-2 spike protein and FPR2 showing a dramatic increase in FPR2 labeling in infected brain areas and cells.

Figure 5. qRT-PCR analysis for Fpr2 gene expression in the cerebral cortex of SARS- CoV-2-infected (Covid) mice versus mock-infected (Mock) controls. *p < 0.01, unpaired t-test.

EXAMPLE 1:

Materials & Methods

Gene expression analysis for ACE 2, TMPRSS2 and correlated genes in the brain

Differential distribution of ACE 2 and TMPRSS2 across brain resions andFPR2 across the hypothalamus

Normalized gene expression values of ACE2 and TMPRSS2 were retrieved from the Allen Human Brain Atlas (AHBA) (57) for nuclei of the hypothalamus, insula, amygdala, paraventricular nucleus of the thalamus, pons and myelencephalon. The frontal lobe and cerebellar cortex as well as the choroid plexus were included for comparison. The probes were mapped to ACE2 and TMPRSS2 genes using the collapseRows function of WGCNA l.69-81 (56) in R ver3.6.3. Probe CUST 16267 PI416261804 for ACE2, probe A 23 P29067 for TMPRSS2 and probe A 23 P 55649 were selected based on maximum variance across all samples (maxRowVariance of WGCNA package). The data were concatenated across all donors for the specified brain structures. To account for missing ACE2 and TMPRSS2 expression values for the brain structures excluded for various technical reasons such as damaged or missing tissue or low-quality RNA (Allen Human Brain Atlas, technical white paper: microarray survey), nonparametric missing value imputation was performed using R- package missForest (57). Median expression values of ACE2 and TMPRSS2 across all donors were presented in the form of a heatmap using GraphPad Prism 8. The same protocol was followed to map FPR2 genes to different hypothalamic nuclei.

ACE2/TMPRSS2/FPR2-correlated senes in key brain structures

For the selected ACE2, TMPRSS2 and FPR2 probes, the “positive” and “anti correlated” genes with their normalized log2intensity gene expression values were retrieved using “ Find Correlates" search utility in the AHBA (with a cut-off of -0.3 > r > 0.3 as in the AHBA, where r is Pearson’s rank correlation coefficient) for the brain structures of interest (ACE2 and TMPRSS2: insula, amygdala, hypothalamus, parabrachial nuclei of pons and myelencephalon; FPR2 - all hypothalamic regions present in the AHBA) sampled in all human donors. After filtering the probes with zero entrez-id, the expression values of the retrieved gene list were used as vector elements to recompute Pearson's correlation coefficients (r) of all gene pairs using the rcorr function of R-package Hmisc (https://cran.r- proiect.org/web/packages/Hmisc/index.html). The rcorr function returns a correlation matrix with the r values and the corresponding asymptotic p value based on t distribution. The function p. adjust and the method ‘fdr’ were applied to control for false discovery rate (fdr). ACE2-, TMPRSS2- and FPR-2 correlated genes were extracted from the correlation matrix and filtered by (i) setting a threshold of -0.3 > r > 0.3 and fdr < 0.25, and (ii) selecting the correlations with the lowest fdr value for probes mapped to multiple genes.

COVID-19 luns RN A sequencing dataset

Raw read counts from COVID-19 infected and uninfected human lung (n=2) RNA-seq dataset were collected from GSE147507 (52) followed by differential expression analysis using DESeq2 (55), as indicated by (52). The differentially expressed genes were checked for the overlapping gene count between the COVID-19 dataset and the ACE2- and TMPRSS2- corr elated gene sets.

KEGG pathway and sene ontology enrichment analyses

KEGG pathway and gene ontology (GO) enrichment analyses of ACE2-, TMPRSS2- and FPR2-correlated genes was performed using Clusterpro filer package in R (59), a widely used R package for enrichment analyses. The package supports enrichment based on both hypergeometric test or gene set enrichment analysis (GSEA). enrichGO and enrichKEGG functions based on hypergeometric distributions were applied to perform the enrichment test for the ACE2, TMPRSS2 and FPR2-correlated gene sets q value cut-off (for fdr) was consistently maintained at 0.25. The enriched pathways for ACE2-, TMPRSS2- and FPR2- correlated genes for the analyzed brain structures were compiled together and visualized using ggplot2 package of R. The enriched pathway-gene networks were generated using cnetplot, while the dot plot for enriched GO terms was generated using the dotplot function of Clusterprofiler package.

Gene-network maps connecting brain regions

The gene network maps connecting brain regions with shared pathways such as neuroactive ligand receptor interaction, olfactory transduction, taste transduction and cAMP signaling enriched for ACE2- and TMPRSS2-correlated genes were generated by curating genes associated with each pathway and projecting them in the form of networks using Cytoscape v3.8.0. Furthermore, these genes were checked for overlap with the COVID-19 differential gene expression dataset.

Functional protein-protein interaction network in the hypothalamus

Common genes between the COVID-19 differentially expressed geneset and ACE2- /TMPRSS2-correlated genes in the hypothalamus, were queried in the STRING database to obtain functional protein-protein (PPI) interaction networks, and the associated GO terms enriched for biological processes. The list of proteins interacting with FPR2 was queried in STRING also.

Immunofluorescence labeling — Animal brains

Animals

Mice: Three C57BL/6J (Charles River) and two NPY::GFP (JAX:006417); POMC::Cre (JAX:005965); tdTomato (JAX:007914) male mice, 8-9 weeks-old were individually housed and given ad libitum access to water and standard pelleted rodent chow (R03-25, Safe diet). Three other C57BL/6J were given ad libitum water and a high-fat diet containing 60% fat (HFD; D 12492 Research Diet) for 9 weeks. Ovariectomy (OVX) in mice: 6 adult female mice against a background of C57BL/6J (ERa flox with VH injection) were subjected to ovarietomy (OVX; N=3) or sham (N=3) surgery. Briefly, OVX was performed under isoflurane anesthesia. A mid- ventral incision was made, the muscle separated gently by forceps to expose ovaries and periovarian fat tissue. Ovaries and ovarian fat were removed bilaterally after ligation of the most proximal portion of the oviduct. In sham animals, the same procedure was carried out except for the removal of the ovaries. The surgical incision was sutured and postsurgical recuperation was monitored daily. Animals were kept for 6 weeks after surgery.

Hamsters: Two 8-week (100 g) male hamsters (Janvier) were fed ad libitum and single- housed. Animal studies were performed with the approval of the Institutional Ethics Committees for the Care and Use of Experimental Animals of the University of Lille and the French Ministry of National Education, Higher Education and Research (APAFIS#2617- 2015110517317420 v5 and APAFIS#25041-2020040917227851), and under the guidelines defined by the European Union Council Directive of September 22, 2010 (2010/63/EU).

Brain Fixation

To fix the brains of mice and hamsters, animals were anesthetized with an intraperitoneal injection of Ketamine/Xylazine (80mg/100mg/Kg body weight). Mice were perfused transcardially with ice-cold NaCl 0.9% solution followed by the fixative solution. For wild type C57BL/6J mice fed standard chow or HFD, a solution of 4% paraformaldehyde in borate buffer (sodium tetraborate decahydrate pH 9.5) was used. For NPY-GFP; POMC::Cre; tdTomato mice and hamsters, a fixative solution of PFA 4% in phosphate-buffered saline (PBS; pH 7.4) was used. Dissected brains were post-fixed for 4h in their respective fixative solutions before cryopreservation in sucrose 30% (sucrose in 0.1M phosphate buffered saline pH7.4) for 48h before cryosectioning. Hamsters were decapitated and the harvested brain immersion-fixed for 24h in 4% paraformaldehyde in phosphate buffer.

Immunohistochemistry

For triple-label immunofluorescence experiments, 30 pm-thick floating sections were rinsed 4 times in 0.1 M PBS pH 7.4 and blocked for 1 hour at room temperature in blocking solution (PBS containing 10% normal donkey serum and 0.3% Triton X-100). Sections were incubated overnight at 4°C with a mix of primary antibodies diluted in blocking solution (goat anti ACE2 1:200; rabbit anti TMPRSS2 1:1,000 and mouse anti FPR2 1:200; see table 1). The sections were then washed three times in 0.1M PBS and incubated for 1.5 hours at room temperature with a biotinylated donkey anti-rabbit secondary antibody to amplify the TMPRSS2 signal (1:500). The sections were then washed three times in 0.1MPBS and incubated at room temperature for 1 hour with Alexa Fluor-conjugated secondary antibodies (1 :500 dilution; all purchased from Molecular Probes, Invitrogen, San Diego, CA) in blocking solution. The sections were rinsed 3 times in 0.1 M PBS. Nuclei were then counterstained by incubating the sections for 1 minute in DAPI.

Immunofluorescence labeling — Human tissues

Tissues were obtained in accordance with French bylaws (Good Practice Concerning the Conservation, Transformation and Transportation of Human Tissue to be Used Therapeutically, published on December 29, 1998). Permission to use human tissues was obtained from the French Agency for Biomedical Research (Agence de la Biomedecine, Saint- Denis la Plaine, France, protocol no. PFS16-002) and the Lille Neurobiobank.

Post-mortem adult human brains

Dissected blocks of the adult brain (2 men and 1 woman) containing the hypothalamus were fixed by immersion in 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 at 4°C for 2 weeks. The tissues were cryoprotected in 30% sucrose/PBS at 4°C overnight, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen in dry ice and stored at -80°C until sectioning. For human hypothalamus immunolabeling, a citrate-buffer antigen retrieval step, lOmM Citrate in TBS-Triton 0.1% pH 6 for 30 min at 70°C, was performed on 20pm sections. After 3 washes of 5 minutes with TBS-Triton 0.1%, sections were blocked in incubation solution (10% normal donkey serum, lmg/ml BSA in TBS-Triton 0.1% pH 7,4) for 1 hour. Blocking was followed with primary antibody incubation (see table 1) in incubation solution for 48h at 4°C. Primary antibodies were then rinsed out, before incubation in fluorophore- coupled secondary antibodies or, in case of amplified immunolabeling, biotinylated secondary antibodies for lh in TBS-Triton 0.1% at room temperature. For classic immunohistochemistry, secondary antibodies were washed and sections counterstained with DAPI (D9542, Sigma). For amplified immunohistochemistry, after secondary antibodies were rinsed, sections were incubated with VECTASTAIN ^® Elite ABC-HRP kit (PK-6100, Vector laboratories) following manufacturer’s instructions. Sections were then incubated with biotinyl-tyramide reagent (SAT700001EA, Perkin Elmer) following manufacturer’s recommendations, washed and incubated with fluorophore-coupled streptavidin (1/500 dilution in TBS-Triton 0.1%) before counterstaining with DAPI. Finally, the sections were incubated with Autofluorescence Eliminator Reagent (2160, Millipore) following manufacturer’s instructions and mounted with Fluoromount™ (F4680, Sigma).

Human embryos/fetuses

Embryos and fetuses were fixed by immersion in 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 at 4°C for 2-5 days depending on sample size. The tissues were cryoprotected in 30% sucrose/PBS at 4°C overnight, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen in dry ice and stored at -80°C until sectioning. For immunolabelling, 20 pm-thick sections of entire heads at GW 11 and GW 14 were processed as follows. Slides first underwent antigen retrieval for 20 minutes in a 5mM citrate buffer heated to 90°C, then were rinsed in TBS and blocked/permeabilized for 2 hours at room temperature in TBS + 0.3% Triton + 0.25% BSA + 5% Normal Donkey Serum (“Incubation solution”, ICS). Sections were then incubated with primary antibodies for two nights at 4°C in ICS. After rinses in TBS, the sections were incubated with secondary antibodies for two hours at RT in ICS, then rinsed again in TBS. Finally, nuclei were stained with DAPI (Sigma D9542, 1:5000 in TBS) for 5 minutes, and sections were rinsed before coverslipping with homemade Mowiol. Table 1: Antibody table

Fluorescence-activated cell sorting and real-time quantitative PCR

Tat-cre infusion

A tat-cre fusion protein produced as detailed previously (60) was stereotaxically infused into the third ventricle (6.37 pg/2 pi at a rate of 0.2pl/ml; AP: -1.7 mm, ML: 0 mm DV: - 5.6 mm) of five isoflurane-anesthetized 4 month-old male C57B1/6 tdTomato ^l0xP/+ reporter mice 2 weeks before experiments, as described before (61).

Sorting Median eminence explants were microdissected from tdTomato ^{Tat Cre} mice, and cell dissociated using the Papain Dissociation System (Worthington Biochemical Corporation). Fresh dissociated cells were sorted by Fluorescence-activated cell sorting (FACS) in an BD FACSAria™ III sorter. Quantitative RT-PCR analyses

For gene expression analyses, lysates of FACS-sorted tanycytes and non-tanycytes were subjected to DNAse treatment (DNasel EN0521, ThermoFisher) and then reverse transcribed using MultiScribe™ Reverse Transcriptase and the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). A preamplification step was performed (TaqMan ^tm PreAmp Master Mix Kit 4488593, Applied Biosystems) before real-time PCR. Real-time PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using TaqMan® Gene Expression Assays listed below. Real-time PCR analysis were performed using the 2 ^~AACt method using as an internal/positive control a sample of 50pg of total RNA obtained from the mediobasal hypothalamus as extensively described previously (62).

Table 2 : RTqPCR primer table

Results

ACE2 and TMPRSS2 are expressed in the human hypothalamus and connected brain regions

The AHBA, comprising 3702 anatomical brain regions from six donors (five males and one female, mean age 42, range 24-57 years), contains microarray gene expression data for >62,000 gene probes, including ACE2 and TMPRSS2. We thus used the atlas to first investigate the occurrence of ACE2 and TMPRSS2 in a number of hypothalamic nuclei and connected regions involved in regulating olfaction, gustation or cardiorespiratory function: the insula, the amygdala, the paraventricular nucleus of the thalamus, the pons and the myelencephalon (Data not shown). The frontal lobe and cerebellar cortex as well as the choroid plexus were included for comparison. The olfactory bulbs and some brainstem regions are unfortunately not represented in the AHBA. We selected probes based on their maximum variance across all samples. Using normalized log2 intensity values to calculate the median expression level and nonparametric missing value imputation for regions where the number or quality of the samples was inadequate (see Methods), we observed that the expression levels of the genes varied widely across and within the brain regions studied (Data not shown and table 3). Of note, as expected from previous studies (63) (He et al, bioRxiv, 2020; https://doi.org/10.1101/2020.05.l l.088500), ACE2 expression levels in the cerebral cortex (frontal lobe, insula) and cerebellar cortex were relatively low, while the paraventricular nucleus of the hypothalamus (PVH) displayed the highest ACE2 levels among hypothalamic nuclei, in keeping with its role in fluid homeostasis through the renin-angiotensin system (64). Surprisingly, the choroid plexus displayed extremely high ACE2 levels. TMPRSS2 was present in all the areas studied, and its levels were, on the whole, higher than those of ACE2. Relatively high levels of both ACE2 and TMPRSS2 were found in a number of regions, including the PVH and several areas of the brainstem, indicating that they are potential targets of SARS- CoV-2.

Genes correlated with ACE2/TMPRSS2 and SARS-CoV-2 infection in key brain structures

We next proceeded to identify genes whose expression was positively or negatively correlated with the selected ACE2 or TMPRSS2 probes, using their normalized log2intensity gene expression values and the Find Correlates option in the AHBA, for 5 regions of interest - the insula, the amygdala, the hypothalamus, the parabrachial nucleus in the pons, and the myelencephalon (Data not shown). Probes with zero entrez-id were eliminated, and if multiple probes represented a single gene, the one with the lowest adjusted p value was retained. The paraventricular thalamic nucleus was sampled in only three brains in the AHBA, and had to be excluded following analysis of Pearson's correlation coefficients. The pontine tegmentum and basal pons were also eliminated from further analysis because the diversity and number of regions yielded results that were hard to interpret. However, the parabrachial nucleus of the pons, which was comparable to the diencephalic and telencephalic regions analyzed and key to the functions studied here, was retained. Out of several thousand genes whose expression was found to be correlated with ACE2 or TMRPSS2 in the 5 regions (Data not shown), we identified 2786 with a Pearson's coefficient (r value) between -0.3 and 0.3, a p value of < 0.01 and a false discovery rate (FDR) < 0.25 for ACE2, and 5369 for TMPRSS2, in the hypothalamus alone (Data not shown). Of these, 1985 were correlated with both ACE2 and TMPRSS2, making them possible candidates of interest for infectious mechanisms or the host cell response. According to the GSEA User Guide, an FDR of <0.25 is reasonable in an exploratory setting, where the aim is to find candidate hypotheses, and where a more stringent FDR cutoff may lead to potentially significant results being overlooked because of the lack of coherence in most expression datasets (see also (65). In order to draw parallels between SARS- CoV-2 infection of the respiratory epithelium and a putative brain infection, we further cross checked our genes against those found to be differentially expressed in the lungs of COVID-19 patients as compared to healthy individuals (52). A total of 140 genes with a variety of known functions were both correlated with ACE2 and TMPRSS2 expression in the hypothalamus according to the AHBA and differentially expressed in COVID-19 patients (Data not shown). The expression of a further 62 and 329 genes, respectively, was correlated with only ACE2 or only TMPRSS2.

ACE2/TMPRSS2-correlated genes are involved in functional networks of interest

To obtain an idea of the possible mechanisms at play if certain ACE2- or TMPRSS2- correlated genes were to be involved in SARS-CoV-2 pathogenesis, we then performed enrichment analyses on these genes (-0.3 < r < 0.3; fdr < 0.25) for KEGG pathways using the Clusterprofiler package in R. The KEGG pathway database, which is manually curated, can be used to identify genes that are known to be implicated in a number of molecular processes and networks in the following categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, human diseases and drug development. Pathway enrichment and selection yielded a number of important functions and interactions among the genes correlated with ACE2 and/or TMPRSS for each brain region analyzed (Data not shown). With regard to ACE2-correlated genes (Data not shown), gene enrichment in the myelencephalon and pons revealed a very large number of KEGG pathways with a small number of significantly correlated genes, even with a stringent q value of < 0.05, perhaps due to the diverse peripheral inputs they integrate. In contrast, the insula yielded 2 enriched pathways for ACE2-correlated genes, and the amygdala 4. The pathways with the most correlated genes were both localized in the hypothalamus, and were for "olfactory transduction" (due to a very large number of OR olfactory receptor genes) and "neuroactive ligand-receptor interaction", in keeping with the variety of neuropeptides and neurotransmitters produced by hypothalamic neural networks.

KEGG pathway enrichment of TMPRSS2-correlated genes (Data not shown) revealed very few pathways at the q value cutoff used for ACE2. We therefore raised the cutoff to q < 0.25, as used in certain studies (the point of the exercise being to identify likely patterns rather than any single significantly correlated pathway). In contrast to ACE2-correlated genes, under these conditions, pathway enrichment for TMPRSS2-correlated genes revealed a lower number of pathways in the hypothalamus, myelencephalon and pons, but a higher number in the insula and amygdala. Here too, the largest pathways were for "neuroactive ligand-receptor interaction" and "olfactory transduction" in the hypothalamus, suggesting that these two pathways could be of unusual importance when taken together with ACE2 and TMPRSS2, and providing some support for our focus on anosmia and neuroendocrine function in SARS-CoV-2 pathogenesis. In addition to these, other common enriched pathways between ACE2- and TMPRSS2- correlated genes were those for "taste transduction", "cAMP signaling", "Cushing syndrome" and "hematopoietic cell lineage". However the latter two were not enriched in the hypothalamus with regard to TMPRSS2-correlated genes. While this is not in itself an indication that genes involved in these networks do not play important roles in SARS-CoV-2 susceptibility or pathogenesis, we chose not to analyze them further in order to keep our study manageable.

We additionally performed enrichment for gene ontology (GO) terms, which are also manually curated and classify genes in identified pathways among the following categories: biological processes, cellular components and molecular function. We limited our analysis to GO terms for "biological processes" , as the number of overlapping terms for individual genes in the other two categories made meaningful interpretation difficult (Data not shown).

Finally, we performed similar pathway enrichment for the genes identified as being differentially expressed in COVID-19 patient lungs (52) (Data not shown). However, in accordance with the differences to be expected between the lung epithelium and the brain, we found few pathways in common, even though this does not rule out the involvement of individual genes as part of a different pathway or pathways depending on the type of tissue analyzed.

Functionally connected brain regions express common ACE2- and TMPRSS2- correlated genes and pathways

While, pathway enrichment allows likely functional links to be pinpointed by identifying groups of genes that are jointly correlated, the relationship between pathways and individual genes that might connect them or that are similarly correlated in several functionally linked areas could also be informative from the point of view of the pathophysiological mechanisms involved. We therefore built gene networks based on the pathways identified above for ACE2- and TMPRSS2-correlated genes in each of the 5 regions (Data not shown). Interestingly, since the gene sets comprising each pathway are manually curated, this exercise allowed us to identify certain genes that we believed to be of interest for more than one function or pathway even though they did not appear in that pathway in the enrichment analysis. Next, we analyzed the four pathways identified above that were correlated with both ACE2 and TMPRSS2 in the hypothalamus as well as other connected regions: "neuroactive ligand- receptor Data not shown) and "cAMP signaling" (Data not shown). For each of these networks, a number of genes that were correlated with ACE2 and/or TMPRSS2 were also expressed as part of the same or linked pathways across multiple brain areas, suggesting that they could be involved in common physiological functions or signaling mechanisms involving ACE2 or TMPRSS2 in these areas. In addition, both among these common genes and genes that were only present in a single region, we found several whose expression was also altered by SARS-CoV-2 infection in the lung, suggesting that regardless of any tissue-specific functional pathways identified, these genes could be involved in susceptibility or response to the virus. Table 3 highlights important genes along with what is known of their function or potential role in SARS-CoV-2 pathogenesis. Intriguingly, this list included the formyl peptide receptor FPR2 (also known as ALX or FPRLl), a molecule that is closely related to vomeronasal receptors (66) but that detects peptides of pathogenic or mitochondrial origin and has either inflammation-resolving or pro-inflammatory actions (53).

FPR2 is differentially expressed in hypothalamic nuclei and correlated with a large number of genes and pathways implicated in viral pathogenesis or the host response

Using similar methods as for ACE2 and TMPRSS2, we found that FPR2 was expressed in the hypothalamus at varying levels depending on the nucleus. Furthermore, gene expression analysis revealed 60 genes in the AHBA whose expression was correlated with FPR2; most of these correlations were negative. We then performed enrichment analysis for these genes using KEGG pathways and GO terms, and came up with 60 KEGG pathways, 114 biological processes, 24 cellular components and 10 molecular functions. Moreover STRING analysis revealed an interactome of 10 other genes, of which most were not found to be specifically correlated with FPR2 in the hypothalamus. However, ANXA1 was found to be negatively correlated with FPR2 and is widely known to be a ligand for FPR2. CLEC4D was found to be positively correlated with FPR2 and involved in a number of functions of interest to risk factors mediated by the hypothalamus as well as infection.

ACE 2, TMPRSS2 and FPR2 are expressed in the mouse and hamster hypothalamus and upregulated by high-fat diet

In order to validate our gene expression data in terms of protein expression and localization, we performed immunofluorescence labeling with available antibodies for ACE2, TMPRSS2 and FPR2 in brain sections from male mice and hamsters, a model that is susceptible to SARS-CoV-2 infection even without genetic modification. Out of curiosity with regard to the increased risk of SARS-CoV-2 infection in certain individuals, we used mice given either a standard or a high-fat diet (HFD). In the brain of mice fed normally, immunofluorescence using an antibody to mouse ACE2 in most parts of the brain appeared to be limited to cells of the capillary walls - pericytes or endothelial cells (Data not shown) - as previously described (63) (He et al., bioRxiv, 2020; https://doi.Org/10.l 101/2020.05.11.088500). However, in the choroid plexus, where ACE2 levels were among the highest seen in our gene expression analysis of human brains (Data not shown), ACE2 immunofluorescence appeared to be present as an apical cap on these cells (Data not shown). In contrast, in the PVH of mice with a normal diet (Data not shown), ACE2 appeared to be present in scattered neurons in addition to vascular cells, as could be expected of a nucleus involved in the angiotensin system. Intriguingly, however, in the median eminence (ME), a circumventricular organ (CVO) at which the traditional blood-brain barrier has been replaced by a fenestrated endothelium and a barrier consisting of tanycytes, specialized hypothalamic ependymoglial cells, it was these tanycytes, in addition to vascular cells, that showed the most labeling (Data not shown). Both the cell bodies of b-tanycytes at the wall of the third ventricle in the ME and the adjacent arcuate nucleus of the hypothalamus (ARH) and their processes extending towards the external capillary bed were labeled for ACE2. TMPRSS2 labeling was also found predominantly in tanycytic cell bodies and processes in the ARH and ME, but was found in astrocyte-like cells at the ventricular wall adjacent to the PVH and some microglia-like cells in the PVH parenchyma (Data not shown). Surprisingly, FPR2, which we identified in several pathways and brain regions above as being correlated with ACE2 and/or TMPRSS2 in addition to being differentially expressed in COVID-19 patient lungs, was found to be strongly expressed in cells with an astrocytic morphology along the ventricular wall at the PVC, where it sometimes colocalized with TMPRSS2, and in both tanycytic cell bodies and processes along the ventricle and microglia-like cells in the ARH and ME (Data not shown) (52). Given the diversity of these results, we next used fluorescence-associated cell sorting (FACS) to sort cells from the mediobasal hypothalamus of mice expressing tdTomato selectively in tanycytes (61) (Data not shown). In the resulting Tomato-positive and Tomato negative cells, we performed RTqPCR for ACE2, TMPRSS2, FPR2 and the tanycytic marker vimentin (bottom panel) to confirm that Tomato-positive and -negative sorted cells were tanycytes and non-tanycytes respectively. While ACE2 was present in both populations as indicated by immunolabeling experiments (Data not shown), TMPRSS2 was expressed almost exclusively in tanycytes in this region and FPR2 was expressed in both tanycytes and non-tanycytic cells.

Next, we confirmed the presence of ACE2 in hypothalamic tanycytes in hamsters, which have higher homology to human ACE2 than mice and are susceptible to SARS-CoV-2 infection without genetic modification. Using an antibody to human ACE2, we found that in the hypothalamus of hamsters, unlike mice, a number of b-tanycytes and a few a-tanycytes, further up along the wall of the 3rd ventricle, expressed abundant ACE2 from their cell bodies and processes (Data not shown). In addition, the same tanycytes also expressed TMPRSS2 (Data not shown).

Finally, we examined the same three markers in obese mice given a HFD for 9 weeks (Data not shown). Strikingly, there was a strong upregulation of ACE2 in vascular cells in the ARH, while at the same time, ACE2 immunolabeling was reduced in the cell bodies of b- tanycytes and completely abolished in their processes (Data not shown). However, apical labeling like that seen in the choroid plexus appeared at the level of the a-tanycytes. ACE2 also increased in the PVH with the HFD, but the pattern and cell types labeled did not change (Data not shown). TMPRSS2 also increased with HFD in both the microglia-like cells of the PVH and the cell bodies of b-tanycytes in the ARH/ME, but to a less remarkable extent (Data not shown). On the other hand, FPR2 immunolabeling in HFD mice showed a dramatic increase in the number and spatial extent of astrocyte-like cells along the ventricular wall and in the PVH parenchyma, as well as in tanycytic cell bodies lining the ventricle and processes reaching the external capillary bed in the ARH and ME, as well as the microglia-like labeling in the latter, in keeping with microglial activation during an inflammatory state (Data not shown) (67). ACE2, TMPRSS2 and FPR2 could therefore form part of a pro-inflammatory pathway by which obesity or other metabolic disorders could alter hypothalamic neural circuits regulating energy balance to increase their susceptibility to SARS-CoV-2 infection or its consequences in terms of patient outcome. ACE 2 and TMPRSS2 are present in olfactory neurons and hypothalamic neurons and tanycytes in humans

To confirm the relevance of our observations to human patients, we performed immunolabeling for the same markers in sections of a control adult human brain and an embryonic head. In the adult brain, the choroid plexus was strongly positive for both ACE2 and TMPRSS2 (Data not shown). In the PVH parenchyma, ACE2 was present in a few capillary walls, as well as cells with a neuronal morphology, in which it was colocalized with FPR2 (Figure 1A). Some FPR2-positive fibers or cell bodies, possibly glial, were visible close to the ventricular wall, but did not appear to be colocalized with vimentin-positive fibers (Figure IB). Lower down the ventricular wall, at the level of the ARH and ME, light ACE2 labeling was seen in a few tanycytic processes radiating from the ventricular wall, colocalized with TMPRSS2. Some capillary walls were also positive for ACE2, as were some cells with a neuronal morphology (Data not shown). TMPRSS2 was strongly colocalized with vimentin- positive tanycytic processes in the ARH and ME at both the ventricular and pial surfaces (Data not shown), which combined with the presence of ACE2 in tanycytes or adjacent cells, even at low levels in the absence of risk factors, may render them susceptible to SARS-CoV-2 infection through either a hematogenous or a CSF route.

In order to investigate the olfactory route, given the absence of olfactory bulb data in the AHBA, we also performed ACE2 and TMPRSS2 labeling in whole head sections of human embryos at gestational week (GW) 11 (Data not shown) or 14 (Data not shown). Both ACE2 (Data not shown) and TMPRSS2 (Data not shown) were strongly colocalized in the olfactory bulb and nerve of the GW11 embryo with TAG-1 (or contactin 2, CNTN2), a marker for olfactory and vomeronasal neurons. At GW14, when the neuronal layer of the olfactory epithelium and the embryonic vomeronasal organ were further developed, strong immunolabeling for both ACE2 and TMPRSS2 was present in the neuronal layer of both epithelia, in addition to the olfactory nerve (Data not shown). FPR2 did not appear to be present in these sections, and is not known to occur in the nose.

SARS-CoV-2 attaches to and infects olfactory sensory neurons and hypothalamic median eminence cells in the COVID-19 patient brain

We obtained the brain of a 63 -year-old obese male patient who tested positive for SARS- CoV-2 at hospitalization, and who died after 33 days in the ICU after severe respiratory distress, complications and multi-organ failure. We observed strong ACE2 labeling around blood vessels, and both ACE2 and TMPRSS2 labeling in the outer layer of the patient's OB (Figure 2A) but not in a control brain from a 36 year-old overweight male patient negative for SARS- CoV-2 (Figure 2A). Immunolabeling for olfactory marker protein (OMP) confirmed that this layer (ONL) is where the olfactory nerve, consisting of fibers from olfactory sensory neurons, enters the OB (Data not shown). Interestingly, these fibers were also highly immunopositive for the SARS-CoV-2 S-protein (Figure 2A), and immunolabeling revealed dsRNA from replicating viruses in numerous cells bordering the ONL and within the glomerular layer (Data not shown), indicating viral entry into the brain through olfactory sensory neuronal fibers from the nose. FPR2 was also found at high levels in the ONL in the patient but not the control brain (Figure 2B), suggesting that SARS-CoV-2 infection itself increases the expression of ACE2, TMPRSS2 and FPR2 in olfactory sensory neurons, potentiating infection.

ACE2 was strongly expressed in the choroid plexus of the COVID-19 patient, where we also found strong labeling for the highly conserved SARS-CoV nucleocapsid N-protein in what appeared to be blood vessels (Data not shown).

In the ME/ARH of the COVID-19 patient hypothalamus, abundant labeling for both N- protein and dsRNA could be observed in a large number of cells (Data not shown), indicating robust viral infection and replication. Neither viral marker was seen in the hypothalamus of controls (Data not shown). However, while diffuse N-protein labeling in the COVID-19 patient was colocalized with vimentin-positive tanycytic fibers in many cases, dsRNA was not present in the cell bodies of tanycytes, which line the wall of the third ventricle (Data not shown). Some parenchymal cells showed high levels of both markers with N-protein aggregation, perhaps indicative of viral assembly. Interestingly, in the ME/ARH, S-protein, present on the outer envelope of assembled viruses, was observed at extremely high levels in the endfeet of vimentin-positive tanycytes at the pial surface of the ME (Figure 2D), where it was colocalized both with ACE2 (Figure 2D) and TMPRSS2 (Data not shown), suggesting that SARS-CoV-2 from the circulation was being internalized by tanycytic endfeet at the level of the fenestrated capillaries, but subsequently transferred to other cell types. In the ventromedial hypothalamus (VMH), however, S-protein was also found in numerous fibers and capillaries close to the ventricular wall, most of which also expressed ACE2; these included some vimentin-positive tanycytes that extended processes into the parenchyma (Data not shown). In addition, some vimentin-negative cells with a neuron-like morphology in the parenchyma of the VMH (Data not shown) and cells with an astrocytic morphology in the lateral hypothalamic area (LHA) (Data not shown) also expressed both ACE2 and S-protein. The widespread and strong expression of ACE2 in the hypothalamus of the SARS-CoV-2 infected patient, unlike the weak expression seen in the control brain, was reminiscent of the increase in ACE2 and TMPRSS2 seen in the ONL of the patient. As for FPR2, at the level of the ME/ARH, it was present both in some vimentin- and TMPRSS2-coexpressing tanycytic processes and in non-vimentin-labeled glial cells that could be microglia, close to the ventricular wall (Figure 2C). More dorsally, it was strongly expressed and colocalized with TMPRSS2 in some cells with an astrocytic morphology in the patient's PVH (Data not shown).

ACE 2, TMPRSS2 and FPR2 are expressed in the hamster and mouse hypothalamus and are upregulated by high-fat diet and ovariectomy

In order to validate our gene expression data in terms of protein expression and localization, we next performed immunofluorescence labeling for ACE2, TMPRSS2 and FPR2 in brain sections from male hamsters, a model that is susceptible to SARS-CoV-2 infection even without genetic modification due to their higher homology with human ACE2 than mice (68). Using the same antibody to human ACE2 that we used in the human brain sections, we found clear confirmation of the presence of ACE2 in the processes of a large number of vimentin-positive tanycytes spanning the hypothalamic parenchyma from the wall of the third ventricle, where their cell bodies are located, to the external surface, where their endfeet are in contact with the capillary bed. In addition to the numerous ME/ARH tanycytes, a few dorsal tanycytes, further up along the wall of the 3rd ventricle, also expressed abundant ACE2 from their cell bodies and processes (Data not shown). In addition, the same tanycytes also expressed TMPRSS2 (Data not shown), again underscoring the fact that tanycytes could provide an entry mechanism for the virus into the brain through either the hematogenous or the CSF route.

Despite the usefulness of the hamster as a model for SARS-CoV-2 infection, pathophysiological mechanisms are easier to elucidate and validate in mice due to the wealth of literature. Out of curiosity with regard to the increased risk of SARS-CoV-2 infection in certain individuals, we therefore next evaluated the effect on ACE2, TMPRSS2 and FPR2 of a standard or high-fat diet (HFD), which induces obesity, and the effect of the gonadal steroid estrogen, given the difference in the risk of developing severe COVID-19 in men and women.

In the brain of mice fed normally, we first validated an antibody to mouse ACE2. Immunofluorescence using this antibody in most parts of the adult mouse brain appeared to be limited to cells of the capillary walls - pericytes or endothelial cells (Data not shown) - as previously described (63) (He et ak, bioRxiv, 2020; https://doi.org/10.1101/2020.05.ll.088500). However, in the choroid plexus, where ACE2 levels were among the highest seen in our gene expression analysis of human brains (Data not shown), ACE2 immunofluorescence appeared to be present as an apical cap on these cells (Data not shown), confirming our observations in the human cortex and choroid plexus. In contrast, in the PVH of male mice with a normal diet (Figure 1C, top left panel), ACE2 appeared to be present in scattered neurons in addition to vascular cells, as seen in the human brain with regard to this nucleus involved in the renin-angiotensin system. Intriguingly, however, in the ME of male (Figure 1C, bottom left panel) and female mice (Figure 1C, left panel), tanycytes, in addition to vascular cells, showed the most labeling. Both the cell bodies of tanycytes at the wall of the third ventricle in the ME and the adjacent ARH, and their processes extending towards the external capillary bed were labeled for ACE2, as was the pars tuberalis, a highly vascularized zone under the ME. TMPRSS2 labeling was also found predominantly in tanycytic cell bodies and processes in the ARH and ME, but was found in astrocyte-like cells at the ventricular wall adjacent to the PVH and some microglia-like cells in the PVH parenchyma (Data not shown). Surprisingly, FPR2 was found to be strongly expressed in cells with an astrocytic morphology along the ventricular wall at the PVH, where it sometimes colocalized with TMPRSS2, and in both tanycytic cell bodies and processes along the ventricle and microglia-like cells in the ARH and ME (Data not shown), where two neuronal populations, expressing NPY and POMC, that control energy metabolism reside (52).

Next, we examined the same three markers in male mice given a HFD for 9 weeks to induce obesity (Figure 1C, right panels) as well as in ovariectomized (OVX) female mice (Figure ID, right panel). Strikingly, in the male HFD mice there was a strong upregulation of ACE2 in vascular cells in the ARH, while at the same time, ACE2 immunolabeling was reduced in the cell bodies of ME/ ARH tanycytes and completely abolished in their processes (Data not shown). However, apical labeling like that seen in the choroid plexus appeared at the level of the tanycytes in the dorsal ARH, while the pars tuberalis was less strongly labeled (Data not shown). ACE2 also increased in the PVH with the HFD, but the pattern and cell types labeled did not change (Data not shown). TMPRSS2 also increased with HFD in both the microglia like cells of the PVH and the cell bodies of tanycytes in the ME/ ARH, but to a less remarkable extent (Data not shown). Importantly OVX female mice displayed the same changes in ACE2 and TMPRSS2 immunolabeling as male HFD mice (Data not shown), i.e. ACE2, present in ME/ARH tanycytic cell bodies and processes under normal conditions, disappeared in OVX females to be replaced by increased labeling at the apical poles of more dorsal tanycytes and vascular cells. Labeling for TMPRSS2 similarly mimicked the changes seen in HFD. On the other hand, FPR2 immunolabeling in HFD mice showed a dramatic increase in the number and spatial extent of astrocyte-like cells along the ventricular wall and in the PVH parenchyma, as well as in tanycytic cell bodies lining the ventricle and processes reaching the external capillary bed in the ARH and ME; in addition, there was an increase in the microglia-like labeling in the latter, in keeping with microglial activation during an inflammatory state (Figure 1C, right panels) (67). However, these observations regarding FPR2 were not evident in OVX females (Figure ID, right panel).

Finally, given the diversity of these results, we next used fluorescence-associated cell sorting (FACS) to sort cells from the median eminence of mice expressing tdTomato selectively in tanycytes (61) (Figure IE, left panel). In the resulting Tomato-positive and Tomato negative cells, we performed RTqPCR for ACE2, TMPRSS2, FPR2 and the tanycytic marker vimentin (bottom panel) to confirm that Tomato-positive and -negative sorted cells were tanycytes and non-tanycytes respectively. While ACE2 was present in both populations as indicated by immunolabeling experiments (Data not shown), TMPRSS2 was expressed almost exclusively in tanycytes in this region (Data not shown) and FPR2 was expressed in both tanycytes and non-tanycytic cells (Figure IE, right panel). These results strongly suggest that altered ACE2 and TMPRSS2 expression could form part of an inflammatory mechanism in hypothalamic circuits through which risk factors such as metabolic diseases and sex hormones could increase susceptibility to and aggravate the outcome of SARS-CoV-2; FPR2 could play a role in this process.

Table 3: Enriched genes common to multiple regions and the COVID-19 lung dataset. The gene sets identified by our enrichment for the 4 KEGG pathways of interest were cross-checked against the geneset obtained from the COVID-19 lung (49), and the common genes annotated below. Discussion

Although viral pneumonia is still the principal symptom in severely ill patients and a large number of complications affecting a variety of organs and physiological processes stem from two accompanying phenomena - the "cytokine storm" and a prothrombotic state (42, 43, 69-71), we consider the possibility that SARS-CoV-2 infiltrates the brain, and specifically the hypothalamus, with functional consequences to disease progression and outcome.

The idea that SARS-CoV-2 could infect the brain, in particular through an olfactory route, has been proposed by other authors (9, 73, 74), both in light of the observation of anosmia in COVID-19 patients and from experimental studies on SARS-CoV, which uses the same receptor and protease as SARS-CoV-2. Additionally, a recent MRI study has revealed changes to the OBs of a patient with anosmia (75), although this could be secondary to changes in the epithelium rather than due to viral invasion. While it has been proposed that it is the sustentacular cells of the OE or secretory cells that express ACE2 and are infected by SARS- CoV-2 and not olfactory sensory neurons themselves (76), in our study, at least in the human embryo, olfactory and vomeronasal sensory neurons do appear to express both ACE2 and TMPRSS2 at high levels. It should be remembered that the ON and vomeronasal nerve also serve as a scaffold to guide gonadotropin-releasing hormone (GnRH) neurons from the nose to the hypothalamus during development (79), and that these GnRH neurons persist as a continuum along this route into adulthood, firmly and directly connecting the olfactory route to the hypothalamus.

Other routes for brain viral infection have also been proposed, notably through other peripheral nerves from sensory or visceral organs that are important targets of infection, or a hematogenous route. A close look at the literature reveals that the virus is not only detected in the blood by PCR in a variable percentage of infected individuals, but that the chances of detecting it are higher in severely ill patients (see for example (82)). Once in the bloodstream, the virus could access the brain through a number of routes, for example, through the leaky endothelial barrier, given the expression of ACE2 by pericytes. The virus could also enter the brain through the fenestrated capillaries of the CVOs, which play key roles in either the risk factors or the physiological functions targeted by SARS-CoV-2. Indeed, our immunolabeling experiments in both animals and humans show that ACE2, while low, is present in ME and ARH tanycytes, whose endfeet contact these fenestrations, in addition to vessels themselves. In addition, viral particles could enter the CSF through the choroid plexus, which we show to be rich in both ACE2 and TMPRSS2, and thereby access other brain regions, including hypothalamic nuclei bordering the third ventricle, where we once more observe ACE2-positive tanycytic cell bodies. Finally, as can be seen by immunolabeling in the hypothalamus of HFD- fed or ovariectomized mice, certain risk factors such as obesity or certain gonadal hormones could increase ACE2 levels or alter the pattern of its expression, and therefore the susceptibility to infection in specific cell types or by specific routes.

Among the four most ubiquitous enriched pathways for genes correlated, positively or negatively, with ACE2 or TMPRSS2, we found the pathways for olfactory and taste transduction, as well as neuroactive ligand receptor interaction. Our network analysis and immunolabeling studies also unexpectedly revealed the inflammatory mediator FPR2, a GPCR, as playing a key role in the potential viral infection of the brain through ACE2. While FPR2 can mediate certain anti-inflammatory effects, it appears to be involved in the replication of double-stranded viral RNA, an important step in the propagation of RNA viruses (86, 87). In addition, ACE2 expression was positively correlated with FPR2 expression in our gene expression analysis from the AHBA, and both ACE2 and FPR2 were found to be higher in HFD-fed animals. The deletion of FPR2 in mice alleviates HFD-induced obesity, insulin resistance and other adverse metabolic indicators by suppressing pro-inflammatory mechanisms in the periphery (93), suggesting that it could potentially exacerbate the effects of SARS-CoV-2 infection in patients with metabolic diseases. Additionally, the changes we observed in ACE2 and FPR2 expression in the hypothalamus, and especially in the ME/ARH, of ovariectomized mice were strikingly similar to those in HFD mice, suggesting that the lack of estrogens in old women or sex differences between men and women, or other conditions in which sex steroid levels differ, trigger the same molecular mechanisms as metabolic imbalances. ACE2 and TMPRSS2 are also themselves responsive to gonadal hormones - estrogens and androgens respectively (95, 96). Indeed protein-protein interaction networks build with some of the molecules found to be correlated with ACE2, TMPRSS2 or FPR2 reveal several other networks that are involved both in the normal physiological and neuroendocrine functions of the hypothalamus and in viral susceptibility or pathogenicity. This is particularly the case with FPR2, which has previously been noted in hypothalamic microglia (97), and which, in addition to being able to bind viral peptides (see for example (87, 98)), could play a role in neurodegenerative disorders, and mediate either pro-inflammatory or anti-inflammatory mechanisms depending on the cellular and molecular pathways used (reviewed in (99-101)).

To summarize, our work provides proof of concept that the brain not only possesses the cellular and molecular machinery necessary to be infected, but that the hypothalamus, which harbors neural circuits regulating a number of risk factors for severe COVID-19 in addition to being linked to brainstem cardiorespiratory centers, expresses the viral receptor ACE2 and could be a preferred port of entry and target for the virus, and that FPR2 could play a role in the host response to the viral infection in the brain.

EXAMPLE 2

Aim

We have previously determined that the hypothalamus is a port of entry for SARS-CoV- 2 into the brain, and identified FPR2 (ALX/FPRL1) as a molecule of interest in determining viral susceptibility and pathogenesis using gene expression and correlation analyses and immunolabeling in the post-mortem brain of Covid-19 patients (Nampoothiri et al., 2020; https://doi.org/10.1101/2020.06.08.139329).

Here using animal models of viral infection, we verified whether FPR2 expression could be increased by SARS-CoV-2 infection, either as part of the viral propagation mechanism or as a host defense, and therefore whether FPR2 antagonists or agonists would reduce brain infection.

Methods

Animals

Adult male K18-hACE2 transgenic mice (JaxMice No. 034680) expressing human ACE2 were housed in pathogen- free conditions at 21-22°C with a 12h light/dark cycle and access to food and water ad libitum throughout the experiment. Viral and pseudoviral infection experiments were performed within facilities at the appropriate biosafety level, and complied with current national and institutional regulations and ethical guidelines. The protocols were approved by the institutional ethics committee.

Drug treatment

Eight K18-hACE2 mice, 6-8 months old, were injected intraperitoneally twice daily with the FPR antagonist Boc-FLFLF (n=2; Boc-2; Bachem) dissolved in PBS, or once daily subcutaneously with the FPR2 agonist BMS-986235 (n=3; LAR-1219; MedChemExpress) dissolved in sesame oil. Mice were administered BMS-986235 or Boc-FLFLF at a dose of 4mg/kg for two days before the infection and given one extra dose on the day after the infection. Control mice were injected subcutaneously with the vehicle, sesame oil (VH) alone (n=2). SARS-CoV-2 infection

The BetaCoV/France/IDF0372/2020 strain of SARS-CoV-2 was supplied by the French National Reference Center for Respiratory Viruses hosted by the Institut Pasteur (Paris, France). Mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and infected intranasally with 50 pi of DMEM containing 104 PFU of SARS-CoV-2 (n=5) or vehicle (n=3). After 7 days, animals were euthanized by intraperitoneal injection of pentobarbital (140 mg/kg), decapitated, brains removed and immersion fixed in 4% paraformaldehyde for 24h before being transferred to PBS.

Detection of Sars-CoV-2 spike (S) protein, nucleocapsid (N) protein and FPR2 immunoreactivity in brain tissue

Mouse brains immersion fixed for 24h were cryoprotected in 30% sucrose/PBS at 4°C overnight, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen in dry ice and stored at -80°C until sectioning. 30 pm-thick floating sections were rinsed 4 times in 0.1 M PBS pH 7.4 and blocked for 1 hour at room temperature in blocking solution (PBS containing 10% normal donkey serum and 0.3% Triton X-100). Sections were incubated overnight at 4°C with one or more primary antibodies diluted in blocking solution: rabbit anti-SARS nucleocapsid protein (Novus Bio; Catalog # NB 100-56576; 1:100) or rabbit anti-SARS-CoV-

2 spike protein (Sino Biologicals; Catalog # 40150-T62-COV2; 1:200), and mouse anti-FPR2 monoclonal (Invitrogen, clone GM1D6, Catalog # MAI-17769; 1:200). The sections were then washed three times in 0.1M PBS and incubated at room temperature for 1 hour with Alexa Fluor-conjugated goat anti-rabbit secondary antibodies (1:500 dilution; all purchased from Molecular Probes, Invitrogen, San Diego, CA) in blocking solution. The sections were rinsed

3 times in 0.1 M PBS before visualization.

Quantitative RT-PCR for FPR2

Total RNA was extracted from cortex of mock and SARS-CoV-2-infected mouse brains with the ReliaPrep FFPE Total RNA Miniprep System (Promega) following kit instructions, and quantified using a NanoDrop spectrophotometer (ThermoScientific). Extracted RNA (10.2 ng) was reverse transcribed using a High capacity cDNA Reverse transcription kit (Applied Biosystems, Ref N°4368814) after DNAse treatment (DNase I, Amplification Grade, Invitrogen, Ref N°18068015). Next, real-time PCR was carried out on an Applied Biosystems 7900HT Fast Real-Time PCR System using the TaqMan probes listed below and TaqMan Universal Master Mix II (Applied Biosystems, Ref N°4440049). Gene expression data were analyzed using the 2-AACt method, and expressed relative to RNA for the control housekeeping gene Actin B. An unpaired t-test was used to compare the groups.

TaqMan probes:

Pseudovirus infection

Lentiviral pseudoviruses used for the infection were constructed by Asis Palazon (CIS bioGUNE, Spain) according to the method detailed in Crawford et al, 2020 (https://dx.doi.org/10.3390%2Fvl2050513) using a five plasmid system: lentiviral backbone (CMV promoter to express ZsGreen Fluorescent Protein), the SARS-CoV-2 spike protein, HDM-Hgpm2, pRC-CMV-Revlb and HDM-tatlb. Infected cells emit green fluorescence due to the expression of ZsGreen, allowing their detection by flow cytometry.

For the infection, mice were stereotaxically injected with 300 nL (-105 transduction units/mL) of the pseudovirus at a rate of 0.25nl/min in the arcuate/mediobasal hypothalamus (stereotaxic coordinates anteroposterior -1.3mm, mediolateral -0.4mm from bregma and dorsoventral -6.1mm from skull surface). For injection, mice were deeply anesthetized with isoflurane (3% in 1 L/min air flow) in an induction chamber, and placed in a stereotaxic apparatus equipped with a mask to maintain anesthesia throughout the experiment (isoflurane between 1 to 1.5% in 1 L/min air flow). Core body temperature was maintained at 37°C with a thermostat-controlled electrical blanket.

Detection of infected cells by flow cytometry

Pseudovirus infection was reported by the expression of the ZsGreen Fluorescent Protein. For flow cytometric quantification, mice were decapitated 36h after injection, brains removed, and the mediobasal hypothalamus microdissected and enzymatically dissociated using the Papain Dissociation System (Worthington, Lakewood, NJ) to obtain a single-cell suspension. Flow cytometry was performed using a CytoFLEX LX flow cytometer device (Beckman Coulter). The sort decision was based on the measurement of green fluorescence by comparing cell suspensions from green fluorescence-positive areas and control non-infected areas. For each animal, green positive events were counted from a total of 300000 events.

Results and Discussion SARS-CoV-2 infection dramatically increases Fpr2 expression in the infected mouse brain

Seven days after infection, the brain of K18-hACE2 mice infected intranasally with SARS-CoV-2 revealed the widespread presence of the viral proteins by immunohistochemistry, while viral proteins were completely absent in mock-infected mouse brains.

Immunohistochemistry for FPR2 and viral proteins showed that FPR2 expression was also dramatically increased in the hypothalamus of infected mice and colocalized with viral spike protein (Figures 3 and 4).

Real-time PCR data from the cortex of SARS-CoV-2-infected mice showed a significant 12-fold increase in Fpr2 expression relative to mock-infected animals (Figure 5).

The presence of high levels of Fpr2 mRNA in infected brains suggests that this gene is induced by SARS-CoV-2 infection, either as part of an immune or inflammation-modulating host defense response, or as part of the host cellular machinery diverted by the virus to facilitate its entry and/or propagation.

An FPR2 antagonist reduced brain infection by a SARS-CoV-2 pseudovirus

Given the distinct biological functions of Fpr2 in the brain and immune system, we then tested the effect of an FPR2 agonist (BMS-986235) and an antagonist (Boc-FLFLF) on brain infection with a replication-deficient SARS-CoV-2 lentiviral pseudovirus. Treating K18- hACE2 mice before and after intracerebral infusion of the Spike-protein-expressing pseudovirus with Boc-FLFLF led to a clear decrease in the number of cells expressing the ZsGreen Fluorescent Protein as compared to vehicle-treated mice, while this number increased in mice treated with BMS-986235. Table 4. ZsGFP-positive cells in the mediobasal hypothalamus of mice treated with vehicle, the Fpr2 antagonist Boc-FLFLF or the Fpr2 agonist BMS-986235 before and after infection with a SARS-CoV-2 pseudovirus.

In light of these results, it seems that Fpr2 induced by the viral infection is not acting as part of the host immune or inflammatory response, but rather as a facilitator of SARS-CoV-2 infection.

Conclusion

These initial data indicate that treatment with Fpr2 antagonists could be of prophylactic or therapeutic value in limiting brain cell entry and propagation of SARS-CoV-2 and other viruses, and thus limiting the severe short- and long-term consequences of neuroinvasion by these viruses.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

References

1. L. Mao et al., Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China. JAMA Neurol, (2020).

2. J. Helms et al., Neurologic Features in Severe SARS-CoV-2 Infection. N Engl J Med, (2020).

3. N. Poyiadji et al., COVID-19-associated Acute Hemorrhagic Necrotizing Encephalopathy: CT and MRI Features Radiology, https://doi.org/10.1148/radiol.2020201187 (2020).

4. Y. Wu et al., Nervous system involvement after infection with COVID-19 and other coronaviruses. Brain Behav Immun, (2020).

5. P. F. Wong et al., Lessons of the month 1 : A case of rhombencephalitis as a rare complication of acute COVID-19 infection. Clin Med (Lond), (2020).

6. Y. C. Li, W. Z. Bai, T. Hashikawa, The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients. J Med Virol, (2020).

7. G. Conde Cardona, L. D. Quintana Pajaro, I. D. Quintero Marzola, Y. Ramos Villegas, L. R. Moscote Salazar, Neurotropism of SARS-CoV 2: Mechanisms and manifestations. J Neurol Sci 412, 116824 (2020). 8. F. G. De Felice, F. Tovar-Moll, J. Moll, D. P. Munoz, S. T. Ferreira, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the Central Nervous System. Trends Neurosci, (2020).

9. Q. Cheng, Y. Yang, J. Gao, Infectivity of human coronavirus in the brain. EBioMedicine 56, 102799 (2020).

10. J. R. Lechien et al., Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study. Eur Arch Otorhinolaryngol, (2020).

11. R. Kaye, C. W. D. Chang, K. Kazahaya, J. Brereton, J. C. Denneny, 3rd, COVID-19 Anosmia Reporting Tool: Initial Findings. Otolaryngol Head Neck Surg, 194599820922992 (2020).

12. L. Zhou, M. Zhang, J. Wang, J. Gao, Sars-Cov-2: Underestimated damage to nervous system. Travel Med Infect Dis, 101642 (2020).

13. T. Moriguchi et al., A first case of meningitis/encephalitis associated with SARS-Coronavirus-2. Int J Infect Dis 94, 55-58 (2020).

14. A. Paniz-Mondolfi et al., Central Nervous System Involvement by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2). J Med Virol, (2020).

15. R. G. Wilkerson, J. D. Adler, N. G. Shah, R. Brown, Silent hypoxia: A harbinger of clinical deterioration in patients with COVID-19. Am J Emerg Med, (2020).

16. R. Lu et al., Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 395, 565-574 (2020).

17. P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020).

18. N. Zhu et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med 382, 727-733 (2020).

19. W. Li et al., Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454 (2003).

20. C. G. K. Ziegler et al., SARS-CoV-2 Receptor ACE2 Is an Interferon- Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell 181, 1016-1035 el019 (2020).

21. M. Y. Li, L. Li, Y. Zhang, X. S. Wang, Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infect Dis Poverty 9, 45 (2020). 22. M. F. Doobay et al, Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system. Am J Physiol Regul Integr Comp Physiol 292, R373-381 (2007).

23. K. M. Elased, T. S. Cunha, F. K. Marcondes, M. Morris, Brain angiotensin converting enzymes: role of angiotensin-converting enzyme 2 in processing angiotensin II in mice. Exp Physiol 93, 665-675 (2008).

24. Y. Ding et al, Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol 203, 622-630 (2004).

25. J. Gu et al, Multiple organ infection and the pathogenesis of SARS. J Exp Med 202, 415-424 (2005).

26. J. Xu et al, Detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis. Clin Infect Dis 41, 1089-1096 (2005).

27. L. Wei et al., Endocrine cells of the adenohypophysis in severe acute respiratory syndrome (SARS). Biochem Cell Biol 88, 723-730 (2010).

28. M. K. Leow et al, Hypocortisolism in survivors of severe acute respiratory syndrome (SARS). Clin Endocrinol (Oxf) 63, 197-202 (2005).

29. W. G. Glass, K. Subbarao, B. Murphy, P. M. Murphy, Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 173, 4030-4039 (2004).

30. P. B. McCray, Jr. et al., Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol 81, 813-821 (2007).

31. J. Netland, D. K. Meyerholz, S. Moore, M. Cassell, S. Perlman, Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2. J Virol 82, 7264-7275 (2008).

32. C. T. Tseng et al, Severe acute respiratory syndrome coronavirus infection of mice transgenic for the human Angiotensin-converting enzyme 2 virus receptor. J Virol 81, 1162-1173 (2007).

33. K. S. Kim, R. J. Seeley, D. A. Sandoval, Signalling from the periphery to the brain that regulates energy homeostasis. Nat Rev Neurosci 19, 185-196 (2018).

34. C. Gizowski, C. W. Bourque, The neural basis of homeostatic and anticipatory thirst. Nat Rev Nephrol 14, 11-25 (2018). 35. M. L. Andermann, B. B. Lowell, Toward a Wiring Diagram Understanding of Appetite Control. Neuron 95, 757-778 (2017).

36. I. Fukushi, S. Yokota, Y. Okada, The role of the hypothalamus in modulation of respiration. Respir Physiol Neurobiol 265, 172-179 (2019).

37. T. E. Holy, The Accessory Olfactory System: Innately Specialized or Microcosm of Mammalian Circuitry? Annu Rev Neurosci 41, 501-525 (2018).

38. R. D. Palmiter, The Parabrachial Nucleus: CGRP Neurons Function as a General Alarm. Trends Neurosci 41, 280-293 (2018).

39. A. Mullier, S. G. Bouret, V. Prevot, B. Dehouck, Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain. J Comp Neurol 518, 943-962 (2010).

40. F. Langlet, A. Mullier, S. G. Bouret, V. Prevot, B. Dehouck, Tanycyte-like cells form a blood-cerebrospinal fluid barrier in the circumventricular organs of the mouse brain. J Comp Neurol 521, 3389-3405 (2013).

41. P. Mo et ak, Clinical characteristics of refractory COVID-19 pneumonia in Wuhan, China. Clin Infect Dis, (2020).

42. W. J. Guan et ak, Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med, (2020).

43. C. Huang et ak, Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497-506 (2020).

44. Y. Yang et ak, Epidemiological and clinical features of the 2019 novel coronavirus outbreak in China. medRxiv, doi: https://doi.org/10.1101/2020.1102.1110.20021675 (2020).

45. M. Madjid, P. Safavi-Naeini, S. D. Solomon, O. Vardeny, Potential Effects of Coronaviruses on the Cardiovascular System: A Review. JAMA Cardiol, (2020).

46. R. E. Jordan, P. Adah, K. K. Cheng, Covid-19: risk factors for severe disease and death. BMJ 368, ml 198 (2020).

47. F. Zhou et ak, Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 395, 1054-1062 (2020).

48. J. Yang et ak, Prevalence of comorbidities in the novel Wuhan coronavirus (COVID-19) infection: a systematic review and meta-analysis. Int J Infect Dis, (2020).

49. J. Clasadonte, V. Prevot, The special relationship: glia-neuron interactions in the neuroendocrine hypothalamus. Nat Rev Endocrinol 14, 25-44 (2018). 50. M. Ishii, C. Iadecola, Metabolic and Non-Cognitive Manifestations of Alzheimer's Disease: The Hypothalamus as Both Culprit and Target of Pathology. Cell Metab 22, 761-776 (2015).

51. E. H. Shen, C. C. Overly, A. R. Jones, The Allen Human Brain Atlas: comprehensive gene expression mapping of the human brain. Trends Neurosci 35, 711-714 (2012).

52. D. Blanco-Melo et al., Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. Cell 181, 1036-1045 el039 (2020).

53. E. Weiss, D. Kretschmer, Formyl-Peptide Receptors in Infection, Inflammation, and Cancer. Trends Immunol 39, 815-829 (2018).

54. S. A. Krepel, J. M. Wang, Chemotactic Ligands that Activate G-Protein-Coupled Formylpeptide Receptors. Int J Mol Sci 20, (2019).

55. H. Q. He, R. D. Ye, The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition. Molecules 22, (2017).

56. P. Langfelder, S. Horvath, WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).

57. D. J. Stekhoven, P. Buhlmann, MissForest— non-parametric missing value imputation for mixed-type data. Bioinformatics 28, 112-118 (2012).

58. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014).

59. G. Yu, L. G. Wang, Y. Han, Q. Y. He, clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012).

60. M. Peitz, K. Pfannkuche, K. Rajewsky, F. Edenhofer, Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci U S A 99, 4489-4494 (2002).

61. F. Langlet et al, Tanycytic VEGF-A Boosts Blood-Hypothalamus Barrier Plasticity and Access of Metabolic Signals to the Arcuate Nucleus in Response to Fasting. Cell Metab 17, 607-617 (2013).

62. A. Messina et al., A microRNA switch regulates the rise in hypothalamic GnRH production before puberty. Nat Neurosci 19, 835-844 (2016).

63. I. Hamming et al, Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 203, 631-637 (2004). 64. C. A. Romero, M. Orias, M. R. Weir, Novel RAAS agonists and antagonists: clinical applications and controversies. Nat Rev Endocrinol 11, 242-252 (2015).

65. J. Reimand et al., Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. NatProtoc 14, 482-517 (2019).

66. S. Riviere, L. Challet, D. Fluegge, M. Spehr, I. Rodriguez, Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors. Nature 459, 574-577 (2009).

67. C. Garcia-Caceres et al., Role of astrocytes, microglia, and tanycytes in brain control of systemic metabolism. NatNeurosci 22, 7-14 (2019).

68. J. F. Chan et al., Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis, (2020).

69. D. Wang et al, Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA, (2020).

70. F. A. Klok et al., Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb Res, (2020).

71. J. Helms et al., High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study. Intensive Care Med, (2020).

72. M. M. Lamers et al, SARS-CoV-2 productively infects human gut enterocytes. Science, (2020).

73. Z. Li et al, Neurological manifestations of patients with COVID-19: potential routes of SARS-CoV-2 neuroinvasion from the periphery to the brain. Front Med, (2020).

74. S. Natoli, V. Oliveira, P. Calabresi, L. F. Maia, A. Pisani, Does SARS-Cov-2 invade the brain? Translational lessons from animal models. Eur J Neurol, (2020).

75. L. S. Politi, E. Salsano, M. Grimaldi, Magnetic Resonance Imaging Alteration of the Brain in a Patient With Coronavirus Disease 2019 (COVID-19) and Anosmia. JAMA Neurol, (2020).

76. K. Bilinska, P. Jakubowska, C. S. Von Bartheld, R. Butowt, Expression of the SARS-CoV-2 Entry Proteins, ACE2 and TMPRSS2, in Cells of the Olfactory Epithelium: Identification of Cell Types and Trends with Age. ACS Chem Neurosci, (2020).

77. E. Milanetti et al., In-Silico evidence for two receptors based strategy of SARS- CoV-2. arXiv, arXiv:2003.11107 (2020).

78. C. J. Sigrist, A. Bridge, P. Le Mercier, A potential role for integrins in host cell entry by SARS-CoV-2. Antiviral Res 177, 104759 (2020). 79. F. Casoni et al., Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains. Development 143, 3969-3981 (2016).

80. J. A. Jaimes, N. M. Andre, J. S. Chappie, J. K. Millet, G. R. Whittaker, Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J Mol Biol, (2020).

81. S. Bertram et al, Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease. J Virol 85, 13363- 13372 (2011).

82. W. Chen et al, Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity. Emerg Microbes Infect 9, 469-473 (2020).

83. I. Ferrer et al., Olfactory Receptors in Non-Chemo sensory Organs: The Nervous System in Health and Disease. Front Aging Neurosci 8, 163 (2016).

84. N. N. Patel, A. D. Workman, N. A. Cohen, Role of Taste Receptors as Sentinels of Innate Immunity in the Upper Airway. J Pathog 2018, 9541987 (2018).

85. A. Salas et al., Whole Exome Sequencing reveals new candidate genes in host genomic susceptibility to Respiratory Syncytial Virus Disease. Sci Rep 7, 15888 (2017).

86. S. Tcherniuk et al, Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections. J Infect Dis 214, 237-247 (2016).

87. P. B. Ampomah, L. A. Moraes, H. M. Lukman, L. H. K. Lim, Formyl peptide receptor 2 is regulated by RNA mimics and viruses through an IFN-beta-STAT3-dependent pathway. FASEB J 32, 1468-1478 (2018).

88. K. Chachlaki et al, Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus. J Comp Neurol 525, 3177-3189 (2017).

89. K. Chachlaki, J. Garthwaite, V. Prevot, The gentle art of saying NO: how nitric oxide gets things done in the hypothalamus. Nat Rev Endocrinol 13, 521-535 (2017).

90. K. Chachlaki, V. Prevot, Nitric oxide signalling in the brain and its control of bodily functions. Br J Pharmacol, doi: 10.1111/bph.14800. (2019).

91. A. Jais, J. C. Bruning, Hypothalamic inflammation in obesity and metabolic disease. J Clin Invest 127, 24-32 (2017).

92. S. Akerstrom et al., Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus. J Virol 79, 1966-1969 (2005).

93. X. Chen et al., Fpr2 Deficiency Alleviates Diet-Induced Insulin Resistance Through Reducing Body Weight Gain and Inhibiting Inflammation Mediated by Macrophage Chemotaxis and Ml Polarization. Diabetes 68, 1130-1142 (2019). 94. M. Bottcher et al., NF-kappaB signaling in tanycytes mediates inflammation- induced anorexia. Mol Metab, 101022 (2020).

95. K. E. Stelzig et al, Estrogen regulates the expression of SARS-CoV-2 receptor ACE2 in differentiated airway epithelial cells. Am J Physiol Lung Cell Mol Physiol, (2020). 96. B. Lin et al, Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2. Cancer Res 59, 4180-4184 (1999).

97. H. Wang et al., Increased hypothalamic microglial activation after viral-induced pneumococcal lung infection is associated with excess serum amyloid A production. J Neuroinflammation 15, 200 (2018). 98. S. Schloer et al., The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model. FASEB J 33, 12188-12199 (2019).

99. M. C. Alessi, N. Cenac, M. Si-Tahar, B. Riteau, FPR2: A Novel Promising Target for the Treatment of Influenza. Front Microbiol 8, 1719 (2017). 100. Y. Le et al, Amyloid (beta)42 activates a G-protein-coupled chemoattractant receptor, FPR-like-1. J Neurosci 21, RC123 (2001).

101. M. A. Panaro et al., Biological role of the N-formyl peptide receptors. Immunopharmacol Immunotoxicol 28, 103-127 (2006).

102. E. Song et al., Neuroinvasion of SARS-CoV-2 in human and mouse brain. J Exp Med 218, e20202035 (2020).

103. Meinhardt et al., Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat Neurosci 24, 168 (2021).