Intranasal vaccine composition and method for boosting using the same

Abstract

To provide a method for vaccinating a subject against mucosal viral infections.SOLUTION: The present disclosure provides a method for vaccinating a subject against a mucosal virus infection, comprising administering to the subject an immunologically effective amount of an intranasal booster, the intranasal booster comprising detoxified Escherichia coli labile toxin (LT) and an antigen from the mucosal virus, and the subject having been previously primed. An intranasal vaccine composition comprising an immunologically effective amount of a mucosal virus antigen adjuvanted with a detoxified Escherichia coli labile toxin (LT) is also provided.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to an intranasal vaccine composition comprising a mucosal viral antigen adjuvanted with detoxified Escherichia coli labile toxin (LT) and a method for preventing mucosal viral infections using the same. In particular, the intranasal vaccine composition is a booster for preventing coronavirus or influenza virus infections. [Background technology]

[0002] The first case of SARS-CoV-2 infection was confirmed in Wuhan, China, in late December 2019. The virus has caused a global pandemic. Since then, several new vaccines against SARS-CoV-2 with different mechanisms have entered Phase III trials or received EUA approval. These vaccines include nucleoside-modified mRNA vaccines, adeno-based vaccines, and recombinant subunit vaccines. The rapid development of vaccines against SARS-CoV-2 represents an unprecedented achievement for biotechnology. However, SARS-CoV-2 mutant strains such as Omicron have shown increased immune evasion, reducing vaccine efficacy (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). Currently, except for one adeno-vector-based intranasal vaccine by Bharat (iNCOVACC), all other SARS-CoV-2 vaccines require intramuscular (IM) administration. Vaccines via the IM route have a proven track record of providing good safety and robust humoral immunity, but efficacy often declines 4 months after the IM booster (Non-Patent Document 1; Non-Patent Document 4; Non-Patent Document 5). Another critical problem with IM vaccination is the poor mucosal immunity, which is likely essential to hinder SARS-CoV-2 infection and prevent transmission (Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10).

[0003] Mucosal immunity is a compartmentalized immune response that plays a crucial role in defense against infections through mucosa. Mucosal vaccination raises infection-specific mucosal immunity to the site of infection (Non-Patent Document 11). Secretory IgA (sIgA) is one marker of mucosal immunity. Unlike the effect of IgG in defense against disease symptom progression, sIgA responds to infectious microorganisms at the site of infection. Despite the crucial role of mucosal immunity against SARS-CoV-2, only one mucosal vaccine, developed by India, has been approved (Non-Patent Document 12). The delay in mucosal vaccine development may be due to concerns over the tolerogenic nature of the epithelium track and neurological adverse events induced by intranasal adjuvants.

[0004] Current vaccines against COVID-19 involve IM administration of SARS-CoV-2-encoding nucleotide or recombinant spike antigen (S-2P) to induce cellular and humoral immunity. Although IM vaccines induce robust spike-specific IgG, clinical evidence indicates that priming and boosting via the IM route is insufficient to stop SARS-CoV-2 transmission. To overcome this deficiency, there is a dire need for an effective mucosal vaccine that terminates viral infection and transmission.

[0005] Heat-labile toxins (LT) of Escherichia coli have been widely studied as effective intranasal (IN) adjuvants for over 30 years (Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16). LTh(αK) is a fully detoxified E. coli LT protein derived from a human isolate. In two IN influenza vaccine clinical trials, inactivated intranasal vaccines (IIV) adjuvanted with LTh(αK) induced both serum IgG and sIgA against hemagglutination (Non-Patent Document 17; Non-Patent Document 18). However, there have been no reports of the application of inactivated intranasal vaccines (IIV) adjuvanted with LTh(αK) to the SARS-CoV-2 epidemic.

[0006] Hybrid vaccination, also known as heterologous vaccination, has been described in the art. For example, Non-Patent Document 1 describes a study of hybrid vaccination involving a systemic subcutaneous prime with LTB subunits followed by an oral boost to enhance the immunogenicity of the vaccine. In this study, the SARS-CoV-2 nucleocapsid, which serves as a T cell antigen, is covalently linked to LTB. The nucleocapsid is required for Th-1 activation and induction of IL-2 expression.

[0007] Another study (Non-Patent Document 2) demonstrated increased protective immunity by systemic prime-mucosal boost immunization. In this study, animals were primed with an mRNA vaccine and boosted with an adeno-vector vaccine via the mucosal route. Adenoviral vectors are replication-defective viruses with one or more target antigens inserted. After administration, adeno-vectors infect cells and release their viral DNA genome into the nucleus for transcription. Adeno-vector entry into host cells also induces innate immune-mediated inflammation, with a burst of pro-inflammatory IL-1, IL-6, TNF-α, etc., driving anti-insert immunity. Complexity also promotes immunogenicity. In addition to the insert targeted by immunity, the entire adenoviral virion complex is targeted by vaccination.

[0008] Vaccines play an important role in preventing infectious diseases. Given the ongoing pandemic of mucosal viruses such as SARS-CoV-2 and seasonal influenza, there remains a need for effective and patient-compliant (e.g., non-invasive) boosters for routine vaccination. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US2008 / 0102078 [Non-patent literature]

[0010]

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[0011] Parenteral vaccines mediate strong systemic immunity against SARS-CoV-2 but show only modest effects on the activation of secretory IgA (sIgA), tissue-resident memory B (BRM) cells, and memory T (TRM) cells. Three years after the outbreak of the pandemic, more than 71% of the world's population has received at least one dose of vaccine against SARS-CoV-2 infection via the parenteral route. Nevertheless, disease transmission is still ongoing due to the high mutation rate of the virus and insufficient protection to upper respiratory epithelial cells by current vaccinations. Thus, upper respiratory tract disease has not been effectively prevented. As mucosal immunity is one promising solution to the current pandemic, we developed an intranasal booster that effectively induces serum IgG, serum IgA, and mucosal IgA against the virus. The booster can be applied to subjects treated with an intranasal prime or an intramuscular prime, the latter being referred to as a hybrid vaccination regimen. Both intranasal prime-intranasal boost vaccination regimes and hybrid vaccination regimes including intramuscular prime and intranasal boost enhance serum neutralizing activity and induce robust mucosal neutralizing activity. LTh(αK)-adjuvanted SARS-CoV-2 spike protein intranasal (IN) booster has been found to significantly increase neutralizing antibody titers in serum and nasal wash samples. This LTh(αK)-adjuvanted IN booster enhances Th1 / Th2 balanced humoral and mucosal immunity in mouse models. IN booster with enhanced mucosal and circulatory immunogenicity provides clinically meaningful mucosal protection against vaccination.

[0012] In addition to inducing immunity against SARS-CoV-2, the IN booster of the present invention is also effective in inducing both humoral and mucosal immunity against influenza virus using influenza antigens adjuvanted with LTh(αK). LTh(αK) has been found to be an effective adjuvant in inducing systemic and mucosal immunity against mucosal viruses in the booster. Compared with conventional IM boosters, LTh(αK) IN boosters are more effective in preventing mucosal virus infection and blocking viral transmission.

[0013] The present invention is described in detail in the following sections. Other features, objects, and advantages of the present invention can be readily found in the detailed description of the invention and in the claims. [Brief description of the drawings]

[0014] [Figure 1A] FIG. 1 shows immunization scheme and immune responses to SARS-CoV-2 spike antigen. Intranasal immunization with S-trimer unadjuvanted or adjuvanted with LTh(αK). [Figure 1B] FIG. 1 shows immune responses to SARS-CoV-2 spike antigen. S-specific serum IgG elicited by immunization is shown, with serum diluted 90-fold for analysis of S-specific IgG titers. [Figure 1C] FIG. 1 shows immune responses to SARS-CoV-2 spike antigen. S-specific serum IgA induced by immunization is shown, with serum diluted 30-fold for analysis of S-specific IgA titers. [Figure 1D] Immune response to SARS-CoV-2 spike antigen. Serum neutralization assays show enhanced neutralizing activity against LTh(αK)-adjuvanted S-trimer. Kruskal-Wallis test results are highlighted in bold. [Figure 2A]FIG. 1 shows that intranasal immunization with LTh(αK)-adjuvanted S-trimer vaccine increases the production of Th1, Th2, and Th17 cytokines. [Figure 2B] FIG. 1 shows that intranasal immunization with LTh(αK)-adjuvanted S-trimer vaccine increases the production of Th1, Th2, and Th17 cytokines. [Figure 2C] FIG. 1 shows that intranasal immunization with LTh(αK)-adjuvanted S-trimer vaccine increases the production of Th1, Th2, and Th17 cytokines. [Figure 2D] FIG. 1 shows that intranasal immunization with LTh(αK)-adjuvanted S-trimer vaccine increases the production of Th1, Th2, and Th17 cytokines. [Figure 2E] FIG. 1 shows that intranasal immunization with LTh(αK)-adjuvanted S-trimer vaccine increases the production of Th1, Th2, and Th17 cytokines. [Figure 3A] FIG. 1 shows the immunization scheme for MVC-COV-1901, showing the immunization combinations of intramuscular prime and intranasal boost, intramuscular prime and intramuscular boost, and intranasal prime and intranasal boost. [Figure 3B] FIG. 1 shows the immune response to MVC-COV-1901. S-2P-specific serum IgG is shown on day 14, with serum diluted 4,000-fold. [Figure 3C] FIG. 1 shows the immune response to MVC-COV-1901, with S-2P-specific serum IgG on days 28 and 42, where serum was diluted 16,000-fold. [Figure 3D] Figure 1. Immune response to MVC-COV-1901. S-2P-specific IgA titers on day 42 from nasal washes diluted 10-fold. The results of the Kruskal-Wallis test are highlighted in bold. [Figure 4A]FIG. 1 shows serum IgG levels against seasonal influenza virus H1N1 antigens from hybrid vaccination. [Figure 4B] FIG. 1 shows serum IgG levels against seasonal influenza virus H3N2 antigens from hybrid vaccination. [Figure 4C] FIG. 1 shows serum IgG levels against seasonal influenza virus B-Victoria antigens from hybrid vaccination. [Figure 4D] FIG. 1 shows serum IgG levels against seasonal influenza virus B-Phuket antigens from hybrid vaccination. [Figure 5A] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus H1N1 antigens from hybrid vaccination using BALF samples. [Figure 5B] FIG. 1 shows the levels of secretory IgA antibodies against seasonal influenza virus H3N2 antigens from hybrid vaccination using BALF samples. [Figure 5C] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus B-Victoria antigen from hybrid vaccination using BALF samples. [Figure 5D] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus B-Phuket antigens from hybrid vaccination with BALF samples. [Figure 5E] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus H1N1 antigens from hybrid vaccination using BALF samples. [Figure 5F] FIG. 1 shows the levels of secretory IgA antibodies against seasonal influenza virus H3N2 antigens from hybrid vaccination using BALF samples. [Figure 5G] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus B-Victoria antigen from hybrid vaccination using BALF samples. [Figure 5H] FIG. 1 shows levels of secretory IgA antibodies against seasonal influenza virus B-Phuket antigens from hybrid vaccination with BALF samples. [Figure 6A] FIG. 1 shows the results of a short interval (½ hour) between IM prime and IN boost on immunogenicity to influenza antigen, where "IM" refers to intramuscular vaccination, all prime doses were administered via IM, "IM / IN (LT only)" refers to intramuscular prime with LTh(αK) and intranasal boost without influenza antigen, and "IM / IN (Flu+LT)" refers to intramuscular prime with LTh(αK) and intranasal boost with influenza antigen. [Figure 6B] FIG. 1 shows the results of a short interval (½ hour) between IM prime and IN boost on immunogenicity to influenza antigen, where "IM" refers to intramuscular vaccination, all prime doses were administered via IM, "IM / IN (LT only)" refers to intramuscular prime with LTh(αK) and intranasal boost without influenza antigen, and "IM / IN (Flu+LT)" refers to intramuscular prime with LTh(αK) and intranasal boost with influenza antigen. [Figure 6C] FIG. 1 shows the results of a short interval (½ hour) between IM prime and IN boost on immunogenicity to influenza antigen, where "IM" refers to intramuscular vaccination, all prime doses were administered via IM, "IM / IN (LT only)" refers to intramuscular prime with LTh(αK) and intranasal boost without influenza antigen, and "IM / IN (Flu+LT)" refers to intramuscular prime with LTh(αK) and intranasal boost with influenza antigen. [Figure 6D]FIG. 1 shows the results of a short interval (½ hour) between IM prime and IN boost on immunogenicity to influenza antigen, where "IM" refers to intramuscular vaccination, all prime doses were administered via IM, "IM / IN (LT only)" refers to intramuscular prime with LTh(αK) and intranasal boost without influenza antigen, and "IM / IN (Flu+LT)" refers to intramuscular prime with LTh(αK) and intranasal boost with influenza antigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Unless otherwise specified herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions.

[0016] As utilized in accordance with the present disclosure, the following terms, unless otherwise specified, shall be understood to have the following meanings:

[0017] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer (or component) or group of integers (or components), but not the exclusion of any other integer (or component) or group of integers (or components).

[0018] The singular forms "a," "an," and "the" include the plural unless the context requires otherwise. Plural terms shall include the singular as well unless the context requires otherwise.

[0019] "Treatment" and "treating" and the like refer to an approach for obtaining beneficial or desired results, 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 occurrence of a condition or reducing the severity of such a condition, such as reducing the number and / or severity of symptoms associated with a condition, improving the quality of life of a person suffering from the condition, reducing the dosage of other medications required to treat the condition, increasing the effectiveness of other medications taken by the patient for the condition, and / or increasing the survival time of a patient suffering from the condition.

[0020] "Prevention," "prophylactic," "prevent," "preventing," "prevention," and the like refer to reducing the chance of developing a condition in a patient who is not suffering from a condition but is at risk of developing the condition. A patient "at risk" may or may not have a detectable condition and may or may not exhibit a detectable condition prior to the treatment methods disclosed herein.

[0021] "Administering" or "administration" of a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intranasally. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, 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 a drug administered by another, and / or who provides a patient with a prescription for a drug, is administering a drug to a patient.

[0022] The term "modulator" as used herein refers to an agent that modulates a state, level, or amount of something. Modulation may be upregulation or downregulation.

[0023] The term "humoral immune response" as used herein refers to the immune response induced in serum. For example, humoral 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 cellular immunity against immunizing antigens.

[0024] As used herein, the term "mucosal immune response" refers to an immune response elicited in 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 cellular immunity against immunizing antigens.

[0025] 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 cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0026] "Effective amount" refers to that amount of a therapeutic agent or a pharma- ceutically acceptable salt thereof that should be effective in a given treatment regimen, based on the knowledge of the treating professional, as well as taking into account the parameters of efficacy and potential toxicity. As is understood in the art, an effective amount can be administered in one or more doses.

[0027] "Hybrid vaccination" refers to a vaccination scheme in which a booster is administered via a different route than the prime vaccine. For example, a hybrid vaccination includes a prime vaccine administered intramuscularly and a booster vaccine administered intranasally.

[0028] "Mucosal virus" refers to a virus that infects a subject through mucous membrane. For example, mucosal virus can infect through eyes, nose, mouth, airway, and other anatomical mucous membranes. Illustrative mucosal viruses are coronavirus, influenza virus, rhinovirus (HRV), adenovirus, respiratory syncytial virus, enterovirus, parainfluenza virus, paramyxovirus, human metapneumovirus, human bocavirus, etc.

[0029] In the present invention, it has been surprisingly found that an intranasal booster comprising a detoxified E. coli labile toxin (LT), such as LTh(αK), and an antigen derived from a mucosal virus, such as coronavirus and influenza virus, can induce both strong humoral and mucosal immune responses. Thus, the present invention relates to an intranasal booster comprising a detoxified E. coli labile toxin (LT) and an antigen derived from a mucosal virus.

[0030] The present disclosure relates to methods for enhancing humoral and mucosal immunity and Th1, Th2, and Th17 cytokine production resulting in Th1 / Th2 balance. Accordingly, the present disclosure provides a method for vaccinating a subject using an intranasal booster comprising detoxified E. coli labile toxin (LT) and an antigen derived from a mucosal virus.

[0031] In one embodiment, the detoxified LT is LTh(αK). LTh(αK) corresponds to LTS61K disclosed in US Pat. No. 5,999,491, which is a detoxified E. coli LT holotoxin having a lysine substitution at position 61 of SEQ ID NO:5 disclosed in US Pat. No. 5,999,491.

[0032] In one embodiment, the coronavirus is a virus of the Orthocoronavirinae family. 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.

[0033] In one embodiment, the antigen from coronavirus is a spike protein. In a preferred embodiment, the spike may be an immunogenic portion of the spike. In a more preferred embodiment, the immunogenic portion of the spike may be recombinant. In a further preferred embodiment, the recombinant antigen may be a multimer, such as a trimer.

[0034] In one embodiment, the influenza virus is a virus of the Orthomyxoviridae family. In a preferred embodiment, the influenza virus is a seasonal influenza virus. In a more preferred embodiment, the influenza virus is an A / Victoria / 2570 / 2019 (H1N1)pdm09-like virus; an A / Darwin / 9 / 2021 (H3N2)-like virus; a B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus; or a B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus.

[0035] In one embodiment, detoxified E. coli labile toxin (LT) is administered to a subject that has been previously primed. In another embodiment, the subject is primed by coronavirus exposure. In another embodiment, the subject is primed by influenza virus exposure. In one preferred embodiment, the subject is primed via the intramuscular route. In one preferred embodiment, the subject is primed via the intranasal route.

[0036] In one embodiment, the intranasal booster is administered one or more times to the subject after the subject has been primed by coronavirus or influenza virus exposure. In a preferred embodiment, the intranasal booster is administered once to the subject after the subject has been primed by coronavirus or influenza virus exposure. In another preferred embodiment, the intranasal booster is administered twice to the subject after the subject has been primed by coronavirus or influenza virus exposure. In another preferred embodiment, the intranasal booster is administered multiple times to the subject after the subject has been primed by coronavirus or influenza virus exposure. In another preferred embodiment, the intranasal booster is administered regularly or periodically to the subject after the subject has been primed by coronavirus or influenza virus exposure.

[0037] In one embodiment, the intranasal booster induces both humoral and mucosal immune responses via enhancing one or more of serum IgG levels, serum IgA levels, and mucosal IgA levels. In one preferred embodiment, the enhancement of the humoral and mucosal immune responses increases neutralizing antibody titers.

[0038] In one embodiment, the intranasal booster enhances Th1, Th2, and Th17 cytokine production by increasing levels of IFN-γ, IL-2, IL-5, IL-13, and IL-17.

[0039] In one embodiment, the increased humoral and mucosal immune response comprises an upregulation of an immune component. In another embodiment, the increased humoral and mucosal immune response comprises a downregulation of an immune component. More preferably, the increased humoral and mucosal immune response provides a prophylactic or preventative benefit.

[0040] The present disclosure further provides a method of vaccinating a subject against a mucosal viral infection, the method comprising administering to the subject an immunologically effective amount of an intranasal booster as described herein, the subject having been previously primed. In one embodiment, the method of the present disclosure provides the efficacy described herein.

[0041] The present disclosure provides an intranasal booster that is non-invasive and convenient to use for subjects who have been intramuscularly or intranasally primed with a coronavirus or influenza virus vaccine, or who have been so primed and received one or more intramuscular or intranasal booster vaccines. Subjects can administer the intranasal booster themselves, which can greatly reduce the burden on the healthcare system to vaccinate the population. Furthermore, because the intranasal booster is non-invasive, subjects will be more willing or compliant to be vaccinated. The present invention provides a new intranasal booster containing simple components (LTh(αK) and antigen) for the prevention of coronavirus or influenza virus infection, and a method of vaccinating a subject using the intranasal booster. The present invention facilitates the development of new booster vaccines for coronavirus or influenza virus infection beyond the traditional intramuscular means.

[0042] Having now generally described the present invention, the same can be more readily understood through reference to the following examples, which provide illustrative protocols for carrying out the method of the present invention in preventing the onset of coronavirus or influenza virus infection. These 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 with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0043] Working Example Materials and Assays Generation of the S-2P protein ectodomain The production of SARS-CoV-2 spike trimers for the S-preinfection form of the Wuhan strain was prepared as previously described by 19. Briefly, the SARS-CoV-2 S protein with the preinfection form was designed to contain a non-functional furin cleavage site and two stabilizing prolines in the hinge loop. The transmembrane domain and the C-terminal intracellular tail were replaced with the trimerization domain IZN4 (S-trimer) and subsequently replaced with histidine (8-mer) at the carboxy terminus for purification. Both S mutant DNA constructs were cloned into the pcDNA3.1(+) plasmid vector by GenScript (Piscataway, NJ, USA). The production of S-trimers was compiled using the ExpiCHO™ Expression System Kit (ThermoFisher Scientific, Carlsbad, CA, USA). Briefly, the S-trimer (or S-2P) was transiently expressed by ExpiCHO cells according to the manufacturer's instructions. The S-trimer-containing medium was centrifuged and dialyzed before purification on a Ni2+-NTA agarose column (GE) with 0.5 M imidazole. This antigen was used in Examples 1 and 2.

[0044] The SARS-CoV-2 spike trimer for the MVC-COV1901 vaccine was prepared as previously described (Non-Patent Document 20). Briefly, a plasmid expressing the recombinant S-2P ectodomain of SARS-CoV-2 was transfected into ExpiCHO-S cells (Thermo Fisher Scientific). The secreted S-2P protein was purified by affinity chromatography and the tag was removed by HRV3C protease, followed by a second S-2P purification. The purified S-2P protein was quantified by BCA assay (Thermo Fisher Scientific), flash frozen in liquid nitrogen, and then stored at -80°C before use. This antigen was used in Examples 3 and 4.

[0045] Collection of mucosal samples Nasal washes were obtained 2 weeks after the boost on day 42. Nasal washes were collected from decapitated animals by refluxing 1 ml of PBS through the trachea. Collected nasal washes were centrifuged and nasal fluids were stored at -20°C prior to assay.

[0046] Measurement of SARS-CoV-2 recombinant S-specific antibodies Anti-SARS-CoV-2S specific serum IgG and nasal sIgA antibodies were determined by enzyme-linked immunosorbent assay (ELISA). 96-well high-binding plates (Thermo 442404) were coated overnight at 4°C with 100 μL of 5 μg / mL recombinant trimeric S protein diluted in carbonate coating buffer (pH 9.5). Plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and wells were blocked with 5% nonfat milk for 1 h at 37°C. Serially diluted sera were added to the plates in a volume of 100 μL / well, followed by incubation at 37°C for 1 h. After three washes, 100 μL of anti-mouse horseradish peroxidase-conjugated anti-IgG (Jackson ImmunoResearch, 115-035-062, 5000X dilution) or IgA antibody (KPL 5220-0367, 2500X dilution) diluted in 5% skim milk was added for 1 hour incubation, followed by rinsing three times with PBST. At the end of the washes, 100 μL of 3,3´,5,5´-tetramethylbiphenyldiamine (TMB) was added to the wells and the color reaction was allowed to continue for 5 minutes. The reaction was quenched with stop solution and the optical density was measured at OD450-655.

[0047] Generation and titration of pseudovirus Pseudoviruses expressing full-length wild-type SARS-CoV-2S protein were prepared as described in Non-Patent Document 20. Briefly, full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein expression plasmids were transduced into HEK293T cells with packaging and reporter plasmids pCMVΔ8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica, Taiwan) using TransIT-LT1 transfection reagent (Mirus Bio). Pseudoviruses were generated by omitting p2019-nCoV spike (WT). 72 hours after transfection, the supernatants were harvested, filtered, and stored at -80°C. The transducing units (TU) of SARS-CoV-2 pseudotyped lentiviruses were calculated by viability assay in response to limiting dilutions of lentivirus.

[0048] Pseudovirus-based neutralization assay Pseudovirus-based neutralization was prepared as described in Non-Patent Document 20. Briefly, HEK293-hAce2 cells (2 × 10 4 Cells / well) were seeded in 96-well plates followed by the addition of further heat-inactivated MEM-diluted mouse serum or nasal wash. The initial dilution factor for serum samples was 20, after which 2-fold serial dilutions were performed. Due to the high dilution for nasal wash samples, nasal wash neutralization was started with 2-fold serial dilutions. The diluted samples were mixed with an equal amount of pseudovirus (1000TU) and incubated before being added to the plate with cells. After incubation, the medium was replaced with fresh medium. The next day, the medium replacement was repeated and 72 hours after infection, the cells were lysed and the relative luciferase units (RLU) were measured. Luciferase activity was detected by Tecan i-control (Infinite 500). The 50% and 90% inhibitory dilution titers (ID50 and ID90) were calculated.

[0049] Cytokine analysis Cytokine analysis was performed as described in Non-Patent Document 19. Briefly, splenocyte cytokine production was evaluated using cytokine ELISA. Spleens were isolated from individual mice in each group 4 weeks after the second IN vaccination. Splenocytes were prepared by centrifugation of mesh-homogenized spleens in RPMI-1640 medium and suspended in LCM with 10% fetal bovine serum. The resulting splenocytes were plated in 24-well plates (5 × 10 6 cells / well) in 5% CO 2 The cells were cultured under reduced pressure at 37° C. After 4 days, the supernatants were harvested and the production levels of Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-5 and IL-13), and Th17 cytokine (IL-17A) were quantified by using different cytokine ELISA kits (ThermoFisher Scientific, Carlsbad, CA, USA) as described by the manufacturer's instructions.

[0050] statistical analysis Statistical analysis was performed using GraphPad Prism 9. Statistical differences between groups were assessed using the Kruskal-Wallis test (bold type) together with Dunn's multiple comparison test. Differences between treatments were assessed using two-tailed unpaired Student's t-test.

[0051] Example 1: Intranasal (IN) immunization of mice with SARS-CoV-2 spike adjuvanted with LTh(αK) significantly enhances spike-specific (S-specific) serum IgG and IgA levels Intranasal vaccination with LTh(αK)-adjuvanted S-trimer vaccine raises both SARS-CoV-2 spike-specific (S-specific) IgG and IgA. The study procedure is outlined in FIG. 1A. In the vaccination scheme described in Example 1 (FIG. 1A), 4-6 week old female mice Balb / c were purchased from BioLASCO Taiwan and randomly divided into 5 groups, 5 mice per group. All mice received 2 IN doses (nasal drops), primed on day 1 and boosted on day 14. Groups 1 to 5 received saline, 5 μg S-trimer, 5 μg S-trimer + 5 μg LTh(αK), 20 μg S-trimer, 20 μg S-trimer + 10 μg LTh(αK), respectively. Mice were bled on days 1, 14, and 28 and sacrificed on day 42. Splenocytes from all animals were harvested on the day of sacrifice. Serum IgG levels on day 42 were analyzed by ELISA. Results showed that the levels of S-specific serum IgG increased with increasing doses of S-trimer, as shown in Figure 1B for group 2 vs. group 4 and group 3 vs. group 5. In addition, adjuvanting the spike with LTh(αK) in the IN vaccine further enhanced anti-S IgG antibody titers, as shown in group 2 vs. group 3 and group 4 vs. group 5. A similar effect was found for serum S-specific IgA (Figure 1C). Enhanced S-specific IgA antibody titers could be seen in groups 3, 4, and 5, with groups 3 and 5 adjuvanted with LTh(αK) in the IN vaccine demonstrating the highest enhancement. On the other hand, groups 3 and 5 showed that the amount of spike antigen did not significantly affect S-specific IgA antibody titers when adjuvanted with LTh(αK), confirming the effectiveness of LTh(αK) as a mucosal adjuvant. Furthermore, serum neutralization analysis (Figure 1D) showed that LTh(αK) enhanced serum S-specific neutralization.

[0052] Example 2: Intranasal boosting with SARS-CoV-2 S-trimer adjuvanted with LTh(αK) activated Th1, Th2, and Th17 A balanced Th1 and Th2 cytokine profile was evident after intranasal instillation of LTh(αK)-adjuvanted vaccine. Following the immunization scheme in Figure 1A, mice were sacrificed on day 42 of the study, and splenocytes were harvested and incubated with S-trimer. After 4 days of incubation, levels of IFN-γ, IL-2, IL-5, IL-13, and IL-17 were analyzed by ELISA. Levels of Th1, Th2, and Th17 cytokines were found to be enhanced after administration of LTh(αK)-adjuvanted intranasal vaccine according to the prime and boost scheme shown in Figure 1A (Figures 2A-E). After IN administration of LTh(αK)-adjuvanted vaccine, levels of IFN-γ, IL-2, IL-5, IL-13, and IL-17 were significantly elevated (Figures 2A-E). All cytokines were significantly higher in animals receiving LTh(αK), but not in the S-trimer only group, confirming that LTh(αK) is a potent IN immune modulator for Th1, Th2, and Th17. Increasing the amount of S-trimer antigen did not result in significant enhancement of cytokines. Levels of IL-2 were significantly enhanced by increasing amounts of LTh(αK) (Figure 2B). Levels of IL-5, IL-13, and IL-17 in group 5 also showed numerical increases with increasing amounts of LTh(αK) (Figures 2C-E). Administration of high or low doses of recombinant S-trimer alone showed minimal effects on the expression of Th1, Th2, and Th17 cytokines.

[0053] Example 3: Intranasal boost with LTh(αK)-adjuvanted SARS-CoV-2 spike in intramuscularly primed mice enhances circulating IgG and secretory IgA IN booster adjuvanted with LTh(αK) enhances spike-specific sIgA and serum IgG. To investigate mucosal immune enhancement without compromising serum IgG, mice were primed with MVC-CoV1901, a SARS-CoV-2 S-2P subunit vaccine adjuvanted with aluminum hydroxide and CpG1018, followed by intranasal boost with S-2P adjuvanted with LTh(αK). In the vaccination scheme described in Example 3 (Figure 3A), 6-8 week-old female Balb / c mice were purchased from BioLASCO Taiwan and randomly divided into 5 groups, 6 mice in each group, and immunized via IM only or combined IM and IN routes as illustrated in Figure 3A. On the first day of the study, animals in all groups were intramuscularly primed with MVC-COV-1901 containing 3 μg of S-2P (Non-Patent Document 21). On day 14 of the study, animals in groups 1 and 2 were boosted intranasally with S-2P adjuvanted with LTh(αK), and animals in groups 3-5 were boosted intramuscularly with MVC-COV-1901 containing 3 μg of S-2P. MVC-COV-1901 is a vaccine approved under EUA by the Taiwan Food and Drug Administration (TFDA) that contains S-2P as an antigen and is adjuvanted with CpG1018+aluminum hydroxide (S-2P / CpG1018 / aluminum hydroxide).

[0054] As shown in the scheme in Figure 3A, mice were randomly divided and vaccinated via IM (prime) and IM (boost) alone, or hybrid vaccination with IM (prime) and IN (boost). IM prime elevated S-specific IgG on day 14 in all groups (Figure 3B). IN and IM boosters were each highly effective in enhancing S-specific IgG titers in IM-primed mice (Figure 3C). On day 28, serum from all animals was collected prior to booster addition. ELISA analysis of serum on day 28 showed that LTh(αK)-adjuvanted IN boosters induced similar (group 2, Figure 3C) or higher (group 1, Figure 3C) anti-S IgG titers compared to IM boosters (groups 3-5, Figure 3C). An additional LTh(αK)-adjuvanted booster on day 28 significantly enhanced S-specific IgG antibody titers in groups 1, 3, and 4 on day 42 (Figure 3C). After an additional IN boost on day 28, mice developed robust S-specific sIgA antibody titers regardless of prior IM or IN boost (groups 1-4, Figure 3D). IgA antibody titers to S-2P were not affected by the number of IN doses or the amount of S-2P in the booster. S-specific sIgA was within background levels for IM-only mice (group 5, Figure 3D).

[0055] Example 4: Intranasal boost with SARS-CoV-2 spike adjuvanted with LTh(αK) induces high levels of neutralizing antibodies in serum samples and nasal washes IN boosters adjuvanted with LTh(αK) are potent in mediating the generation of S-specific neutralizing antibodies in serum and nasal mucosa. Inhibition of pseudovirus carrying wild-type spike protein by sera from mice primed and boosted IM, IN, or IM and IN was tested. Sera and nasal washes from all mice were collected on day 42 and analyzed for pseudovirus-blocking activity. Neutralizing antibody titers by pseudovirus assay were calculated as TCID 90 ) and nasal wash (TCID50 ) were determined. The results are shown in Table 1 below. On day 14, two weeks after the prime, serum neutralizing antibody titers did not increase in most mice, except for some slight increases in group 1. On day 28, two weeks after the first boost, significant increases in serum neutralizing antibody titers were evident among different groups. The pseudovirus neutralizing activity via IN boost (groups 1 and 2) was not different from that of the IM boost groups (groups 3-5). The serum neutralizing antibody titers in groups 1-4 on day 42 were much higher than those on day 28. The results show that the IN booster is as effective as the IM booster, and the second IN booster effectively further increases the serum neutralizing antibody titers.

[0056] [Table 1] Analysis of the neutralizing capacity of nasal washes is a straightforward approach to demonstrate the superiority of sIgA-mediated antiviral activity by the IN booster. The results shown in Table 2 below demonstrate the significant enhancement of nasal neutralizing activity induced by the IM prime and IN boost vaccination regimen. Mucosal neutralizing capacity from one IN boost (groups 3 and 4) is as effective as group 2, which received two IN boosts. No nasal neutralizing activity was observed in IM only mice (group 5).

[0057] [Table 2] Example 5: Hybrid vaccination against influenza viruses Hybrid vaccination against influenza viruses that are also transmitted via mucosa was further evaluated. The experimental materials, methods, and assays were essentially the same as those described above, except for the antigens used in the vaccine. The antigens were the following influenza virus strains: H1N1: A / Victoria / 2570 / 2019(H1N1)pdm09-like virus; H3N2: A / Darwin / 9 / 2021 (H3N2)-like virus; B-Victoria: B / Austria / 1359417 / 2021 (B / Victoria lineage)-like viruses; and B-Phuket: B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus; Obtained from.

[0058] The vaccination procedure for the hybrid vaccine is outlined in Table 3 below.

[0059] [Table 3] Briefly, all animals received a primary inoculation of a quadrivalent seasonal influenza vaccine containing 1 μg of hemagglutinin (HA) antigen per strain via the intramuscular (IM) route for a total of four strains. Three animals were sacrificed 28 days after the prime, and serum, nasal washes, and bronchoalveolar lavage fluid (BALF) were collected and frozen prior to analysis. Two, four, or eight weeks after the prime, animals were randomly selected and equally divided into three regimens to receive an IM booster, an intranasal (IN) booster (HA only), or an IN booster adjuvanted with LTh(αK) (HA+LTh(αK)) at each interval (see Table 3 above). Four animals were included in each regimen for each interval; 28 days after the booster administration, animals were sacrificed, and serum, nasal washes, and BALF were collected for analysis. The results (Figures 4A-4D) show that IN boosters adjuvanted with LTh(αK) increase the levels of antigen-specific IgG in serum. Although boosters without LTh(αK) can increase the levels of antigen-specific IgG, the increase is smaller than that induced by boosters adjuvanted with LTh(αK). Furthermore, increases in the levels of influenza antigen-specific IgG were observed throughout the 2- to 8-week intervals.

[0060] Additionally, nasal washes and BALF from animals in the hybrid vaccination were evaluated for levels of secretory IgA against influenza antigens. Results (Figures 5A-5H) show that secretory IgA was highly enhanced in BALF from animals treated with LTh(αK)-adjuvanted IN booster. In addition, enhanced levels of influenza antigen-specific IgA, as well as serum IgG levels, were observed throughout the 2- to 8-week interval. The ability of the IN booster to enhance IgG and sIgA levels up to 8 weeks after prime indicates that the IN booster can be applied with a flexible time window in the hybrid vaccination scheme.

[0061] Example 6: A short interval (½ hour) between IM prime and IN boost does not show a boosting effect on immunogenicity to vaccine antigens In this study, all animals received a primary inoculation as described in Example 5 above. As shown in Figures 6A-6D, serum IgG against influenza viruses H1N1, H3N2, B-Victoria, and B-Phuket were induced by IM prime, respectively. IN booster administration was performed 30 min after prime administration. IgG levels were not elevated by IN booster, regardless of whether the booster contained Flu antigen, LTh(αK), or both. The results suggest that the interval between IM prime and IN boost to successfully boost the immune response is unpredictable.

Claims

1. A method for vaccinating a subject against a mucosal viral infection, comprising administering to the subject an immunologically effective amount of an intranasal booster, the intranasal booster comprising detoxified Escherichia coli labile toxin (LT) and an antigen derived from the mucosal virus, the subject having been previously primed with an antigen.

2. The method of claim 1 , wherein the mucosal virus is selected from a coronavirus and an influenza virus.

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

4. 4. The method of claim 3, wherein the coronavirus is SARS-CoV-2.

5. The method of claim 4, wherein the antigen is a spike antigen.

6. 3. The method of claim 2, wherein the influenza virus is a seasonal influenza virus.

7. The method of claim 1, wherein the detoxified E. coli labile toxin (LT) is LTh(αK).

8. 3. The method of claim 2, wherein the subject has been pre-primed by intramuscular or intranasal vaccination.

9. 10. The method of claim 1, wherein the intranasal booster is administered multiple times.

10. 10. The method of claim 1, wherein the intranasal booster enhances one or more of serum IgG, serum IgA, and mucosal IgA levels.

11. 10. The method of claim 1, wherein the intranasal booster enhances one or more of Th1, Th2, and Th17 cytokine production.

12. The method of claim 11, wherein the cytokine is selected from IFN-γ, IL-2, IL-5, IL-13, and IL-17.

13. An intranasal vaccine composition comprising an immunologically effective amount of a mucosal viral antigen adjuvanted with detoxified Escherichia coli labile toxin (LT).

14. The vaccine composition of claim 13, wherein the mucosal virus is selected from a coronavirus and an influenza virus.

15. 15. The vaccine composition of claim 14, wherein the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

16. The vaccine composition of claim 15, wherein the coronavirus is SARS-CoV-2.

17. 17. The vaccine composition of claim 16, wherein the antigen is a spike antigen.

18. The vaccine composition of claim 14, wherein the influenza virus is a seasonal influenza virus.

19. The vaccine composition of claim 13, wherein the detoxified E. coli labile toxin (LT) is LTh(αK).

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