METHOD FOR THE PROPHYLAXIS OR TREATMENT OF CORONAVIRUS INFECTION USING AN IMMUNOMODULATOR AND VACCINE COMPOSITIONS COMPRISING THE SAME

Abstract

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

Claims

1. A method for the treatment or prophylaxis of 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.

2. The method of claim 1, wherein the immunomodulator is a detoxified Escherichia coli labile toxin (LT), a Toll-Like Receptor (TLR) agonist or antagonist, Vaxfectin, or a pattern recognition receptor (PRR) agonist or antagonist.

3. The method of claim 2, wherein the LT is LTh(αK).

4. The method of claim 1, wherein the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

5. The method of claim 1, wherein the immunomodulator is administered multiple times.

6. The method of claim 1, further comprising administering an anti-coronavirus antigen to the subject.

7. A vaccine composition comprising a pharmaceutically effective amount of an immunomodulator.

8. The vaccine composition of claim 7, wherein the immunomodulator is a detoxified Escherichia coli labile toxin (LT), a Toll-Like Receptor (TLR) agonist or antagonist, Vaxfectin, or a pattern recognition receptor (PRR) agonist or antagonist.

9. The vaccine composition of claim 8, wherein the LT is LTh(αK).

10. The vaccine composition of claim 7, further comprising an anti-coronavirus antigen.

11. The vaccine composition of claim 10, wherein the anti-coronavirus antigen is an attenuated virus, an inactivated whole virus, a split coronavirus, a recombinant coronavirus, a subunit of coronavirus, a peptide or protein from coronavirus, or a biological entity.

12. The vaccine composition of claim 10, wherein the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

13. A method for the treatment or prophylaxis of coronavirus infection in a subject in need thereof or at risk of coronavirus infection, comprising administering to the subject the vaccine composition of claim 7.

14. A method for inducing IFNα production in epithelial cells of a subject, comprising administering the epithelial cells of the subject a therapeutically effective amount of an immunomodulator.

15. The method according to claim 14, wherein the immunomodulator is a detoxified Escherichia coli labile toxin (LT), a Toll-Like Receptor (TLR) agonist or antagonist, Vaxfectin, or a pattern recognition receptor (PRR) agonist or antagonist.

16. The method according to claim 15, wherein the LT is LTh(αK).

17. The method according to claim 14, wherein the epithelial cells are respiratory tract mucosal epithelial cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1A shows the serum anti-RBD IgG titers after SARS CoV-2 vaccination by LTh(αK)-adjuvanted S1 via IM route. The left bar corresponds to Group 1 and the right bar corresponds to Group 2 in Table 1.

[0029] FIG. 1B shows the serum anti-RBD IgG titers after SARS CoV-2 vaccination by LTh(αK)-adjuvanted S1 via IN route. The left bar corresponds to Group 3 and the right bar corresponds to Group 4 in Table 1.

[0030] FIG. 1C shows the serum anti-RBD IgA titers after SARS CoV-2 vaccination by LTh(αK)-adjuvanted S1 via IM route. The left bar corresponds to Group 1 and the right bar corresponds to Group 2 in Table 1.

[0031] FIG. 1D shows the serum anti-RBD IgA titers after SARS CoV-2 vaccination by LTh(αK)-adjuvanted S1 via IN route. The left bar corresponds to Group 3 and the right bar corresponds to Group 4 in Table 1.

[0032] FIG. 2 shows the SARS-CoV-2 neutralization IgG antibody titers after intranasal vaccination by LTh(αK)-adjuvanted recombinant RBD vaccine.

[0033] FIG. 3 shows the SARS-CoV-2 Spike recombinant (rA1)-specific IgA titers after intranasal vaccination by LTh(αK)-adjuvanted recombinant RBD vaccine.

[0034] FIG. 4 shows the histopathological scores of the lungs of the SARS-CoV-2-infected Syrian golden hamsters with or without the treatment with LTh(αK).

[0035] FIG. 5A shows the effect of LTh(αK) treatment on IFNα production. FIG. 5B shows phosphorylation of p65 induced by treatment with LTh(αK) to epithelial cells. FIG. 5C shows the inhibition of p65 phosphorylation by an inhibitor to NF-κB (pyrrolidine dithiocarbamate, PDTC). FIG. 5D shows the results of chromatin immunoprecipitation (ChIP) assay, which demonstrated recruitment of p-p65 to IFNα promoter in LTh(αK) mediated epithelial activation. FIG. 5E shows that treatment of PDTC blocked the production of IFNα by epithelial cells.

[0036] FIG. 6 shows a proposed mechanism of LTh(αK) in the treatment or prophylaxis of coronavirus infection.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning 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 latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition.

[0038] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0039] 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 components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0040] The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise. Unless otherwise required by context, plural terms shall also include the singular.

[0041] “Treatment,” “treating,” and the like refer to an approach for obtaining a beneficial or desired result, including clinical results. For purposes of this disclosure, beneficial or desired results include but are not limited to inhibiting and/or suppressing the onset and/or development of a condition or reducing the severity of such condition, such as reducing the number and/or severity of symptoms associated with the condition, increasing the quality of life of those suffering from the condition, decreasing the dose of other medications required to treat the condition, enhancing the effect of another medication a patient is taking for the condition, and/or prolonging survival of patients having the condition.

[0042] “Prophylaxis,” “prophylactic,” “prevent,” “preventing,” “prevention,” and the like refer to reducing the probability of developing a condition in a patient who does not have, but is at risk of developing a condition. A patient “at risk” may or may not have a detectable condition, and may or may not have displayed a detectable condition prior to the treatment methods disclosed herein.

[0043] “Administering” or “administration of” a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered sublingually or intranasally, by inhalation into the lung or rectally. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0044] The term “modulating” and “modulation” used herein refer to the regulation of a condition, level, or amount. The regulation may be upregulation or downregulation.

[0045] The term “mucosal immune response” used herein refers to the immune responses that are induced at the mucosa. For example, mucosal immune response includes, but is 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 immunized antigens.

[0046] The term “mucosal site” as used herein refers to any anatomical mucosa covered with mucosal epithelium. For example, the mucosal site may be sublingual mucosa, intranasal mucosa, respiratory track mucosa, oral mucosa, vaginal mucosa, rectal mucosa, or other anatomical mucosa.

[0047] The term “immunomodulator” as used herein refers to a pharmacological or immunological agent that modifies the immunity and ultimately changes the outcome of immunogenicity to specific antigens/allergens. For example, an immunomodulator may be detoxified LT or Toll-Like Receptor (TLR) agonists.

[0048] The terms “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.), and rodents (e.g., mice and rats).

[0049] “Effective amount” refers to such amount of a therapeutic agent or a pharmaceutically acceptable salt thereof which in combination with its parameters of efficacy and potential for toxicity, as well as based on the knowledge of the practicing specialist, should be effective in a given therapeutic form. As is understood in the art, an effective amount can be administered in one or more doses.

[0050] The present invention surprisingly finds that immunomodulators, such as LTh(αK), are capable of providing protective immune response against coronavirus infection. The immunomodulator may be administered prior to or following coronavirus challenge, or at both time periods. In addition, it is found that immunomodulators, when used as an adjuvant in combination with an anti-coronavirus antigen, can elicit the production of SARS CoV-2 neutralizing IgG and IgA. It is further found that the above effects of immunomodulators involve activation of the p65 subunit of NF-κB and enhanced production of IFNα. The present invention provides a novel use of immunomodulator in the treatment or prophylaxis of coronavirus infection to facilitate the development of novel therapy vaccine for coronavirus infection beyond traditional means.

[0051] Having now generally described the invention, the same may be more readily understood through reference to the following examples, which provide exemplary protocol for performing the method of the present invention in the treatment or prophylaxis of coronavirus infection. The examples are offered 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 with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

EXAMPLES

Example 1

Evaluation of the Efficacy of LTh(αK) on Serum Anti-RBD IgG and IgA through Intramuscular (IM) Route or Intranasal (IN) Route

[0052] S1 is a recombinant protein from Spike of SARS CoV-2. Animals used in the studies were female Balb/c mice aged 8 weeks. Dosing and timing of serum collections were conducted according to the animal study protocol shown in Table 1 below. Serum samples were respectively diluted as indicated in FIGS. 1A to 1D.

TABLE-US-00001 TABLE 1 Dosing (μg) Week 1 Week 2 Week 3 Assayed date Administration (Day 0) (Day 7) (Day 0) 2 weeks post Groups routes S1 LTh(αK) S1 LTh(αK) S1 LTh(αK) final vaccination 1 Intramuscular 2 0 2 0 2 0 0, 30 2 Intramuscular 2 1 2 1 2 1 0, 30 3 Intranasal 2 0 2 0 4 0 0, 30 4 Intranasal 2 1 2 1 4 2 0, 30

Example 2

Evaluation of SARS-CoV-2 Neutralization IgG Antibody Titers After Intranasal Vaccination by LTh(αK)-adjuvanted Recombinant RBD Vaccine

[0053] Balb/c mice received three 20 μg doses, two weeks apart, of Fc-conjugated SARS-CoV-2 Spike recombinants (rA1) adjuvanted by 10 μg LTh(αK) via intranasal route. The serum specimens were collected at the end of the study (week 10) and serum neutralizing titers to wild type SARS-CoV-2 were analyzed as described in Manenti A, et al., J Med Virol 2020, 92(10):2096-2104 with minor modifications. The results shown in FIG. 2 indicate that the intranasal vaccine induced 100%, 94%, 87% and 65% of viral neutralization at 320, 640, 1280 and 2560-fold dilutions, respectively.

Example 3

Evaluation of SARS-CoV-2 rA1-Specific IgA Titers Followed by Intranasal Vaccination by LTh(αK)-Adjuvanted Recombinant RBD Vaccine

[0054] The rA1-specific IgA titers from nasal wash or bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. The results shown in FIG. 3 indicate that rA1 intranasal vaccination induced rA1-, Spike-1 (S1)- and RBD-specific IgA antibodies from nasal wash and BALF. Nasal wash and BALF specimens were collected and measured by ELISA as described in Lin IP, et al., PLoS One 2014, 9(3):e90293.

Example 4

Evaluation of the Effects of LTh(αK) in the Treatment and Prophylaxis of the SARS-CoV-2 Induced Pneumonia

[0055] Table 2 below shows the scheme for determining the effects of LTh(αK) in treating and preventing SARS-CoV-2 induced pneumonia in Syrian golden hamsters. The hamsters of groups 1 and 2 were housed in a specific pathogen free (SPF) environment and transferred to a biosafety level three (P3) facility on Day 20. The hamsters of group 3 were kept in SPF environment and prophylactically treated by LTh(αK), 10 μg/animal intranasally, on Days 0, 7 and 14, and then transferred to the P3 facility with the hamsters of groups 1 and 2 on Day 20. On Day 21, all hamsters received SARS-CoV-2 virus (10.sup.5 TCID50) via nasal route. On Day 22, the hamsters of group 1 received a formulation buffer treatment via intranasal route, while the hamsters of groups 2 and 3 received an LTh(αK) treatment, 10 μg/animal, via intranasal route. On Day 24, all the hamsters were sacrificed, and the tissues were collected for histopathological analysis.

TABLE-US-00002 TABLE 2 Days 0 Days 7 Days 14 Days 20 Days 21 Days 22 Days 24 Groups Treatment (prophylactic) Transfer Challenge Treatment Sacrifice 1 Placebo — — — T/P3 SARS CoV-2 Buffer Specimens 2 Treatment — — — T/P3 SARS CoV-2 LTh(αK) Specimens 3 Prevent/Treat LTh(αK) LTh(αK) LTh(αK) T/P3 SARS CoV-2 LTh(αK) Specimens Buffer: Formulation buffer T: Transfer P3: Biosafety level 3 facility Challenge: intranasal challenge by wild-type SARS-CoV-2 virus (10.sup.5 TCID50) Specimens: biological specimens from serum and lung

[0056] Treatment by LTh(αK) reduced SARS-CoV-2 induced histopathological scores of the lungs of Syrian golden hamster (FIG. 4). The hamsters of group 1, which received placebo, showed the most severe histopathological score (2.33). The hamsters of group 2, which received a single dose of LTh(αK) (LTh(αK)-single dose) after challenge by SARS-CoV-2, showed an improved histopathological score (1.83). The hamsters of group 3, which received a combination of three prophylactic treatments and one therapeutic treatment by LTh(αK) (LTh(αK)-3+1), showed significant improvement to SARS-CoV-2 induced pneumonia (1.42) (p≤0.05).

Example 5

Evaluation of the Effects of LTh(αK) Treatment on IFNα Production and Relevant Signal Transduction Pathway

[0057] IFNα is a critical player in inflammatory and anti-inflammatory responses during respiratory infection. During treatment of epithelial cells (BEAS-2B) by LTh(αK), elevated IFNα protein was detected in the culture medium (FIG. 5A). Treatment of LTh(αK) to epithelial cells also induced phosphorylation of p65 (FIG. 5B). This phosphorylation was inhibited by pyrrolidine dithiocarbamate (PDTC), an inhibitor to NF-κB (FIG. 5C), suggesting that NF-κB pathway is involved, not the ERK. In chromatin immunoprecipitation (ChIP) assay (FIG. 5D), recruitment of p-p65 to IFNα promoter revealed the key role of NF-κB in LTh(αK) mediated epithelial activation. Further, treatment of PDTC blocked the production of IFNα by epithelial cells in the medium (FIG. 5E).

Example 6

Proposed Mode of Action

[0058] From the examples described above, the mechanism of LTh(αK) in the treatment or prophylaxis of coronavirus infection is proposed as shown in FIG. 6. When LTh(αK) is adminstered to nasal cavity, it directly contacts epithelial cells. GM1 on the surface of epithelial cells binds to LTh(αK) and signals the secretion of type 1 interferon (IFNα). It is known that IFNα can modulate the inflammatory pathway. A recent publication reveals that crosstalk between inflammatory macrophage and CD8 cells within aveolar of SARS-CoV-2 infected lung sustains the inflammatory response. IFNα is also known to respond to CD8 and induce IL10 secretion that can interrupt the crosstalk. IFNα activates monocyte with Ly6C.sup.1o phenotype, which promotes anti-inflammatory outcomes. IFNα then activates DCs (pDC and mDC) and induces further activities and cytokine activations. LTh(αK) may directly contact the dendrites of DC exposed to nasal cavity. Transcytosis of AB5 via epithelum has also been reported, which further provides a pathway for LTh(αK) induced DC activation. The pDC within the nasal associated lymphoid tissue (NALT) is a major source of IFNα, which provides another wave of IFNα to modulate immune responses. IFNα under inflammatory environment promotes anti-inflammatory responses as described above. DCs activated by LTh(αK) down regulate proinflammatory cytokines, such as IL6 and IL5. IFNα produced by 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.

[0059] Interferon alpha (IFNα) plays an essential role in innate immunity against infection including SARS-CoV-2. IFNα belongs to type 1 interferon family and is secreted by many cell types at an enhanced level following infection. Treatment with LTh(αK) to nasal epithelial cells induced elevated levels of IFNα, suggesting an activation of innate defensing cascade following treatment. In addition to viral eradication, IFNα plays an important role in anti-inflammatory responses, and SARS-CoV-2 challenged hamsters tested in the examples further verified this hypothesis. In conclusion, we propose that LTh(αK) is a potent immunomodulator and innate immunity enhancer for the treatment of COVID-19 patients.