Methods for preventing or treating coronavirus infection using immunomodulators and vaccine compositions containing the same

Abstract

The present disclosure provides a method for treating or preventing coronavirus infection, comprising administering a therapeutically effective amount of an immunomodulator to a subject in need thereof or at risk of coronavirus infection. Also provided is a vaccine composition comprising a pharmaceutically effective amount of an immunomodulator.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Application No. 63 / 022,017, filed May 8, 2020, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the treatment or prevention of coronavirus infection, and in particular to the treatment or prevention of coronavirus infection by administering an immunomodulatory agent. Background Art

[0004] SARS-CoV, MERS-CoV, and SARS-CoV-2 are deadly coronavirus pathogens that have caused widespread mortality in specific populations. SARS-CoV-2 attacked its human host in late 2019. The infection spread silently, with up to 80% of infected individuals showing no symptoms. However, the elderly and those with underlying medical conditions such as diabetes, hypertension, and immunosuppression are particularly vulnerable, with mortality rates exceeding 15%. In China alone, SARS-CoV-2 infected over 80,000 people within three months, claiming over 3,200 lives.

[0005] Current treatment options for SARS-CoV-2 are diverse and experimental, including antiviral agents such as remdesivir, hydroxychloroquine, ritonavir / lopinavir, and ritonavir / lopinavir with interferon beta. Remdesivir is an anti-infective drug for Ebola infection. In animal studies (SARS models) and a limited number of human individuals with SARS-CoV-2, remdesivir has shown encouraging efficacy in inhibiting coronavirus and reducing fever. Hydroxychloroquine is believed to alleviate infection in a large number of patients. However, due to a lack of understanding of its exact mechanism and adverse events caused by high doses, the actual application of hydroxychloroquine remains controversial. The combination of hydroxychloroquine and azithromycin has been reported as a disruptive change in SARS-CoV-2 therapy due to its high efficacy, but this study has not yet been verified. Both ritonavir / lopinavir and ritonavir / lopinavir plus interferon beta are not suitable for the treatment of SARS-CoV-2.

[0006] Bacillus Calmette-Guérin (BCG) has been recommended as a treatment option for SARS-CoV-2 due to its immunomodulatory properties. BCG is a preventive vaccine against Mycobacterium tuberculosis, but it is also an immunotherapeutic agent against SARS-CoV-2. BCG is believed to nonspecifically enhance innate and adaptive immune responses through innate pattern recognition receptors on dendritic cells (DCs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), leading to nonspecific / broad-spectrum CD4 T cell activation. These immunological implications give BCG a unique role in cancer immunotherapy (Nishida S et al., "Immune adjuvant therapy using Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) in advanced malignancies: A phase 1 study of safety and immunogenicity assessments," Medicine (Baltimore) 2019, 98(33): e16771).

[0007] Many academic institutions or pharmaceutical companies are currently developing other therapeutic drugs and preventive treatments for SARS-CoV-2. Vaccines are a typical preventive method involving the use of one or more antigens, recombinant microorganisms or nucleic acids encoding viral proteins to enhance anti-SARS-CoV-2 immunity. Currently, the receptor binding domain (RBD) of SARS-CoV-2 is the most commonly tested antigen target. Viral antigen epitopes do not always promote the desired quantitative and qualitative immune responses. In order to optimize vaccine efficacy, adjuvants can be incorporated. Adjuvants are usually co-administered with vaccines to enhance the antigen-specific immune response qualitatively or quantitatively via innate immune activation.

[0008] Vaccines are key to stopping the spread of SARS-CoV-2 infection. SARS-CoV-2 evolved from bats and pangolins and then infected humans through an unknown intermediate host. Currently, there is no effective treatment or vaccine available for SARS-CoV-2 infection. Many vaccine strategies have been proposed for SARS CoV-2, including recombinant viral proteins, subunit vaccines, DNA or mRNA vaccines. Most SARS-CoV-2 vaccines under development use the viral spike protein (S protein) as the primary antigen and intramuscular (IM) injection as the route of administration. The S protein mediates the fusion of SARS-CoV-2 with host cells carrying the angiotensin converting enzyme 2 (ACE2) receptor, and the receptor binding domain (RBD) on the S protein has been identified. Both human and animal studies have revealed that the IgG titer of the S protein is positively correlated with antibody-dependent enhancement (ADE), which is the pathogenic result after the immune response to the S protein and the production of non-RBD blocking IgG. In a new season or pandemic, pre-existing non-RBD-blocking IgG may bind to the S protein of different strains of circulating SARS-CoV-2 and enhance infection. Solutions to address ADE include changing the immunization route to induce mucosal sIgA with improved neutralizing capacity in the absence of ADE burden.

[0009] Mucous membranes cover the surfaces of body passages, such as the intestinal tract, respiratory tract, and urogenital tract. Mucous membranes are epithelial layers that are covered with sIgA and antimicrobial molecules on the lumen side to keep the mucous membranes from infection. sIgA is the main defense molecule for polymeric immunoglobulins and the adaptive immunity of mucous membranes. Compared with IgG, the polymeric structure of IgA provides a wider range of specificity and improves neutralization ability to genetically drifted virus strains. In addition, compared with other Ig isotypes, IgA is more resistant to proteases from the host or microorganisms.

[0010] Escherichia coli heat-labile toxin (LT) is suitable as an adjuvant or immunomodulator for intranasal (IN) vaccination due to its ability to enhance the immune mucosal response to co-administered vaccines. Tamura S et al., "Intranasal Inactivated Influenza Vaccines: a Reasonable Approach to Improve the Efficacy of Influenza Vaccine?" Jpn J Infect Dis 2016, 69(3):165-179 reported that inactivated influenza vaccines combined with adjuvants via the intranasal route induced an increase in both serum hemagglutination inhibition (HI) antibodies and secretory IgA (sIgA) antibodies. Some adverse events have been reported, including fever, fatigue, and headache; and local reactions such as rhinitis, nasal stiffness, and rhinorrhea, but they are generally mild. Diarrhea and local inflammation are attributed to an ADP-ribosylase enzyme on one subunit that causes the efflux of water and electrolytes from epithelial cells. Therefore, genetically detoxifying LTs with reduced or completely absent ADP-ribosylase activity have been constructed to better address the unmet medical need for mucosal adjuvants.

[0011] Unlike wild-type LT and LTK63, developed by Chiron, which induce the secretion of proinflammatory cytokines such as IL-6 in neuronal phenotype SK-N-SH cells, LTh(αK), also known as LTS61K (U.S. application Ser. No. 13 / 097,218), does not promote IL-6 secretion. In clinical trials enrolling more than 350 individuals, both healthy and allergic rhinitis subjects aged 20 to 75 years have received multiple doses of LTh(αK) via the nasal route, and none of them reported signs of facial paralysis.

[0012] Studies support the intranasal administration of LTB, a subunit of LT, to alleviate inflammation induced by autoimmunity. Both regulatory T cells and IL-10 are hallmarks of anti-inflammatory responses. Treatment with LTB may involve crosslinking of the GM1 receptor complex with TrkA or integrins, thereby triggering a signaling cascade that leads to regulatory T cell activation and IL-10 secretion.

[0013] The type I IFN family is a multigenic cytokine family that includes IFNα, IFNβ, and several less well-defined single gene products. IFNα and IFNβ are the best-defined and most widely expressed type I IFNs. Type I IFNs are best known for inducing antiviral activity in both virus-infected and uninfected bystander cells by inducing host gene transcripts, interfering with viral replication through multiple mechanisms. Type I IFNs have diverse functions, regulating both innate and adaptive immune responses against viruses as well as bacterial pathogens. The outcome of IFNα / β responses during infection is highly context-dependent. Different tissue-specific conditions during infection influence when and where IFNα / β signals are transmitted and trigger signaling pathways downstream of the type I IFN receptor (IFNAR). This outcome determines the activation or repression of IFN-stimulated genes (ISGs). Overall, type I IFNs promote the initiation of immune responses.

[0014] Given the ongoing pandemic and high mutation rate of coronaviruses, particularly SARS-CoV, MERS-CoV, and SARS-CoV-2, there remains a need for effective therapies for the treatment and / or prevention of coronavirus infections. Summary of the Invention

[0015] In the present invention, it was unexpectedly discovered that immunomodulators such as LTh(αK) can be used alone or as adjuvants in combination with anti-coronavirus antigens to treat or prevent coronavirus infection. Therefore, the present invention relates to the use of immunomodulators / adjuvants such as LTh(αK) as therapeutic agents.

[0016] The present disclosure relates to the discovery that immunomodulators, such as LTh(αK), can be used as therapeutic or prophylactic agents for treating or preventing coronavirus infection in a subject. Accordingly, the present disclosure provides a method for treating or preventing coronavirus infection in a subject in need thereof or at risk of coronavirus infection, comprising administering to the subject a therapeutically effective amount of an immunomodulator.

[0017] In one embodiment, the immunomodulator can signal through the mucosal epithelium. Preferably, the immunomodulator is a toxin or toxoid, and more preferably, the immunomodulator is a toxin or toxoid of bacterial origin. In a preferred embodiment, the immunomodulator is a detoxifying LT, LTh (αK), a Toll-like receptor (TLR) agonist or antagonist, Vaxfectin, or a pattern recognition receptor (PRR) agonist or antagonist. In another preferred embodiment, the immunomodulator does not induce the production of cytokines IL6 from contacted cells, including epithelial cells, Langerhans cells, resident monocytes, and neuronal cells. In another other preferred embodiment, the immunomodulator is LTh (αK). LTh (αK) corresponds to LTS61K as disclosed in US 2008102078, which is a detoxifying E. coli LT holotoxin with a lysine substitution at a position corresponding to position 61 of SEQ ID NO: 5 as disclosed in US 2008102078.

[0018] In one embodiment, the coronavirus is a virus of the subfamily Orthocoronavirinae. In a preferred embodiment, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2. In a more preferred embodiment, the coronavirus is SARS-CoV-2.

[0019] In one embodiment, the immunomodulator is administered to the individual after the individual is challenged with the coronavirus. In another embodiment, the immunomodulator is administered to the individual before the individual is challenged with the coronavirus. In another embodiment, the immunomodulator is administered to the individual before and after the individual is challenged with the coronavirus. In another embodiment, the immunomodulator is administered to the individual one or more times before and / or after the individual is challenged with the coronavirus. In a preferred embodiment, the immunomodulator is administered to the individual three times before the individual is challenged with the coronavirus. In another preferred embodiment, the immunomodulator is administered to the individual once after the individual is challenged with the coronavirus.

[0020] In one embodiment, an immunomodulatory agent is administered to a subject in combination with an anti-coronavirus antigen.

[0021] In one embodiment, the mucosal site can be any anatomical mucosa. In a preferred embodiment, the mucosal site is the sublingual mucosa, intranasal mucosa, respiratory mucosa, oral mucosa, vaginal mucosa, rectal mucosa or other anatomical mucosa. In another preferred embodiment, the antigen is administered to the sublingual mucosa. In another preferred embodiment, the immunomodulator is administered to the intranasal mucosa that may extend to the pharynx.

[0022] In one embodiment, the immune response involves the production of antigen-specific IgG and its subclasses, antigen-specific IgA and its subclasses, antigen-specific IgM and its subclasses, and / or cell-mediated immunity. In another embodiment, the immune response involves the upregulation of immune components. In another embodiment, the immune response involves the downregulation of immune components. In another embodiment, the immune response involves the production of immunoglobulins in response to the antigen. More preferably, the immune response provides a therapeutic benefit.

[0023] The present disclosure further provides a vaccine composition for treating or preventing coronavirus infection, comprising a pharmaceutically effective amount of an immunomodulator as described herein.

[0024] In one embodiment, the vaccine composition further comprises an anti-coronavirus antigen. In one embodiment, the anti-coronavirus antigen is an attenuated virus, an inactivated whole virus, a split coronavirus, a recombinant coronavirus, a coronavirus subunit, a peptide or protein from a coronavirus, or a biological entity. In one embodiment, the anti-coronavirus antigen is an antigen derived from a virus of the subfamily Orthocoronavirus. In a preferred embodiment, the anti-coronavirus antigen is an antigen derived from SARS-CoV, MERS-CoV, or SARS-CoV-2. In a more preferred embodiment, the anti-coronavirus antigen is an antigen derived from SARS-CoV-2. In a more preferred embodiment, the anti-coronavirus antigen is an antigen derived from the SARS-CoV-2 spike protein (S protein), the recombinant SARS-CoV-2 spike protein (rA1), or the receptor binding domain (RBD) of SARS-CoV-2.

[0025] The present disclosure further provides a method for treating or preventing a coronavirus infection in an individual in need thereof or at risk of coronavirus infection, comprising administering to the individual a therapeutically effective amount of an immunomodulatory agent or vaccine composition as described herein.

[0026] The mechanism by which LTh(αK) achieves its efficacy in treating or preventing coronavirus infection may be as follows: After LTh(αK) is administered to the nasal cavity, LTh(αK) directly contacts epithelial cells and binds to GM1 on the epithelial cell surface. GM1 then signals the secretion of type 1 interferon (IFNα). IFNα activates DCs (pDCs and mDCs) and induces further cytokine activation. LTh(αK) can also directly contact DC dendrites exposed in the nasal cavity or induce DC activation through transcytosis of AB5 via the epithelium. pDCs within the nasal-associated lymphoid tissue (NALT) are the main source of IFNα, providing another wave of IFNα to regulate the immune response. DCs activated by LTh(αK) downregulate proinflammatory cytokines such as IL6 and IL5. IFNα produced by the mucosa drives class switching of IgA, which neutralizes SARS-CoV-2. Class switching of IgA is TGFβ-dependent. TGFβ and IL10 upregulate Treg activity to attenuate inflammation.

[0027] LTh(αK) promotes the secretion of antigen-specific IgA, which has the potential to block and neutralize infectious agents such as SARS-CoV-2 before they can infect the epithelium, eliminating the possibility of viral shedding. Furthermore, compared to circulating IgG, mucosal surfaces offer significantly less contact with cells expressing Fc receptors, suggesting a reduced risk of ADE through viral targeting of IgA. Therefore, LTh(αK), alone or as an adjuvant co-administered with vaccine candidates, can reduce the risk of ADE and may be suitable for the prevention or treatment of coronavirus infection.

[0028] The present invention is described in detail in the following sections. Other features, objects and advantages of the present invention can be easily found in the detailed description and claims of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A Shown are serum anti-RBD IgG titers after SARS CoV-2 vaccination with LTh(αK)-adjuvanted S1 via the IM route. The left bar corresponds to Group 1 in Table 1, and the right bar corresponds to Group 2.

[0030] Figure 1B Shown are serum anti-RBD IgG titers after SARS CoV-2 vaccination with LTh(αK)-adjuvanted S1 via IN route. The left bar corresponds to Group 3 in Table 1, and the right bar corresponds to Group 4.

[0031] Figure 1CShown are serum anti-RBD IgA titers after SARS CoV-2 vaccination with LTh(αK)-adjuvanted S1 via the IM route. The left bar corresponds to Group 1 in Table 1, and the right bar corresponds to Group 2.

[0032] Figure 1D Shown are serum anti-RBD IgA titers after SARS CoV-2 vaccination with LTh(αK)-adjuvanted S1 via IN route. The left bar corresponds to Group 3 in Table 1, and the right bar corresponds to Group 4.

[0033] Figure 2 Shown are SARS-CoV-2 neutralizing IgG antibody titers after intranasal vaccination with LTh(αK)-adjuvanted recombinant RBD vaccine.

[0034] Figure 3 Shown are SARS-CoV-2 spike protein recombinant (rA1)-specific IgA titers after intranasal vaccination with LTh(αK)-adjuvanted recombinant RBD vaccine.

[0035] Figure 4 Shown are lung histopathology scores of Syrian golden hamsters infected with SARS-CoV-2 with or without LTh(αK) treatment.

[0036] Figure 5A Shown are the effects of LTh(αK) treatment on IFNα production. Figure 5B Shown is the phosphorylation of p65 induced by LTh(αK) treatment of epithelial cells. Figure 5C The inhibition of p65 phosphorylation by NF-κB inhibitor (pyrrolidine dithiocarbamate, PDTC) was shown. Figure 5D Shown are the results of chromatin immunoprecipitation (ChIP) analysis demonstrating the recruitment of p-p65 to the IFNα promoter in LTh(αK)-mediated epithelial activation. Figure 5E It was shown that treatment of PDTC blocks IFNα production by epithelial cells.

[0037] Figure 6 Shown is the proposed mechanism of LTh(αK) in treating or preventing coronavirus infection. DETAILED DESCRIPTION

[0038] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0039] As used in accordance with this disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated.

[0040] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer (or component) or group of integers (or groups of components) but not the exclusion of any other integer (or component) or group of integers (or groups of components).

[0041] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Unless the context requires otherwise, plural terms will also include the singular.

[0042] "Treatment," "treating," and the like are methods of obtaining beneficial or desired results, including clinical results. For the purposes of this disclosure, beneficial or desired results include, but are not limited to, inhibiting and / or arresting the onset and / or development of a condition or reducing the severity of such a condition, such as reducing the number and / or severity of symptoms associated with the condition, increasing the quality of life of a patient suffering from the condition, reducing the dose of other medications required to treat the condition, enhancing the patient's response to another medication for the condition, and / or prolonging the survival of a patient suffering from the condition.

[0043] "Prophylaxis," "prophylactic," "prevent," "preventing," "prevention," and the like refer to reducing the probability of developing a condition in a patient who does not have the condition but is at risk of developing the condition. A patient "at risk" may or may not have had a detectable condition prior to the treatment methods described herein, and may or may not have had the detectable condition shown prior to the treatment methods disclosed herein.

[0044] "Administering" or "administration of" a substance, compound, or medicament can be performed using one of a variety of methods known to those skilled in the art. For example, a compound or medicament can be administered sublingually or intranasally, by inhalation into the lungs or rectum. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some aspects, administration includes direct administration (including self-administration) and indirect administration (including the act of prescribing a medication). For example, as used herein, a physician who instructs a patient to self-administer a medication or to have the medication administered by another and / or provides a patient with a prescription for a medication is administering a medication to a patient.

[0045] As used herein, the terms "modulating" and "modulation" refer to the regulation of a condition, level, or amount. Modulation can be upregulation or downregulation.

[0046] The term "mucosal immune response" as used herein refers to an immune response induced at the mucosa. For example, mucosal immune responses include, but are not limited to, antigen-specific immunoglobulin G and its subclasses, immunoglobulin A and its subclasses, immunoglobulin M and its subclasses, and cell-mediated immunity to the immunizing antigen.

[0047] As used herein, the term "mucosal site" refers to any anatomical mucosa covered with mucosal epithelium. For example, the mucosal site can be the sublingual mucosa, intranasal mucosa, respiratory mucosa, oral mucosa, vaginal mucosa, rectal mucosa, or other anatomical mucosa.

[0048] As used herein, the term "immunomodulator" refers to a pharmacological or immunological agent that modifies immunity and ultimately changes the immunogenic outcome against a specific antigen / allergen. For example, an immunomodulator can be a detoxifying LT or a Toll-like receptor (TLR) agonist.

[0049] The terms "patient," "subject," and "individual" are used interchangeably and refer to humans or non-human animals. These terms include mammals, such as humans, primates, livestock animals (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0050] "Effective amount" refers to that amount of a therapeutic agent or a pharmaceutically acceptable salt thereof, which, in combination with its efficacy and toxicity parameters, and based on the knowledge of the practitioner, should be effective in a given treatment format. As is understood in the art, an effective amount can be administered in one or more doses.

[0051] The present invention unexpectedly discovered that immunomodulators, such as LTh(αK), can provide a protective immune response against coronavirus infection. The immunomodulator can be administered before or after the coronavirus attack, or at both time periods. In addition, it was found that when used as an adjuvant in combination with an anti-coronavirus antigen, the immunomodulator can induce the production of SARS CoV-2 neutralizing IgG and IgA. It was also found that the above effects of the immunomodulator involve activation of the p65 subunit of NF-κB and enhanced IFNα production. The present invention provides a novel use of an immunomodulator in the treatment or prevention of coronavirus infection to promote the development of novel therapeutic vaccines for coronavirus infection that go beyond traditional means.

[0052] The present invention has now been generally described, and the present invention can be more easily understood by reference to the following examples, which provide exemplary schemes for carrying out the method of the present invention in treating or preventing coronavirus infection. Examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy (such as amount, temperature, etc.) about the numerals used, but some experimental errors and deviations are allowed to be taken for granted.

[0053] Examples

[0054] Example 1: Evaluation of LTh(αK) efficacy on serum anti-RBD IgG and IgA by intramuscular (IM) or intranasal (IN) routes

[0055] S1 is a recombinant protein of the spike protein from SARS CoV-2. The animals used in the study were 8-week-old female Balb / c mice. The timing of dosing and serum collection was performed according to the animal study protocol shown in Table 1 below. Figure 1A Serum samples were diluted as indicated in 1D.

[0056] Table 1

[0057]

[0058] Example 2: Evaluation of SARS-CoV-2 neutralizing IgG antibody titers after intranasal vaccination with LTh(αK)-adjuvanted recombinant RBD vaccine

[0059] Balb / c mice received three 20 μg doses of Fc-binding SARS-CoV-2 spike protein recombinant (rA1) adjuvanted with 10 μg LTh (αK) via the intranasal route, two weeks apart. Serum samples were collected at the end of the study (week 10) and analyzed for serum neutralization titers against wild-type SARS-CoV-2 as described in Manenti A et al., J Med Virol 2020, 92(10):2096-2104 (with minor modifications). Figure 2 The results shown in indicate that the intranasal vaccine induced 100%, 94%, 87% and 65% virus neutralization at 320-, 640-, 1280- and 2560-fold dilutions, respectively.

[0060] Example 3: Evaluation of SARS-CoV-2 rA1-specific IgA titers after intranasal vaccination with LTh(αK)-adjuvanted recombinant RBD vaccine

[0061] rA1-specific IgA titers from nasal washes or bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Figure 3 The results shown in indicate that rA1 intranasal vaccination induces rA1, spike protein-1 (S1), and RBD-specific IgA antibodies from nasal washes and BALF. Nasal wash and BALF samples were collected and measured by ELISA as described in Lin IP et al., PLoS One 2014, 9(3):e90293.

[0062] Example 4: Evaluation of the effect of LTh (αK) in the treatment and prevention of SARS-CoV-2-induced pneumonia

[0063] Table 2 below shows the protocol for determining the effect of LTh(αK) in the treatment and prevention of SARS-CoV-2-induced pneumonia in Syrian golden hamsters. Hamsters in groups 1 and 2 were housed in a specific pathogen-free (SPF) environment and transferred to a biosafety level 3 (P3) facility on day 20. Hamsters in group 3 were placed in an SPF environment and treated prophylactically with LTh(αK), 10 μg / animal intranasally on days 0, 7, and 14, and then transferred to a P3 facility on day 20 along with hamsters in groups 1 and 2. On day 21, all hamsters received SARS-CoV-2 virus (10 μg / animal) via the nasal route. 5 TCID50). On day 22, hamsters in group 1 received formulation buffer via intranasal route, while hamsters in groups 2 and 3 received LTh(αK) via intranasal route, 10 μg / animal. On day 24, all hamsters were sacrificed and tissues were collected for histopathological analysis.

[0064] Table 2

[0065]

[0066] Buffer: Formulation buffer

[0067] T: Transfer

[0068] P3: Biosafety Level 3 Facility

[0069] Challenge: By wild-type SARS-CoV-2 virus (10 5 TCID50) for intranasal challenge

[0070] Samples: Biological samples from serum and lung

[0071] Treatment with LTh (αK) reduced the lung histopathology scores of Syrian golden hamsters induced by SARS-CoV-2 (Figure 4). The first group of hamsters receiving placebo showed the most severe histopathology score (2.33). The second group of hamsters receiving a single dose of LTh (αK) (LTh (αK)-single dose) showed improved histopathology scores (1.83) after SARS-CoV-2 attack. The third group of hamsters receiving a combination of LTh (αK) (LTh (αK)-3+1) of three preventive treatments and one therapeutic treatment showed significant improvement in pneumonia (1.42) (p≤0.05) induced by SARS-CoV-2.

[0072] Example 5: Evaluation of the effects of LTh(αK) treatment on IFNα production and related signal transduction pathways

[0073] IFNα is a key factor in the inflammatory and anti-inflammatory responses during respiratory infections. During treatment of epithelial cells (BEAS-2B) with LTh(αK), elevated IFNα protein ( Figure 5A LTh(αK) treatment of epithelial cells also induced the phosphorylation of p65 ( Figure 5B This phosphorylation is inhibited by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB ( Figure 5C ), indicating that the NF-κB pathway, rather than ERK, is involved. In chromatin immunoprecipitation (ChIP) analysis ( Figure 5D ), p-p65 recruitment to the IFNα promoter revealed a key role for NF-κB in LTh(αK)-mediated epithelial activation. In addition, treatment with PDTC blocked IFNα production by epithelial cells in culture ( Figure 5E ).

[0074] Example 6: Proposed Behavior Pattern

[0075] Based on the examples described above, the LTh(αK) mechanism is proposed in the treatment or prevention of coronavirus infection as follows Figure 6 As shown in . When LTh(αK) is administered to the nasal cavity, it directly contacts the epithelial cells. GM1 on the surface of the epithelial cells binds to LTh(αK) and signals the secretion of type 1 interferon (IFNα). IFNα is known to regulate inflammatory pathways. Recently published literature reveals that crosstalk between inflammatory macrophages and CD8 cells in the alveoli of SARS-CoV-2 infected lungs maintains the inflammatory response. IFNα is also known to respond to CD8 and induce IL10 secretion, which can interrupt the crosstalk. IFNα activates Ly6C loPhenotype of monocytes, which promote anti-inflammatory results. IFNα then activates DC (pDC and mDC) and induces other activities and cytokine activation. LTh (αK) can directly contact DC dendrites exposed to the nasal cavity. AB5 has also been reported to transcytose via the epithelium, which further provides a path for (αK)-induced DC activation. pDC in the nasal-associated lymphoid tissue (NALT) is the main source of IFNα, which provides another wave of IFNα to regulate the immune response. As described above, IFNα in an inflammatory environment promotes anti-inflammatory responses. DC activated by LTh (αK) downregulates proinflammatory cytokines such as IL6 and IL5. IFNα produced by the mucosa drives the class switching of IgA, which neutralizes SARS-CoV-2. The class switching of IgA is TGFβ-dependent. TGFβ and IL10 upregulate Treg activity to attenuate inflammation.

[0076] Interferon alpha (IFNα) plays an important role in innate immunity to infections including SARS-CoV-2. IFNα belongs to the type 1 interferon family and is secreted at increased levels by many cell types after infection. Treatment of nasal epithelial cells with LTh(αK) induced elevated levels of IFNα, indicating activation of the innate defense cascade after treatment. In addition to viral eradication, IFNα plays an important role in anti-inflammatory responses, and the hamsters challenged with SARS-CoV-2 tested in the examples further validated this hypothesis. In summary, we propose that LTh(αK) is an effective immunomodulator and innate immunity enhancer for the treatment of COVID-19 patients.

Claims

1. Use of an immunomodulator in the preparation of a medicament for treating or preventing SARS-CoV-2 infection in an individual in need or at risk of SARS-CoV-2 infection, wherein the immunomodulator is LTh (αK). The use according to claim 1 , wherein the drug is administered multiple times.

3. The use according to claim 1, wherein the drug further comprises a coronavirus antigen or is administered in combination with a coronavirus antigen.

4. A vaccine composition for use in the preparation of a medicament for treating or preventing SARS-CoV-2 infection in an individual in need or at risk of SARS-CoV-2 infection, wherein the vaccine composition comprises a pharmaceutically effective amount of an immunomodulator, wherein the immunomodulator is LTh(αK) and is the sole active ingredient of the vaccine composition.

5. Use of an immunomodulator in the preparation of a medicament for inducing IFNα production in epithelial cells to treat or prevent SARS-CoV-2 infection, wherein the immunomodulator is LTh(αK). The use according to claim 5 , wherein the epithelial cells are respiratory mucosal epithelial cells.

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