WO2023060078A1

WO2023060078A1 - Vaccines against coronavirus infection using lipid a analogs as advuvants

Info

Publication number: WO2023060078A1
Application number: PCT/US2022/077531
Authority: WO
Inventors: Fredrick Heath DAMRON; Justin R. BEVERE; Ting Yu WONG
Original assignee: West Virginia University Board of Governors on behalf of West Virginia University
Priority date: 2021-10-04
Filing date: 2022-10-04
Publication date: 2023-04-13

Abstract

A mammalian patient may be immunized against infection by a viral pathogen, where the viral pathogen is a coronavirus, by administering a coronavirus antigen to the mammalian patient, where the coronavirus antigen may be a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof. The coronavirus antigen may be administered in combination with an adjuvant which may be a bacterial enzymatic combinatorial chemistry (BECC)-derived Lipid A analog, monophosphoryl lipid A, or a combination thereof. The coronavirus antigen may be conjugated to a carrier protein or a virus-like particle (VLP).

Description

VACCINES AGAINST CORONAVIRUS INFECTION

USING LIPID A ANALOGS AS ADVUVANTS

TECHNICAL FIELD

[0001] Various exemplary embodiments disclosed herein relate generally to preparation of vaccines against coronavirus infection.

BACKGROUND

[0002] Coronaviruses are RNA viruses that cause diseases in mammals and birds, including humans. Some coronaviruses cause respiratory tract infections, including mild infections such as the common cold, while other coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), may cause potentially lethal infections.

[0003] Coronaviruses have protein spikes projecting from their surface. The viral spike proteins attach to complementary host cell receptor, facilitating infection. The coronavirus spike proteins include a Receptor Binding Domain (RBD) which binds to the host cell, and is made of two protein subunits, the SI spike protein and the S2 spike protein. The spike protein, as well as subunits of the spike protein, including RBD and the SI and S2 proteins, are attractive targets for development of a vaccine against coronavirus infections.

[0004] The virus SARS-CoV-2 is responsible for the COVID-19 pandemic accounting for over 4 million deaths around the world. As of January 2020, there are 216 vaccines being developed worldwide to combat SARS-CoV-2, the causative agent of COVID-19. More than 3 billion COVID- 19 vaccines that have been given worldwide. Approved vaccinations for COVID-19 around the world have been designed to be administered through the intramuscular route. Intramuscular vaccination produces a systemic immune response directed mostly by serum IgG, but no mucosal immune responses found at the site of infection. Since SARS-CoV-2 is a respiratory infection, an immune response in the respiratory tract would be advantageous.

[0005] In view of the foregoing, it would be desirable to produce mucosal immune responses in the respiratory tract. SUMM RY

[0006] In light of the present need for improved vaccines against coronavirus infection, a brief summary of various exemplary embodiments is presented. Some simplifications and omissions may be made in the following summary, which is intended to highlight and introduce some aspects of the various exemplary embodiments, but not to limit the scope of the invention. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the inventive concepts will follow in later sections.

[0007] Various embodiments disclosed herein relate to a method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method including administering a coronavirus antigen to the mammalian patient, wherein the coronavirus antigen is least one of a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; and wherein the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof.

[0008] The viral pathogen may be severe acute respiratory syndrome coronavirus (SARS- CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Canine coronavirus HuPn-2018 (CCoV-HuPn-2018), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKUl (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63), or a combination thereof.

[0009] The viral pathogen may be severe acute respiratory syndrome coronavirus (SARS- CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0010] In various embodiments, the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC).

[0011] In various embodiments, the coronavirus antigen is a recombinant coronavirus RBD, where the recombinant coronavirus RBD has at least 80% sequence identity to an RBD from the viral pathogen; or a recombinant coronavirus spike protein, where the recombinant coronavirus spike protein has at least 80% sequence identity to a spike protein from the viral pathogen.

[0012] In various embodiments, the coronavirus antigen is administered intranasally, intravenously, intramuscularly, subcutaneously, intradermally, or intraperitoneally. The coronavirus antigen may be administered intranasally or intramuscularly.

[0013] Various embodiments disclosed herein relate to a vaccine composition, including a coronavirus antigen conjugated to a carrier protein, where the coronavirus antigen comprises a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof. The carrier protein may be detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); the outer membrane protein complex of serogroup B meningococcus (OMPC) , or a combination thereof. The vaccine composition includes an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier.

[0014] Various embodiments disclosed herein relate to a vaccine composition, including a coronavirus antigen conjugated to a carrier protein, where the coronavirus antigen comprises a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof. The vaccine composition includes an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier. The coronavirus antigen may be conjugated to a carrier protein, where the carrier protein is a detoxified diphtheria toxin produced in E. coli.

[0015] Various embodiments disclosed herein relate to a method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method comprising intranasally administering a coronavirus antigen to the mammalian patient, wherein the coronavirus antigen is least one of a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; wherein the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus, an inactivated virus, and a virus-like particle.

[0016] In various embodiments, the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (IT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC); and administered intranasally in combination with an adjuvant selected from the group consisting of a live attenuated virus, an inactivated virus, and a virus -like particle.

[0017] In various embodiments, the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus and an inactivated virus, and the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of DT, TT; a detoxified diphtheria toxin produced in E. coli; PD; and OMPC); or the coronavirus antigen is conjugated to a virus-like particle.

[0018] In various embodiments, the coronavirus antigen is administered in combination with a live attenuated virus as an adjuvant, wherein the adjuvant is a live attenuated virus obtained from a live attenuated influenza vaccine.

[0019] In various embodiments, the coronavirus antigen is administered in combination with an inactivated virus as an adjuvant. In certain embodiments, the inactivated virus is obtained from an inactivated influenza vaccine.

[0020] In various embodiments, the coronavirus antigen is administered in combination with a virus -like particle as an adjuvant.

[0021] Various embodiments disclosed herein relate to a vaccine composition, including a coronavirus antigen. The coronavirus antigen may be a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; and the coronavirus antigen may be conjugated to a virus-like particle (VLP). The vaccine composition may contain an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier. The vaccine composition may be used in a method of immunizing a mammalian patient against infection by a viral pathogen, where the viral pathogen is a coronavirus, by intranasally administering the vaccine composition to the mammalian patient. [0022] In various embodiments, a vaccine composition including a coronavirus antigen conjugated to a VLP may be used in a method of immunizing a mammalian patient against infection by a coronavirus by intranasally administering the vaccine composition to the mammalian patient. The coronavirus may be severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Canine coronavirus HuPn-2018 (CCoV-HuPn-2018), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKUl (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), or Human coronavirus NL63 (HCoV-NL63).

[0023] The coronavirus antigen may be a recombinant coronavirus RBD, wherein the recombinant coronavirus RBD has at least 80% sequence identity to an RBD from the viral pathogen; or a recombinant coronavirus spike protein, wherein the recombinant coronavirus spike protein has at least 80% sequence identity to a spike protein from the viral pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A and IB illustrate vaccination protocols for testing vaccines with RBD or RBD- CRM-197 complexes in CD1 mice or in an hACE2 -transgenic mouse model for SARS-CoV-2 infection, respectively. The procedure of FIG. 1 A is used for testing vaccine immunogenicity, without a step of viral challenge. The procedure of FIG. IB is used for testing vaccine efficacy, and includes a step of viral challenge following administration of a vaccine boost.

[0025] FIGS. 2A and 2B show COVID-19 vaccine immunogenicity screens. 12 COVID-19 vaccine formulations were administered intranasally (FIG. 2A) or intramuscularly (FIG. 2B) in CD-I mice in two doses. A heat map depicts the AUC450 values from RBD-IgG titers at 2 weeks post prime (left) and 2 weeks post boost (right). The maximum AUC450 value is set at 300,000, and the minimum is at 0. The tested formulations include:

An RBD antigen with a whole cell pertussis adjuvant (wP), an acellular pertussis vaccine adjuvant (DTaP), BECC 438, or BECC 470; and

An RBD-CRM-197 complex antigen with a whole cell pertussis adjuvant (wP), an acellular pertussis vaccine adjuvant (DTaP), an irinotecan (IRI) adjuvant, a CpG oligonucleotide adjuvant, a combination of DTaP and a CpG oligonucleotide adjuvant, BECC 438, or BECC 470.

[0026] FIG. 3 shows how mice were monitored for disease symptoms and overall health. [0027] FIG. 4 shows that RBD-EcoCRM adjuvanted with BECC 470 elicited robust antibody responses in GDI mice. CD-I mice were IN or IM vaccinated with BECC470 with RBD or RBD- EcoCRM in two doses. Serum was taken at 2 weeks post prime, 2 weeks post boost, and 22 weeks post boost. Logio AUC450 values are depicted for each vaccine. Naive represents the group that received no vaccine, 470 represents BECC 470 adjuvant, RBD represents receptor binding protein, and RBD-CRM denotes RBD-EcoCRM®.

[0028] FIGS. 5 and 6 show that intranasal administration of BReC-CoV2 in K18-ACE2 mice reduced symptoms arising from SARS-CoV2 challenge. FIG. 5 shows weight change from 100% initial weight for the NVC (no vaccine, challenged), IN and IM groups of mice. FIG. 6 shows temperature as a function of time for the NVC, IN and IM groups of mice.

[0029] FIGS. 7A to 7D show clinical health scores the NVC, IN and IM groups of mice, where a higher score correlates with poor health. If a mouse reached a clinical health score of 5 or above, the mouse was euthanized, but the score was retained downstream for health score analysis.

[0030] FIG. 8 shows the Kaplan Meier survival curve for NVC mice (n=8), mice vaccinated with IN BreC-CoV-2 (n=9), and mice vaccinated with IM BreC-CoV-2. NVC mice had 31% survival, IN mice had 89% survival, and IM mice had 60% survival.

[0031] FIGS. 9A to 9C show that IN vaccination with BreC-CoV-2 decreases viral burden in the lung and brain. 100 ng of lung and brain was used to perform qPCR analysis on the SARS-CoV- 2 nucleocapsid RNA in the lung. FIG. 9A shows a violin plot depicting the SARS-CoV-2 viral RNA copies in the right lobe of the lung, with white dotted line representing the median for each group plot. Ordinary one-way ANOVA with Sidak’s multiple comparisons test was used to perform statistical analysis. Viral burden in the lung was significantly lower in mice vaccinated intranasally than in:

NVC mice (P=0.0007), and mice vaccinated intramuscularly (P=0.0436).

[0032] FIG. 9B shows a violin plot depicting the SARS-CoV-2 viral RNA copies in the left lobe of the brain, with white dotted line representing the median for each group plot. An unpaired T- test was performed for statistical analysis. Viral burden in the brain was significantly lower in mice vaccinated intranasally than in NVC mice (P=0.023). FIG. 9C shows a violin plot depicting the SARS- CoV-2 viral RNA copies in nasal wash (NW). 500pL of NW was assessed for qPCR quantification of viral nucleocapsid RNA.

[0033] FIGS. 10A and 10B show serum RBD IgG antibody levels after vaccination with BreC-CoV-2, but prior to viral challenge before challenge. Blood was collected at 2 weeks post prime (FIG. 10A) and 2 weeks post boost (FIG. 10B), and tested for IgG antibody levels.

[0034] FIGS. 11A to 11C show RBD IgA antibody levels after vaccination with BreC-CoV-2 in the lung supernatant, nasal wash, and serum post challenge.

[0035] FIG. 1 ID shows neutralizing antibody (nAb) levels in the serum following vaccination with BreC-CoV-2.

[0036] FIGS. 12A to 12F show levels of pro-inflammatory proteins in the serum, following IN and IM vaccination with BreC-CoV-2.

[0037] FIGS. 13A to 13E show levels of pro-inflammatory proteins in the lung, following IN and IM vaccination with BreC-CoV-2.

[0038] FIGS. 14A to 14F show that IM vaccination with BreC-CoV-2 decreases both chronic and acute inflammation in the lung, whereas IN vaccination decreases acute inflammation only.

FIGS. 14A and 14B show 40X magnification and 100X magnification of the lung of NVC mice, respectively. Inflammation in the parenchyma is denoted by the asterisk, inflammation surrounding the blood vessel is marked by an arrowhead, and inflammation in the airways are denoted by an arrow.

FIGS. 14C and 14D show 40X magnification and 100X magnification of the lung of an IN BReC-CoV-2 vaccinated representative mouse, respectively. Arrows show inflammation in the airways.

FIGS. 14E and 14F show 40X magnification and 100X magnification of the lung of an IM BReC-CoV-2 vaccinated representative mouse, respectively. Inflammation in the parenchyma is denoted by the asterisk, surrounding the blood vessels marked by an arrowhead and inflammation in the airways represented by arrows.

[0039] FIGS. 14G to 141 show the chronic inflammation score of each mouse, acute inflammation score of each mouse, and total inflammation score (chronic + acute inflammation scores) of each mouse. All statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparisons test. P=0.0449 (chronic); P=0.0143, 0.150*, 0.0004*** (acute); P=0.0488, 0.0320* (total).

[0040] FIGS. 15A to 15D show that IN vaccination with BReC-CoV-2 induced antibodies that can neutralize the RBD protein of multiple SARS-CoV-2 variants of concern (VOCs). An MSD neutralization assay with RBD and the Spike protein of the variants of concern with ACE2 was performed. All values are represented by the logw AUG of the electrochemiluminescence emitted from the MSD plate reader. Results from IN vaccination led to statistically significant neutralization of RBD. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed for statistical analysis. P=0.0261* (RBD-Wu), P=0.0322* (RBD-B.1.1.7), P=0.0062**,

P=0.0009***(RBD-B.1.351) P=0.0361* (RBD-P.l).

[0041] FIGS. 15E to 15H show that IN vaccination with BReC-CoV-2 induced antibodies that may neutralize the Spike protein of some SARS-CoV-2 VOCs. An MSD neutralization assay with RBD and the Spike protein of the variants of concern with ACE2 was performed. All values are represented by the loglO AUG of the electrochemiluminescence emitted from the MSD plate reader. Results from IN vaccination led to statistically significant neutralization of RBD from the Wuhan coronavirus (WU). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed for statistical analysis. P=0.0376 * (Spike -Wu).

[0042] FIG. 16A shows that neither IN nor IM vaccination with BReC-CoV-2 produces a significant change in IgM antibodies to RBD in hACE2 mice, relative to NVC hACE2 mice or hACE2 mice administered only a prime dose.

[0043] FIG. 16B shows that IN and IM vaccination each produce a significant increase in IgG antibodies to RBD in hACE2 mice, relative to NVC mice and mice administered only a prime dose.

[0044] FIG. 16C shows that IN and IM vaccination each produced a significant increase in IgG antibodies to RBD in the lung supernatant of hACE2 mice, relative to NVC mice and mice administered only a prime dose.

[0045] FIG. 17A shows serum IgG 2a antibodies, represented by loglO AUC450, in challenged mice. IN and IM vaccination with BReC-CoV-2 produce a significant increase in IgG2a antibodies to RBD in hACE2 mice, relative to NVC hACE2 mice. Ordinary one-way ANOVA with Tukey’s multiple comparisons testwas performed for statistical analyses. P=0.0047 (IgG2a). [0046] FIG. 17B shows serum IgGl antibodies, represented by loglO AUC450, in challenged mice. IN and IM vaccination with BReC-CoV-2 produce a significant increase in IgG2a antibodies to RBD in hACE2 mice, relative to NVC hACE2 mice. Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed for statistical analyses. P<0.0001**** and P=0.0063** fed)-

[0047] FIGS. 18A, 18B and 18C show that IM whole cell pertussis with RBD (wP+RBD) generated robust Ab response in mice, but did not protect the mice from viral challenge. K18-ACE2 mice were primed and boosted with PBS or wP+RBD, then challenged with 10⁵ SARS-CoV-2 WA-1. FIG. 18A shows Kaplan Meier survival curves. NVNC mice (No vaccine, no challenge) mice showed 100% survival (black line); NVC (No vaccine, challenged) mice (grey) and mice vaccinated with wP+RBD (blue) each showed 0% survival across 7 days of challenge. FIG. 18B shows results of analyzing SARS-CoV-2 nucleocapsid RNA copies in 100 ng of supernatant from the right lobe of the lung using qPCR. Unpaired T-testwas used for statistical analysis. P=0.0099**. FIG. 18C shows RBD- IgG titers represented by loglO AUC492 values at days 14 (pre-challenge), 28 (pre-challenge) and 41 (challenged). Naive baseline is represented by a dotted line.

[0048] FIGS. 19A to 19C show IgG levels in serum from intramuscular mRNA-immunized mice and from intranasally RBD-VLP+BECC immunized mice post-prime, post-boost, and 10 days post-challenge with SARS-COV-2.

[0049] FIGS. 19D and 19E show IgA levels in nasal wash and lung supernatant from intramuscular mRNA-immunized mice and from intranasally RBD-VLP+BECC immunized mice.

[0050] FIG. 20 shows RBD binding by serum antibodies from vaccinated mice prior to viral challenge, including binding to RBD from WA-1, Alpha, Beta, Gamma, Delta, and Omicron variants of SARS-CoV-2.

[0051] FIG. 21 shows reduction of plaque formation in serum from vaccinated mice.

[0052] FIGS. 22A, 22B, and 22C show disease scores, weight loss, and temperature change, respectively, for vaccinated mice.

[0053] FIG. 22D shows survival rates of vaccinated mice.

[0054] FIG. 23A shows viral replication in the lung of vaccinated mice.

[0055] FIG. 23B shows viral nucleocapsid RNA burden in the nasal wash of vaccinated mice.

[0056] FIG. 23C viral RNA burden in the lung of vaccinated mice; [0057] FIG. 23D shows detection of viral RNA in the brain of vaccinated mice.

[0058] FIGS. 24A and 24B show the inflammation score in the lungs of vaccinated mice.

[0059] FIGS. 25A to 25F show binding of serum antibodies from vaccinated mice to RBD antigens from various SARS-COV-2 variants.

[0060] In the above figures, statistical significance in terms of P-value is shown by the following scale:

* (P<0.0445);

** (P<0.0066);

*** (P=0.0003); and

**** (p<0.0001).

DETAILED DESCRIPTION

[0061] Certain broad aspects of various embodiments disclosed herein will now be addressed.

[0062] As of June 4, 2021, SARS-CoV-2 has killed 3.7 million people and infected at least 172 million. Recently it is becoming clear that SARS-CoV-2 is mutating, and the current variants of concern are able to infect fully vaccinated people.

[0063] Intramuscular vaccines dominate today’s COVID-19 vaccine market. As COVID-19 becomes an endemic threat worldwide, discussion has picked up surrounding the necessity of seasonal and SARS-CoV-2 variant specific booster shots, on top of a completed COVID-19 vaccine course consisting of one prime dose and two boosters. The intramuscular route is a popular option for vaccine delivery strategies due to its low incidence of site-specific adverse reactions, and optimal immunogenicity for a systemic immune response. Blood supply to the muscles, compared to adipose tissues, clears adjuvants and vaccine formula inclusions better, which also increases the uptake and spread of antigen through the periphery. Subcutaneous injections are attractive as they may be less painful and easier to administer but have a higher risk of local inflammation, necrosis, and granuloma formation. This route also delays the systemic response to antigen due to low vascularity throughout the fat layer and increases the chances of antigen breakdown by enzymes. Even more attractive to recipients than subcutaneous vaccines, are those that are administered intranasally. There is only one intranasal vaccine currently approved for human use worldwide-FluMist Quadrivalent. With eight orally administered vaccines (the United States utilizes oral vaccines against rotavirus, cholera, typhoid, and adenovirus), this together makes only nine whole-virus vaccines that directly target the mucosal arm of the immune system. A protective mucosal immune response may be the key to protection against pathogens in general, and SARS-CoV-2, in particular, that primarily target and replicate in the upper respiratory tract’s mucosal surfaces.

[0064] SARS-CoV-2 causes a respiratory disease, and infects through the respiratory system. All current vaccines are delivered through an intramuscular (IM) route, which offers simplicity of administration. However, intranasal (IN) immunization may overcome some of the shortcomings of muscular vaccination. Muscular vaccination results in immunoglobulin G (IgG) production. IgG is produced by cells in the bone marrow, and it circulates through the bloodstream. However, nasal vaccination or viral infection results in IgA production, which is a critical antibody for controlling viruses and bacterial pathogens at mucosal surfaces, particularly mucosal surfaces in the respiratory system where the virus enters the body. Furthermore, nasal vaccination would induce mucosal immune responses, such as resident T cell infiltration, that would protect better than antibodies alone.

[0065] Intranasal COVID-19 vaccines are highly desirable as the COVID-19 pandemic evolves and persists. Not only would they circumvent the discomfort that the intramuscular COVID- 19 vaccines have become known for causing, but they may also induce a superior level of protection. Vaccines prepare the immune system to defend against disease-causing agents through a simulated exposure. Attenuated or inactivated whole-viruses, viral vectors, pathogen proteins, mRNA, and bacterial toxoids, are all utilized to expose the host immune system to the pathogen without an authentic exposure or infection. The resulting immunological response and memory can effectively prevent against severe disease and oftentimes death. Still, the vaccine-elicited immune response differs from a natural response to exposure, and may not always produce the most superior cellular responses. In the case of COVID-19 vaccines, studies have shown that vaccinated hosts have relatively weak neutralizing antibody responses against multiple SARS-CoV-2 variants compared to convalescent patients. Specifically, the utilization of intramuscular mRNA vaccines now and into the future as seasonal boosters poses an even bigger problem as it does little to induce respiratory immunity that is integral to stopping viral replication and therefore preventing high transmission rates from highly transmissible variants like Omicron. None of the nine COVID-19 vaccines that have been administered to the global populace as of summer 2022 is an intranasally administered vaccine. By vaccinating intranasally from the beginning, or by introducing intranasal boosters on top of a completed intramuscular vaccine schedule, the primed immune response for SARS-CoV-2 can evolve to include pathogen specific IgA antibodies as well as greater B cell and T cell responses throughout the respiratory tract.

[0066] Most vaccines used now are based on full spike(S) protein antigens, or on the SI and/ or S2 subunits of the S protein. The full spike protein is difficult to produce, and the receptor binding domain (RBD) is more easily produced and purified. However, the RBD antigen alone is not as immunogenic as the full spike. In order to improve the RBD immunogenicity, the RBD antigen may be fused to a carrier protein. Suitable carrier proteins include detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC). The detoxified diphtheria toxin produced in E. coli may be Cross Reacting Material 197 (CRM-197). CRM-197 is commonly used in polysaccharide vaccines such as

Haemophilus influenza Hib vaccines.

[0067] Upon fusing RBD to commercially available CRM-197, e.g., Economical CRM-197 (EcoCRM®), a high molecular weight antigen containing 7 RBD antigens bound to each CRM-197 molecule was developed. This novel antigen, hereinafter referred to as RBD-CRM, was used to immunize mice against various strains of SARS CoV-2. Vaccination was carried out using a variety of adjuvants, both by the intramuscular (IM) and intranasal (IN) routes.

[0068] Suitable adjuvants included lipid A analogs, including bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof. The BECC-derived lipid A analogs BECC 438 and BECC 470 were found to be suitable adjuvants, as they were bacterial derived lipid A TLR4 agonists. When BECC 470 was paired with RBD-CRM and administered by IN or IM vaccination, strong antibody responses to the RBD protein antigen occurred. To test the efficacy of these novel vaccines, the hACE2 mouse model of COVID-19 was used; such mice are highly susceptible to the SARS CoV-2 virus. The transgenic hACE2 mice used herein express angiotensin-converting enzyme 2 (hACE2), which functions as the SARS-CoV-2 receptor protein in humans. Thus, hACE2 mice are particularly susceptible to SARS- CoV-2 infection. After infection, hACE2 mice develop a respiratory disease similar to CoVid-19 in humans; this disease is generally lethal in the infected mice. [0069] The hACE2 mice were immunized and boosted with RBD-CRM/BECC 470 by IN or IM routes. The mice were challenged with 10⁴ PFU of SARS-CoV-2 strain Washington-1 (WA-1). Only 30% of non-vaccinated (naive; NVA) mice survived the challenge, whereas the IM vaccine allowed for 60% survival and the IN vaccine allowed 90% survival. IN vaccination protected and decreased viral load in the brain, lung and nasal lavage. IN vaccination showed increased IgA, IgG, and responses that helped clear the virus. Overall, decreased inflammation was observed in vaccinated mice. IN immunization with RBD-CRM/BECC 470 could provide superior protection, when compared to IM immunization with RBD-CRM/BECC 470. For protection against respiratory pathogens, nasal vaccines, unlike muscular vaccines, can offer both localized protection at the site of infection and activate systemic responses.

[0070] Various embodiments disclosed herein relate to a recombinant coronavirus vaccine that includes a Receptor Binding Domain (RBD) or a spike protein as a coronavirus antigen, conjugated to a carrier protein. The antigen may be a complete spike protein (S protein), an SI subunit of the S protein, an S2 subunit of the spike protein, or a combination thereof. The antigen may be a complete spike protein (S protein), an SI subunit of the S protein, an S2 subunit of the spike protein, or a combination thereof. Suitable carrier proteins include detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); Economical CRM-197 (EcoCRM®), a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC). Alternatively, RBD or a spike protein may be conjugated to DT, TT, PD, or OMPC. The coronavirus antigen may be an RBD or a spike protein may be a protein from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome- related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Canine coronavirus HuPn-2018 (CCoV-HuPn-2018), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKUl (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), and Human coronavirus NL63 (HCoV-NL63). In various embodiments, the coronavirus antigen is a protein from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome- related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2).

[0071] Various embodiments disclosed herein relate to a recombinant COVID-19 vaccine that includes a spike protein or an RBD as a SARS-CoV-2 antigen, conjugated to a carrier protein. Suitable carrier proteins include DT, TT, EcoCRM®, PD, and OMPC. In various embodiments, EcoCRM® may be conjugated to an RED from SARS-CoV-2 to produce a product with approximately one EcoCRM® fused to 7 RBD antigen moieties (RBD-EcoCRM™). This increases both the size and immunogenicity of the SARS-CoV-2 RBD. Alternatively, RBD may be conjugated to DT, TT, PD, or OMPC.

[0072] Optimal coronavirus vaccine immunity requires the activation of both cellular and humoral responses in regard to: activation of CD4 T cells to activate B-cell maturation to produce functional antibodies to neutralize SARS-COV-2 and decrease replication as well as B-memory responses, and stimulation of CD8 T cell production to eliminate virus infected cells, and the activation of CD8 T memory cells.

[0073] CD4 T cells include Thl and Th2 cells, and produce are known as Thl-type cytokines and Th2-type cytokines, respectively. Thl-type cytokines produce proinflammatory responses responsible for killing intracellular parasites. However, excessive Thl proinflammatory responses may produce tissue damage, so there needs to be a mechanism to counteract this. Th2-type cytokines provide an anti-inflammatory response, and counteract Thl responses. Lipid A analog adjuvants offer a balanced combination of Thl and Th2 responses, and help avoid excessive tissue damage from inflammation.

[0074] To achieve optimal vaccine protection, RBD-EcoCRM™ was supplemented with a variety of adjuvants. In various embodiments, RBD-EcoCRM™ was supplemented with a variety of adjuvants. Suitable adjuvants may include lipid A mimetics, CpG oligodeoxynucleotides, an acellular pertussis vaccine, a whole cell pertussis vaccine, and combinations thereof. Suitable lipid A mimetics include bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof.

[0075] Lipid A analogs used in vaccines disclosed herein include TLR4-agonist adjuvants produced by Bacterial Enzymatic Combinatorial Chemistry (BECC), a methodology developed to synthesize lipid A mimetics in attenuated Yersinia pestis strains with the addition and subtraction of lipid A modifying enzymes grown at different temperatures. BECC470 and BECC438, which have the structures below, were found to be efficacious when paired with coronavirus antigens. Monophosphoryl lipid A may also be used as a lipid A analog.

Monophosphoryl lipid A

[0076] In various embodiments, lipid A analogs may be used as an adjuvant to deliver a strong combined Thl/Th2 immune response and generate high titers of neutralizing antibodies and a robust cellular response against a coronavirus, e.g., SARS-CoV, MERS-CoV, and/or SARS-CoV-2. A K18- hACE2 transgenic mouse model was used to evaluate intramuscular and intranasal vaccination of mice with RBD-EcoCRM™ and an adjuvant, e.g., the lipid A analog BECC 470, against the coronavirus SARS-CoV-2. K18-hAC2 mice use a human keratin 18 promoter to drive the expression of human Ace2 in the respiratory epithelial cells, liver, kidney, brain, and GI tract. The expression of human ACE2 in their epithelial cells make these mice vulnerable for lethal SARS-CoV-2 infection after inoculation. Therefore, this SARS-CoV-2 mouse challenge model allows for the study of vaccine efficacy in regard to challenge.

[0077] A combination of RBD-EcoCRM™ and BECC 470 (where the combination is referred to herein as BReC-CoV2) was evaluated as a vaccine in the K18-ACE2 mouse SARS-CoV-2 challenge model using nasal and muscular routes of administration. The mucosal and systemic immune response induced by nasal vaccination may lead to improved outcomes in mammals exposed to SARS- CoV-2, when compared to intramuscular vaccination. Other combinations of adjuvants and the RBD- EcoCRM™ antigen have also been tested. Intranasal vaccination with RBD or RBD-EcoCRM™ in combination with a lipid A mimetic offered protection against SARS-CoV-2 challenge, compared to intramuscular vaccination.

[0078] Nasal vaccination may offer a unique protection profile and advantages to muscular vaccination. To test this hypothesis, numerous antigens and adjuvants were evaluated to identify a vaccine formulation for use in a hACE2-transgeneic mouse model. Before assessing vaccines in hACE2 mice, GDI outbred mice were primed and boosted through intranasal or intramuscular administration of the Receptor Binding Domain (RBD) of SARS-CoV-2 with various adjuvants, including acellular pertussis vaccine (DTaP) and whole cell pertussis vaccine (wP) by the protocol of FIG. 1A.

[0079] There was a strong intramuscular antibody response to RBD antigen when combined with a whole cell pertussis (wP) adjuvant, a BEGG 438 adjuvant, and a BEGG 438 adjuvant (FIG. 2B). wP has TRL4 agonist properties that promote a robust Thl immune response. Intramuscular antibody responses to RBD were superior when wP vaccine was used, compared to DTaP adjuvanted with alum (FIG. IB). The RBD-EcoCRM vaccine product showed a strong intramuscular antibody response when combined with CpG oligodeoxynucleotides, a combination of DTaP (an acellular pertussis vaccine product) and CpG oligodeoxynucleotides, a BECC 438 adjuvant, and a BECC 470 adjuvant (FIG. 2B).

[0080] There was a strong intranasal antibody response to either the RBD antigen or the RBD-EcoCRM antigen when the antigen was combined with a BECC 470 adjuvant (FIG. 2A). The RBD-EcoCRM vaccine product also showed a strong intranasal antibody response when combined with wP, a combination of DTaP and CpG oligodeoxynucleotides, or a BECC 438 adjuvant (FIG. 2A).

[0081] Due to the robust antibody responses, the intramuscular wP+RBD vaccine formulation was evaluated in a SARS-CoV-2 challenge model with hACE2 mice. The intranasal response to the RBD antigen when combined with wP or DTaP was comparatively weak (FIG. 2A).

Vaccination with wP+RBD

[0082] Despite a high anti-RBD IgG response, the wP+RBD combination failed to protect mice against SARS-CoV-2 challenge. Nine-week-old mice hACE2 mice were immunized through the intramuscular route with wP+RBD and were compared to IM saline as a control (NVC: No Vaccine Challenged). Three weeks post initial prime, the mice were boosted with the same vaccine formulations. At 2 weeks post boost, mice were challenged intranasally with the 105 USA-WA1 / 2020 (WA-1) strain of SARS-CoV-2. Mice were monitored for disease symptoms and overall health for 7 days. Weight loss, temperature change, appearance and activity, eye closure, and respiratory function were observed (FIG. 3). Each mouse was assigned a daily disease score that reflected their overall health on a defined scale. For example, no weight loss corresponded to a health score of 0, while >20% weight loss corresponded to a health score of 5. Similarly, 80-200 breaths per minute corresponded to a health score of 0, while <80 or >200 breaths per minute with gasping corresponded to a health score of 2. When the total health score reached or exceeded 5 from adding all parameters, the animal was euthanized.

[0083] Mice vaccinated with wP+RBD displayed limited weight loss or temperature change from 1 to 5 days post challenge; however, they experienced a decrease in weight and temperature at day 6, as well as an increase in disease scores, similar to the NVC mice. At day 6, all vaccinated mice were moribund and met the requirements for euthanasia, as shown in FIG. 18A. The NVC mice had 11% survival on day 6, and 0% survival on day 7, as shown in FIG. 18A. Overall, disease monitoring data showed that IM wP+RBD vaccination was not effective in protecting mice from 105 SARS-CoV- 2 challenge.

[0084] Interestingly, wP+RBD significantly decreased SARS-CoV-2 viral RNA compared to NVC, as shown in FIG. 18B. This suggests that wP+RBD was efficacious at clearing virus but not at preventing death from challenge.

[0085] Submandibular bleeds were performed throughout the immunization schedule at 2 weeks post prime and at 2 weeks post boost to collect serum for antibody analysis. Blood was also collected at euthanasia to determine anti-RBD titers at time of death. Mice that received the second dose of wP+RBD (2 weeks post boost) had significantly increased titers of anti-RBD IgG, compared to the NVC mice, as shown in FIG. 18C. Even though wP+RBD generated high anti-RBD IgG titers, this did not help elicit protection against SARS-CoV-2 challenge. Thus, intramuscular wP+RBD did not protect hACE2 mice from SARS-CoV2 challenge.

Vaccination with RBD- EcoCRM™ and adjuvants.

[0086] In an effort to improve responses to RBD, RBD was conjugated to a genetically detoxified diphtheria toxin carrier protein, economical CRM197(EcoCRM™). The purpose behind the fusion of RBD with EcoCRM™ was to enhance the immunogenicity and subsequent recognition of RBD by the immune system. Based on the wP+RBD immunogenicity and strong antibody responses, a TLR4-agonist was used to induce robust antibody responses. Bacterial Enzymatic Combinatorial Chemistry (BECC) offers adjuvants which induce a strong Thl response that can help clear viral infections. Various different adjuvants were evaluated, including Alum, wP, CpG and IRI- 1501 Beta-glucan, BECC 438 and BECC 470 (Table 1). In these preliminary vaccine studies, female CD1 outbred mice were immunized with the vaccine formulations stated (Table 1), either through the intranasal or intramuscular route. Mice were boosted 3 weeks later with the same formulation through the same routes (FIG. 1A). Serological analysis was performed at 2 weeks post prime and 2 weeks post boost (FIG. 1A). Overall, mice demonstrated modest improvement in immunogenicity with RBD-EcoCRM compared to RBD supplemented with different adjuvant combinations, both through the IM and IN routes.

[0087] Intranasally, BECC470 induced similar RBD-IgG responses formulated with RBD or RBD-EcoCRM. RBD-EcoCRM adjuvanted with CpG+DTaP elicited similar RBD-IgG responses compared to BECC 470 (FIG. 2A). However, in humans, CpG is only administered IM and would not likely be given intranasally. Intramuscularly, both RBD and RBD-EcoCRM combined with BECC 470 induced robust RBD IgG titers, with CpG and CpG+DTaP eliciting similar responses (Fig. 2B). Nevertheless, RBD-EcoCRM adjuvanted with BECC470 generated a robust RBD-IgG response both intranasally and intramuscularly compared to other adjuvants tested.

Table 1.

[0088] RBD-EcoCRM adjuvanted with BECC 470 (BReC-CoV2) elicits robust antibody responses in GDI mice. IN and IM administration of BReC-CoV2 produced robust RBD-IgG titers in the serum after boost (FIG. 4). The IN BReC-CoV2 generated a 3-log increase of anti-RBD IgG 1-week post boost from 2 weeks post prime, whereas the IM BReC-CoV2 produced a 2-log increase of anti-RBD IgG from 2 weeks post prime to 1-week post boost. An ideal CO VID-19 vaccine would need to protect long-term; therefore, we measured RBD IgG titers at 22 weeks post boost. At 22 weeks post boost, RBD-IgG titers were consistent with 2 weeks post boost in both IN and IM BReC- CoV2 vaccinated groups (Fig. 4). In addition to serological analyses, antibodies generated were tested, and found to be able to neutralize RBD binding to ACE2 in vitro at 2 weeks post boost. Overall, IM and IN BReC-CoV2 vaccines produced strong anti-RBD IgG responses that were functional at neutralizing RBD binding to ACE2.

[0089] Intranasal administration of BReC-CoV2 protected mice from SARS-CoV2 challenge.

We next aimed to test the protective capacity of IN and IM BReC-CoV-2 in a SARS-CoV-2 challenge model. K18-hACE2 mice were vaccinated with IN and IM formulations of BReC-CoV2 using the same vaccine timeline by the protocol of FIG. IB. At 2 weeks post boost, IN BReC-CoV2 (n=9), IM BReC-CoV2 (n=10), and mock vaccinated (NVC) (n=8) groups were challenged with a 10⁴PFU/ dose of the WA-1 variant of SARS-CoV-2 and monitored for disease outcomes for a 14-day period. The same disease manifestations as the wP+RBD study were assessed, including weight loss, appearance, activity, eye closure, respiration, and hypothermia. Mice were immediately euthanized if they achieved a disease score 5 or greater, which determined that they were morbid and required humane euthanasia. The daily disease score was calculated by adding the total scores of each mouse in one group. When animals became morbid and required euthanasia, the animal score was retained in the sum of the remaining days of the experiment. This disease scoring system helps predict when mice will become morbid and is inverse to the falling Kaplan Meier curve. Throughout this 14-day period, NVC and IM mice began decreasing in weight at day 7 post challenge, whereas the IN vaccinated mice gradually gained weight (FIG. 5). IN vaccinated animals, compared to NVC and IM, maintained stable rectal temperatures throughout the 2-week monitoring period, which corroborated their disease scores (FIGS. 6, 7A). However, NVC and IM vaccinated mice rectal temperature plummeted at days 7 and 8 post challenge (FIG. 6), accompanied by a large increase in clinical disease scores (FIG. 7A). Unlike the IM vaccinated mice, NVC mice were not able to recover in temperature. When evaluating the groups based on their disease scores, NVC began to increase in disease scores at day 7 and continually increased in disease scores until day 10 (FIG. 7B). IM BReC-CoV2 vaccinated mice peaked in disease scores at day 8, but then returned to normal health scores throughout the rest of the challenge trial (FIG. 7D). IN BReC-CoV2 vaccinated mice maintained low disease scores throughout the entirety of the 14-day monitoring period compared to both the NVC and IM vaccinated mice (FIG. 7C). As the weight loss, temperature loss, and disease scores illuminated, NVC mice succumbed to infection and this led to a survival of 33% (FIG. 8). IM vaccinated mice portrayed a better disease outcome than NVC, with 66% survival (FIG. 9), and IN vaccinated mice experienced significant survival compared to NVC (P=0.0332) with 90% survival (FIG. 8). Overall, a protection profile comprised of weight loss, temperature change, disease scores, and survival all indicate that IN vaccination with BReC-CoV2 compared to IM and NVC protected mice from SARS-CoV-2 challenge, suggesting that the mucosal immune response may play a role in driving protection from SARS-CoV2.

[0090] IN vaccination with BReC-CoV2 decreases viral burden in the lung and brain. As the disease monitoring data suggested, IN vaccination with BReC-CoV2 was superior in protection compared to IM and NVC mice. To corroborate the observed disease monitoring data, the viral burden of the vaccinated mice compared to the NVC was determined. Therefore, RNA copies of nucleocapsid to SARS-CoV-2 in the lung (FIG. 9A), brain (FIG. 9B) and nasal wash (NW) (FIG. 9C) were measured. In the lung, intranasal vaccination of BReC-CoV2 significantly decreased viral RNA compared to NVC mice and intramuscular vaccinated BReC-CoV2 mice, indicating that IN vaccination limits viral replication in the lung.

[0091] K18-ACE2 mice succumb to SARS-CoV-2 brain infection during challenge. IN vaccination with BreC-CoV-2 significantly decreased viral RNA in the brain compared to NVC mice, suggesting that IN vaccination prevented the dissemination of virus into the brain (FIG. 9B). The NW study showed that IN vaccination decreased viral copies, compared to NVC and IM vaccination; however, these differences were not statistically significant (FIG. 9C). Overall, there was a significant reduction of viral RNA copies in the lung of IN vaccinated mice compared to IM vaccinated mice and NVC mice, a significant decrease of viral RNA in the brain of IN vaccinated mice compared to NVC mice, as well as fewer viral RNA copies in the NW. Decreased detection of viral RNA suggested that IN BReC-CoV-2 vaccination was the superior route, compared to IM BReC-CoV-2 vaccination.

[0092] Both IM and IN BReC-CoV2 RBD antibody responses increase during SARS-CoV-2 challenge. To investigate the antibody responses generated by BReC-CoV2 vaccination, RBD specific IgG production was analyzed systemically and then locally in the lung. Systemic RBD IgG was measured before challenge and after challenge with SARS-CoV-2. In order to measure serum RBD IgG before challenge, blood was collected at 2 weeks post prime (FIG. 10A) and 2 weeks post boost (FIG. 10B). At 2 weeks post prime, both IN and IM vaccination begin to generate detectable RBD IgG titers, with the IM vaccination generating higher RBD titers than both NVC and IN (FIG. 10A). Both IN and IM BReC-COV2 vaccinated groups induced a robust response to boost vaccination, but IM vaccination elicited a higher RBD-IgG titer than either IN vaccination or NVC control mice (FIG. 10B). Post challenge, serum RBD IgG was significantly elevated in both IN and IM vaccinated groups compared to NVC (FIG. 10B). Interestingly, before SARS-CoV-2 challenge, IM vaccinated mice generated 1.06 log more RBD IgG titers at 2 weeks post boost than IN vaccinated mice at 2 weeks post boost, indicating that challenge increased antibody production in IN vaccinated mice. In the lung supernatant, similar to the serum, RBD IgG were significantly increased in both the IN and IM vaccinated mice compared to the NVC mice, indicating no difference between the IN and IM RBD IgG titers in the lung. The similarities between the IgG levels between both vaccines hinted that the immune response occurring during IN vaccination is more dependent on a localized immune response rather than a systemic response. Therefore, anti-RBD IgA levels in the lung, NW, and serum were investigated to assess mucosal antibody response to IN and IM vaccination.

[0093] IN BReC-CoV2 generated a robust localized IgA response compared to IM vaccination. In the lung supernatant, NVC and IM vaccinated mice did not generate RBD specific IgA compared to IN BReC-CoV2 vaccination, demonstrating that the mucosal antibody response was playing a large role in clearing SARS-CoV-2 infection (FIG.11A). To further confirm the findings that the mucosal antibody response was contributing significantly to protection, anti-RBD IgA in the nasal wash was analyzed. Similar to the lung supernatant, IN vaccination significantly increased RBD-IgA compared to the undetectable IgA amounts in the NVC and IM vaccinated groups (FIG.1 IB). Serum RBD IgA titers were elevated in IN vaccinated mice, but not in NVC and IM mice. However, post challenge, there was no change in the serum RBD IgA titers in any groups (FIG. 11C). In summary, both IN and IM vaccination generated similar IgG responses in the lung and serum; however, IN vaccination induced a stronger IgA response in the lung and NW compared to IM, suggesting that the mucosal antibody response is potentially important in facilitating clearance of SARS-CoV-2 in the respiratory tract.

[0094] However, IN and IM vaccination with BReC-CoV2 each lead to a significant increase (P<0.05) in neutralizing antibodies (nAbs) in the serum, as shown in FIG. 11D. Antibody analysis of IN and IM BReC-CoV-2 vaccination detected high levels of RBD specific IgG and IgA; therefore, these antibodies were tested to determine if they were functional in neutralizing RBD binding to ACE2. Both IN and IM vaccinated mice had significant neutralizing antibody titers compared to NVC in the serum, demonstrating that both IN and IM vaccination generated functional antibodies that can neutralize RBD.

[0095] BECC470 induces Thl/Th2 responses in both IN and IM BReC-CoV-2 vaccination. Pre-clinical vaccine studies using BECC470 as an adjuvant have shown that BECC470 generates a balanced Thl/Th2 immune response. To investigate the Thl and Th2 immune response elicited by IN and IM BReC-CoV-2 vaccination, IgGl (Th2) and IgG 2a (Thl) subtypes were analyzed in the serum. Both IN and IM BReC-CoV-2 vaccination induced significant RBD specific IgG2a and IgGl responses compared to NVC, as shown in FIGS. 17A and 17B. Intranasal and intramuscular vaccination each generated a significant increase in IgGl antibodies. As shown in FIG. 17B, intramuscular BReC-CoV-2 vaccination generated a significant increase in IgGl, indicating a Th2 biased response in IM vaccination. Also as shown in FIG. 17B, IN vaccination generated a significant increase in IgGl; however, IgGl antibody titers generated by IN vaccination are reduced relative to IgGl antibody titers generated by IM vaccination by about an order of magnitude. NVC mice did not show a significant increase in IgGl antibodies. As shown in FIG. 17B.

[0096] Referring now to FIGS. 17A and 17B, NVC mice had an expected increase in IgG2a compared to IgGl indicating a Thl response to viral infection. Referring to FIG. 17A, intranasal and intramuscular vaccination each generated a significant increase in IgG2a antibodies, relative to NVC mice. There was no significant difference between IgG2a antibodies generated by intranasal vaccination and IgG2a antibodies generated by intramuscular vaccination. Overall, both IN and IM vaccination induced IgGl/IgG2a ratios indicative of a balanced Thl/Th2 immune response.

[0097] The proposed BReC-CoV2 vaccine product was tested against several SARS CoV-2 variants, in addition to the WA-1 strain. An MSD (Meso Scale Discovery) COVID-19 ACE2 neutralization multiplex assay was used to analyze neutralization of the RBD and spike protein of Wuhan coronavirus (WU), and several variants of concern (VOC), including the B.l.1.7, B.1.351, and P.l variants. Neutralization of RBD or Spike binding to ACE2 was measured through electrical chemiluminescent (ECL) signal intensity for NVC, IN and IM vaccinated mice. The higher the signal the less neutralization and the less intense the signal the more neutralization capability. NVC mice, as expected, had no neutralization between the VOCs, as shown in FIGS. 15A to 15H.

[0098] IN vaccinated mice had significantly higher neutralization capacity than either IM vaccinated mice or NVC mice for the RBD protein of the WU and B.l.1.7 coronavirus variants (FIGS. 15A and 15B); and comparable neutralization capacity against the RBD protein to IM vaccination against the B.1.351 and P.l variants (FIGS. 15C and 15D).

[0099] For whole spike neutralization, IN vaccination generated higher levels of neutralizing titers against the Wuhan spike or the B.l.1.7 spike, compared to IM vaccination or NVC mice (FIGS. 15E and 15F). IN vaccination was less effective against the spike proteins of the B.1.351 and P.l variants (FIGS. 15G and 15H).

[00100] Increased levels of serum CXCL13 in NVC and IM BReC-CoV-2 vaccination indicate poor disease prognosis. CXCL13 is an important chemokine marker for germinal center activity, B- cell maturation, and memory B-cell and plasma cell formation. Therefore, it is a potential biomarker for long-term vaccine efficacy. It has been shown that COVID-19 patients that have a poor clinical prognosis, had increased circulating CXCL13 compared to patients who survived COVID-19. CXCL13 potentially can be a biomarker for strong antibody responses during either vaccination or during infection. Therefore, CXCL13 levels were measured to vaccination before and after SARS- CoV-2 challenge to observe if vaccinated and challenged mice followed the same trend. At prechallenge, CXCL13 was evaluated at 2 weeks post prime and 2 weeks post boost. CXCL13 levels were significantly increased at both prime and boost in the IM BReC-CoV2 compared to the IN vaccinated mice, indicating germinal center formation and high affinity antibody development after vaccination. For both IN and IM vaccination, there are no changes between IN prime and IN boost or IM prime and IM boost, suggesting that CXCL13 levels peaked after prime. After challenge, NVC mice with a 33% survival had increased CXCL13 serum levels, compared to naive, unchallenged (NVNC) mice (FIG. 12A). Post challenge, mice with IM vaccination with BreC-CoV2 (66% survival) exhibited significantly increased levels of CXCL3 in the serum, compared to NVC, naive, and IN vaccinated mice (FIG. 12A). Surprisingly, even though IN vaccinated and IM vaccinated mice generated similar titers of RBD IgG, IN vaccination (90% survival) induced significantly less CXCL13 than IM vaccination and NVC (FIG.12A), suggesting antibody responses limited viral replication and progression to mortality. Intranasal vaccination with BReC-CoV-2 did not lead to significant changes in serum levels of IFNy, TNFa, IL6, or C-reactive protein (CRP) (FIGS. 12B-12E). Intramuscular vaccination with BReC-CoV-2 produced a significant decrease in C-reactive protein (CRP) (FIG. 12E). As shown in FIG. 12F, CXCL13 levels (measured as loglO pg/mL) in pre-challenged serum were increased from baseline after a prime with either IM or IN BReC-CoV2 vaccination, and then further increased after a boost vaccination. Two-way ANOVA was performed for statistical analysis; the difference in CXCL13 levels after boost was significantly different increased from levels after the prime (P<0.0001). The baseline value for naive mice is represented as dotted line at 1.861183.

[00101] Both vaccinated animals showed increased CXCL13 in the lung (FIG. 13A). These results implicate that the germinal center antibodies generated during IN vaccination were able to protect mice from challenge. The antibodies generated from IM vaccination were not sufficient in clearing infection, therefore prolonging the secretion of CXCL13 and leading to more production of high affinity antibodies. [00102] IN vaccination with BReC-CoV-2 significantly decreased IFNy in the lung, as compared to IM vaccination. The difference in IFNy in the lung in IM vaccinated mice and NVC mice was not significant. SARS-CoV-2 is known to cause inflammation in the lung and induce interferon responses. IN vaccination may help decrease inflammatory markers in the lung. Inflammatory cytokines in the lung supernatant were measured after SARS-CoV-2 challenge. Compared to the NVC and IM vaccination, IN vaccination significantly lowered IFNy in the lung supernatant (FIG. 13D) compared to intramuscular vaccination, while other pro-inflammatory cytokines, including TNFa, IL6, and C-reactive protein (CRP) remained similar between IN vaccinated mice and IM vaccinated mice (FIGS. 13B, 13C, and 13E). CRP was significantly decreased to a similar extent in both IN and IM vaccinated groups in the lung, compared to NVC mice. IL6 and TNFa levels in the lung were not significantly different between IN and IM vaccinated groups, or between either vaccinated group and NVC mice. Overall, vaccination decreased inflammation in the lung, with:

IN vaccination decreasing both IFN-y and CRP compared to NVC; and IM vaccination decreasing CRP compared to NVC.

[00103] IM vaccination decreases both chronic and acute inflammation in the lung whereas IN vaccination decreases acute inflammation only. The left lobe of the lung was subjected to H&E staining, for histopathological analysis for chronic and acute inflammation. Chronic inflammation was scored by the presence of recruited lymphocytes, plasma cells, and macrophages in the parenchyma and blood vessels. Acute inflammation was denoted by the infiltration of neutrophils and the presence of edema in the parenchyma, blood vessels and airways. NVC mice scored the highest in acute inflammation, and an average overall inflammation score of 4.5, compared to NVNC mice with an inflammation score of 0.3 (FIGS. 14G and 14H). IN vaccinated mice had increased chronic inflammation scores (3.8) compared to NVNC mice (0.33), NVC mice (3.1), and IM vaccinated mice (2.7) (FIGS. 141), with the presence of plasma cells, lymphocytes, and macrophages localized around blood vessels in IN vaccinated mice (FIGS. 14C and 14D). IN mice scored an average inflammation score of 4.1, lower than NVC (FIG.141). Mice with IM vaccination with mBreC-CoV2 had lower chronic and acute inflammation scores than either NVC or IN vaccinated mice, with an overall total inflammation score of 2.8 (FIGS.14G, 14H and 141). IM mice had mostly chronic inflammation found in the parenchyma, blood vessels and bronchi (FIGS.14E and 14F). Overall, IM vaccinated mice had less acute and chronic inflammation than NVC or IN vaccinated mice, suggesting that IN vaccination mimicked natural infection by recruiting cells into the lung to fight viral infection.

[00104] An intensive immunogenicity screen for immunogenic vaccine antigen and adjuvant combinations in outbred mice was performed. Twenty-four different vaccine combinations were tested through both the intranasal and intramuscular route. RBD with a whole cell pertussis adjuvant (wP+RBD) administered through the muscular route was the first vaccine candidate that produced robust RBD IgG titers compared to other vaccines that were screened. However, wP+RBD did not protect KI 8 hACE2 mice that were challenged with SARS-CoV-2. Since IM wP+RBD was not protective, an intranasal route utilizing RBD-EcoCRM and an adjuvant (BECC470) that induced similar responses was tested.

[00105] Using the K-18 hACE2 mouse model, it was determined that intranasal vaccination with BReC-CoV-2 offered greater protection against SARS-CoV-2 than to IM vaccination. IN vaccination with BReC-CoV2 increased survival rate, decreased disease scores, and maintained weight and temperature in the IN group throughout infection, when compared to IM vaccinated mice and NVC mice. Nasal vaccination decreased viral burden in the lung compared to IM vaccination and NVC subjects, as well as increasing RBD IgA titers in the lung and nasal wash compared to IM vaccination and NVC. Intranasal vaccination with BReC-CoV2 decreased IFNy in the lung compared to IM and NVC. However, histopathological analyses showed an increase of recruitment of lymphocytes, macrophages and plasma cells to blood vessels in the lung compared to IM vaccination. [00106] The ancestral SARS-CoV-2 WA-1 strain was used to challenge mice in preparing this disclosure. Serum from IN BReC-CoV-2 vaccination was able to significantly neutralize RBD-ACE2 from several SARS-CoV-2 variants of concern. IN BReC-CoV-2 vaccination was superior to or comparable to IM vaccination for each variants of concern, suggesting that IN BReC-CoV-2 vaccination is effective in SARS-CoV-2 vaccination against multiple variants.

[00107] CD4+ and CD8+ T-cells play a large role in clearing and controlling SARS-CoV-2 infection. Studies have shown that in humans, resident T-cells in the lung instead of in circulation were linked with better disease prognosis and survival. In other intranasal vaccination studies for bacterial and viral pathogens, T resident memory cells are elevated in the lung and nasal associated lymphoid tissue. Since BECC470 is a strong driver of Thl immune responses, IN BReC-CoV-2 may also elicit robust T resident memory responses that will contribute to protection. [00108] Bacterial components can serve as potent adjuvants for either bacterial or viral vaccines. Adjuvants such as ASO4 (Monophosphoryl lipid A + aluminum salt) and ASO1 (Monophosphoryl lipid A + QS-21 (Chilean soapbark tree extract)), are currently being used in vaccines for HPV and shingles. As disclosed herein, BECC 470 was used as an adjuvant to supplement the RBD-EcoCRM vaccine product. BECC 438 and monophosphoryl lipid A (MPLA) are similar to BECC 470, with each molecule being generated from lipid A, each molecule being a TLR4-agonist, and each driving a robust Thl immune response. BECC 438 and BECC 470 are different from MPLA in the way that they are synthesized. Bacterial enzyme combinatorial chemistry (BECC) was developed as an alternative route to produce lipid A mimetics. It uses methodology that generates products that are cost effective, easy to produce, and malleable to changes in the immune response. Additionally, BECC molecules with Aluminum hydroxide have been shown to be efficacious ASO4 biosimilar adjuvants. Currently, most vaccine compositions administered to humans contain an Alum adjuvant. Therefore, it is important to investigate adjuvants that can help drive not only the Th2 response that Alum induced by Alum, but also a Thl response as generated by BECC 470, especially for intracellular pathogens.

[00109] EcoCRM®, the carrier protein of used for the RBD antigen, is also derived from bacteria. EcoCRM®, or CRM197, is detoxified diphtheria toxin obtained from Corynebacterium diphtheriae. The vaccine DTaP (Diphtheria Tetanus Pertussis) is a widely used vaccine for prevention infection of three bacterial diseases. Primary vaccination with DTaP followed by booster vaccination with RBD-EcoCRM increases Diphtheria IgG compared to a single dose of DTaP or a single vaccine of RBD-EcoCRM. EcoCRM as a carrier protein could increase diphtheria specific antibodies; thus, potentially increasing the protective capacity against Diphtheria infection. Overall, utilizing bacterial components as adjuvants in vaccines has strong advantages such as increasing immunogenicity of weakly immunogenic antigens, inducing strong cellular responses to promote clearance of intracellular pathogens, and allowing for the possibility of a bacterial-viral combination vaccine.

[00110] Various embodiments disclosed herein relate to a method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method comprising intranasally administering a coronavirus antigen to the mammalian patient, wherein the coronavirus antigen is least one of a coronavirus Receptor Binding Domain (RBD, a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; and the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus, an inactivated virus, and a virus-like particle.

[00111] In various embodiments, the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (IT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC); and administered intranasally in combination with an adjuvant selected from the group consisting of a live attenuated virus, an inactivated virus, and a virus -like particle.

[00112] In various embodiments, the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus and an inactivated virus, and the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of DT, TT; a detoxified diphtheria toxin produced in E. coli; PD; and OMPC); or the coronavirus antigen is conjugated to a virus-like particle.

[00113] In various embodiments, the coronavirus antigen is administered in combination with a live attenuated virus as an adjuvant, wherein the live attenuated virus is obtained from at least one of a live attenuated influenza vaccine; a live attenuated Japanese encephalitis vaccine; a live attenuated measles vaccine; a live attenuated mumps vaccine; a live attenuated measles and rubella (MR) vaccine; a live attenuated measles, mumps, and rubella (MMR) vaccine; a live attenuated measles, mumps, rubella and varicella (MMRV) vaccine; a live attenuated polio vaccine; a live attenuated rotavirus vaccine; a live attenuated rubella vaccine; a live attenuated smallpox vaccine; a live attenuated varicella vaccine; a live attenuated yellow fever vaccine; and a live attenuated zoster/ shingles vaccine.

[00114] In various embodiments, the coronavirus antigen is administered in combination with a live attenuated virus as an adjuvant, wherein the adjuvant is a live attenuated virus obtained from a live attenuated influenza vaccine. In various embodiments, the live attenuated influenza vaccine is a live attenuated influenza vaccine strain which may be A/ Ann Arbor/6/60 (H2N2), A/H1N1, A/H3N2, or Type B influenza virus. The live attenuated influenza vaccine may be Nasovac-S as the influenza strain, where Nasovac-S contains three live attenuated virus strains, including A/H1N1, A/H3N2 and Type B influenza virus, cultivated on embryonated hen eggs. The live attenuated influenza vaccine may be FluMist, which contains two type A influenza virus strains (H1N1 and H3N2), and one type B (Victoria or Yamagata) influenza virus strain. The live attenuated influenza vaccine may be FluMist Quadrivalent, which contains two type A influenza virus strains and two type B influenza virus strains.

[00115] In various embodiments, the coronavirus antigen is administered in combination with an inactivated virus as an adjuvant, wherein the inactivated virus is obtained from at least one of an inactivated polio vaccine, an inactivated hepatitis A vaccine, an inactivated rabies vaccine, an inactivated influenza vaccine, and an inactivated tick-bome encephalitis vaccine. In certain embodiments, the inactivated virus is obtained from an inactivated influenza vaccine.

[00116] In various embodiments, the coronavirus antigen is administered in combination with a virus -like particle as an adjuvant.

[00117] In summary, the present disclosure demonstrates that intranasal administration of BReC-CoV-2 confers superior protection against SARS-CoV-2 challenge in hACE2 mice, compared to intramuscular vaccination. Not only did IN vaccination protect mice against challenge, but IN vaccination produced high neutralizing titers against VOCs, induction of IgA in the lung and nasal wash and a substantial reduction of viral burden in the lung. Overall, intranasal vaccination with BReC- CoV-2 offers protection at the site of infection unlike conventional intramuscular vaccination, indicating that this is a vaccine candidate to pursue in future studies.

[00118] Various embodiments disclosed herein relate to a virus-like particle (VLP) vaccine using SARS-CoV-2 WA-1 Receptor Binding Domain (RBD) proteins conjugated to a Hepatitis B surface antigen (HbsAg).

[00119] Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. E. co was used to express VLPs from the bacteriophage AP205, where the VLPs were genetically fused to SpyCatcher. SpyCatcher- VLPs were conjugated to HbsAg, and then mixed with SpyTag-linked RBD antigens. The RBD-SpyTag antigens were incubated overnight with HBsAg-SpyCatcher VLP. Administering SpyCatcher- VLPs tagged with an RBD antigen induced antibody responses.

[00120] This vaccine, like BReC-CoV-2, was then adjuvanted with the TLR4-agonist and lipid BECC 470 to enhance both the cellular and humoral immune responses. Intranasal administration of two doses of the RBD-VLP vaccine in combination with the BECC 470 adjuvant (RBD- VLP+BECC470) to K18-hACE2 mice provided equal protection against disease manifestation and morbidity to two intramuscular administrations of an mRNA vaccine when the mice were challenged intranasally with a lethal dose of the SARS-CoV-2 Delta variant. RBD-VLP+BECC470 limited viral replication in the upper airway at two days post-challenge and maintained a reduction in viral RNA in the nasal wash, lung, and brain, between days two and 10. IN RBD-VLP+BECC470 elicited greater IgA titers in the lung and nasal cavity than mRNA or challenge alone. Compared to intramuscular mRNA, RBD-VLP+BECC470 consistently maintained low histopathological inflammation scores in the lung tissue.

Examples.

Example 1. Mouse vaccination.

[00121] Female outbred GDI mice were obtained from Charles River Laboratories at 4weeks old and vaccinated at 8 weeks of age. Both male and female B6.Cg-Tg(K18-ACE2)2Prlmn/J (I<18- hACE2) mice were purchased from Jackson Laboratory at 8weeks old and vaccinated at 10 weeks old. Both CD1 and K18-hACE2 mice were given 50 L immunizations through either the intramuscular route or intranasal route. For intranasal immunization, mice were anesthetized through intraperitoneal injection with ketamine(80mg/kg)/xylazine(8.3mg/kg), then administered 25uL of vaccine into each nostril. The adjuvant BECC 470 was obtained from Dr. Robert Ernst at the University of Maryland.

Example 2. Vaccine antigen and adjuvant composition.

[00122] RBD-EcoCRM®. The receptor binding domain of the Wuhan variant of SARS-CoV- 2 (SARS-CoV-2 Wu RBD) was recombinantly produced by transient transfection in HEK293T cells using a pCAGGS expression vector with RBD construct with a C-terminal hexahistidine tag and codon optimized for mammalian expression (pCAGGS vector catalog #: NR-52309 BEI Resources). RBD was then chemically conjugated to the carrier protein EcoCRM® by Fina Biosolutions. 20pg of RBD-EcoCRM® was used in the vaccine formulations. The RBD sequence of the Wuhan variant of SARS-CoV-2 is shown in Table 2, identified as SEQ ID NO: 1 (RBD). Other SARS-CoV-2 RBD sequences that may be used include: SEQ ID NO: 2 (Delta-RBD), the sequence of the receptor binding domain of the Delta variant of SARS-CoV-2 (B.l.617.2 strain), shown in Table 2 with a C-terminal hexahistidine tag;

SEQ ID NO: 3 (Delta+Beta-RBD), the sequence of a receptor binding domain containing mutations found in the Alpha variant of SARS-CoV-2 (B.l.1.7 strain), the Beta variant of SARS-CoV-2 (B.1.351 strain), and the Delta variant of SARS-CoV-2 (B.l.617.2 strain), shown in Table 2 with a C-terminal hexahistidine tag; and

SEQ ID NO: 8 (Omicron-RBD), the sequence of the receptor binding domain of the Omicron variant of SARS-CoV-2 (B.l.617.2 strain), shown in Table 2 with a C-terminal hexahistidine tag.

The RBD sequence may include mutations from any desired variant of SARS-CoV-2, including the Alpha variant (B.l.1.7), the Beta variant (B.1.351, B.l.351.2, or B.1.351.3), the Gamma variant (P.l, P.1.1, or P.1.2), the Delta variant (B.l.617.2 and AY.l sublineages), the Epsilon variant (B.1.427 and B.1.429), the Eta variant (B.l.525), the Iota variant (B.l.526), the Kappa variant (B.l.617.1), the B.l.617.3 strain, the Mu variant (B.l.621 or B.l.621.1), the Omicron variant (B.l.1.529), and the Zeta variant (P.2). The RBD sequence may include multiple mutations found in any combination of two or more SARS-CoV-2 variants.

Table 2. RBD Sequences

[00123] Bacterial enzymatic combinatorial chemistry (BECC). 50pg BECC470 was sonicated in a water bath sonicator for 15 minutes prior mixing with RBD-EcoCRM™ for 2 hours before vaccination.

Example 3. SARS-CoV-2 challenge.

[00124] Previously sequenced SARS-CoV-2 USA-WA-1/2020 (NR-52281) was the challenge strain used in this K18-hACE2 vaccine study. K18-hACE2 mice were challenged with a 10⁴ PFU/dose. Viral dose was prepared from the first passage of WA-1 at a concentration of 3.7xl0⁶ PFU/mL diluted to a working concentration of 10⁶ PFU/mL. Mice were anesthetized with IP injection of ketamine/xylazine, and a total of 50pL of 10⁴PFU SARS-CoV-2 WA-1 was administered intranasally (25 .L per nostril).

Example 4. Disease score of SARS-CoV-2 challenged mice.

[00125] Challenged K18-hACE2 mice were evaluated daily through both in-person health assessments in the BSL3 and SwifTAG Systems video monitoring for 14 days. Flealth assessments of the mice were scored based on six criteria:

1) weight loss (scale 0-5),

2) appearance (scale 0-2),

3) activity (scale 0-3), 4) eye closure (scale 0-2),

5) respiration (scale 0-2), and

6) rectal temperature (scale 0-2).

[00126] All six criteria were scored based off a scaling system where 0 represents no symptoms and the highest number on the scale denotes the most severe symptoms. Additive health scores of the six criteria were assigned to each mouse after evaluation. Mice that scored 5 or above on the health assessment required immediate euthanasia. Cumulative disease scoring was calculated by adding the disease scores of each mouse from the group. Morbid mice that were euthanized during the study, before day 14, retained their disease score for the remainder of the experiment.

Example 5. Euthanasia and tissue collection.

[00127] Challenged mice that were assigned a health score of 5 or above or reached the end of the experiment were euthanized with an IP injection of Euthasol (390mg/kg) (Pentobarbital) followed by secondary measure of euthanasia with cardiac puncture. Blood from cardiac puncture was collected in BD Microtainer gold serum separator tubes, centrifuged at 15,000 x g for 5 minutes and serum collected for downstream analysis. Nasal wash was acquired by pushing ImL of PBS through the nasal pharynx. 500 ;JL of nasal wash was added to 167 ;JL of TRI reagent for RNA purification and the remainder of the nasal wash was frozen for serological analysis. Lungs were separated into right and left lobes. The right lobe of the lung was homogenized in ImL of PBS in gentleMACS C tubes using the m_l iing^O 2 program on the gentleMACS Dissociator. 300pL of lung homogenate was added to 167 pl . of TRI Reagent (Zymo research) for downstream RNA purification and 300 ;JL of lung homogenate was centrifuged at 15,000 x g for 5 minutes and the lung supernatant was collected for downstream analyses. The brain was excised from the skull and separated into the right and left hemispheres. The right hemisphere was homogenized in ImL PBS in gentleMACS C tubes using the same setting as the lung on the gentleMACS Dissociator. 167;JT . of TRI Reagent was added to 500 ;JL of brain homogenate for RNA purification.

Example 6. Serological analysis

[00128] ELISAs were performed to assess the total IgM (NB7497), IgG (NB7421), and IgA (NB7504) in the serum, lung supernatant, and nasal wash. Total IgM and IgG titers were quantified in the serum. High binding plates (Pierce 15041) were coated overnight at 4° C with 2pg/ mL of RED. Plates were then blocked with 3% non-fat milk in PBS-0.1% Tween 20 overnight in the 4°C. After blocking, 1:20 dilution of serum from mice was added in the first row and diluted 1:2 down two plates in 1% non-fat milk in PBS-0.1% Tween 20. Plates were incubated for 10 minutes at room temperature with shaking. Plates were then washed with PBS-0.1% Tween20 4 times, then either goat-anti-mouse secondary IgG HRP (1:2000 dilution) or goat-anti-mouse IgM HRP (1:20000 dilution) was added to the plates and incubated as above (Novus Biosolutions). ELISAs were developed using 3, 3', 5,5'- Tetramethylbenzidine (TMB) reagent (Biolegend 421101) (1:1 ratio) in the dark for 10 minutes, and the reaction was stopped using 25 ;JL 2N sulfuric acid. ELISAs were read using the Synergy Hl plate reader at 450nm.

[00129] Nasal wash, serum, and lung supernatant IgA titer quantification was performed using the same coating and blocking procedures as mentioned above. In separate ELISA assays, 100 .L of nasal wash, 1:20 dilution of serum and 1:5 dilution of lung supernatant were added to the first rows of high binding plates, and diluted down 2 plates at 1:2 dilution in 1% non-fat milk in PBS-0.1% Tween 20. Serum, nasal wash and lung supernatant samples were incubated for 2 hours at room temperature with shaking. Plates were washed according to the protocol mentioned above. Secondary goat-anti -mouse IgA HRP (1:10000) (Novus biologicals) was used in these assays and incubated for 1 hour at room temperature with shaking. IgA ELISAs were developed with TMB substrate (1:1) for 20 minutes in the dark before adding stopping solution and read on the Synergy Hl plate reader at 450nm. Titers were represented as Area Under the Curve values calculated via GraphPad Prism.

[00130] ELISAs were performed on challenged serum to assay IgGl (NB7511) and IgG2a (NB7516) titers. ELISAs were coated with RED following the same concentration and procedures mentioned above. Plates were blocked with 3% non-fat milk in PBS-0.1% Tween 20 for one hour at room temperature with shaking at 480rpm. Serum concentration (1:20) was used as above following a 10-minute incubation period. Secondary IgGl -HRP and IgG2a-HRP were used at a 1:10000 dilution in 1% non-fat milk in PBS-0.1% Tween 20 with a 10-minute incubation period. ELISAs were developed and stopped using the same protocol as above. Titers were represented as Area Under the Curve values calculated via GraphPad Prism.

[00131] As seen in FIG. 16A, neither IN nor IM vaccination with both a BReC-CoV-2 prime and boost produced a significant change in IgM antibodies to RBD in hACE2 mice, relative to NVC hACE2 mice or hACE2 administered only a prime dose. As seen in FIG. 16B, IN and IM vaccination each produced a significant increase in IgG antibodies to RBD in hACE2 mice, relative to NVC mice and mice administered only a prime dose. As seen in FIG. 16C, IN and IM vaccination each produced a significant increase in IgG antibodies to RBD in the lung supernatant of hACE2 mice, relative to NVC mice and mice administered only a prime dose. Referring back to FIGS. 11A and 11B, IN vaccination produced a significant increase in IgA antibodies to RBD in the lung supernatant and nasal wash of hACE2 mice; no such increase was seen from IM vaccination.

[00132] As seen in FIG. 17A, IN and IM vaccination of hACE2 mice with both a BReC-CoV- 2 prime and boost produced a significant change in IgG2a antibodies to RBD in hACE2 mice, relative to NVC hACE2 mice; there was no significant difference between IgG 2a antibody titers generated by IN vaccination and IgG2a antibody titers generated by IM vaccination. As seen in FIG. 17B, IN and IM vaccination each produced a significant increase in IgGl antibodies to RBD in hACE2 mice, relative to NVC mice. Also, IM vaccination produced a significant increase in IgGl antibodies to RBD in hACE2 mice, relative to IgGl antibodies generated by IN vaccination.

Example 7. Luminex Neutralization assay.

[00133] A neutralization assay was developed using the Luminex Magpix platform. 1:2 dilution of pre-challenged mouse serum was added to Greiner black non-binding 96 well plates. Serum was diluted 1:5 down the plate. Luminex Magpix® Microspheres (MC10012-YY) conjugated to RBD were added to the serum dilutions. After a 2-hour incubation period, plates were washed 2X with IX PBS- TBN on a 96 well magnet, ACE2 -biotin was added to the plates and incubated for 1 hour. Plates were washed again 2X on the magnet, and Streptavidin-phycoerythrin was added to the plates and incubated for 30 minutes at room temperature at 700rpm. After the Streptavidin-phycoerythrin incubation, plates were washed again, and 100pL of 1XPBS-TBN was added to plates and analyzed on the Magpix to measure neutralizing ability of serum antibodies. IC50 values of the mean fluorescent intensities generated were determined by calculating the non-linear regression on GraphPad Prism v9.

Example 8. Meso Scale Discovery COVID-19 ACE2 Neutralization assay

[00134] SARS-CoV-2 challenged serum was analyzed using the SARS-CoV-2 Plate 7 MultiSpot 96-well, 10 spot plate following the manufacturer protocol (catalog # N05428A-1) on the MSD QuickPlex SQ120. The 10 spots contained: 1) CoV-2 Spike, 2) RED from the B.1.351 VOC, 3) CoV- 2 nucleocapsid (N) protein, 4) RBD from the P.l VOC, 5) Bovine serum albumin (BSA), 6) RBD from the B.l.1.7 VOC, 7) Spike from the P.l VOC, 8) Spike from the B.l.1.7 VOC, 9) Spike from the B.1.351 VOC, and 10) the CoV-2 receptor-binding domain (RBD) in the SI spike subunit. Three dilutions of serum, 1:5, 1:50, and 1:500 were analyzed on the MSD neutralization assay for each mouse to perform Area Under the Curve analysis on the electrochemiluminescence using GraphPad Prism.

Example 9. qPCR SARS-CoV-2 viral copy number analysis of lung, brain and nasal wash.

[00135] RNA purification of the lung, brain and nasal wash was performed using the Direct- zol RNA miniprep kit (Zymo Research R2053) following the manufacturer protocol. SARS-CoV-2 copy numbers were assessed through qPCR using the Applied Biosystems TaqMan RNA to CT One Step Kit. We utilized nucleocapsid primers (F: SEQ ID NO: 4, ATGCTGCAATCGTGCTACAA; R: SEQ ID NO: 5, GACTGCCGCCTCTGCTC); and TaqMan probe (IDT:/56- FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/). The TaqMan probe is labeled: at the 5'-end with the reporter molecule 6-carboxyfluorescein (FAM); with a ZEN™ Internal Quencher (ZEN) positioned between the ninth (9th) and tenth (10th) nucleotide base in the oligonucleotide sequence; and with an Iowa Black® FQ (3IABkFQ) located at the 3’-end.

The FAM and ZEN moieties in the TaqMan probe are joined by the sequence TCAAGGAAC. The ZEN and 3IABkFQ moieties in the TaqMan probe are joined by SEQ ID NO: 6, AACATTGCCAA. [00136] The following final concentrations were used according to the Applied Biosystems TaqMan RNA to CT One Step Kit manufacturer protocol: TaqMan RT-PCR Mix 2X, Forward and reverse primers 900nM final, TaqMan probe 250nM final, TaqMan RT enzyme mix 40X and RNA template lOOng (with the exception of nasal wash). Nasal wash RNA concentrations were not quantifiable on the Qubit 3 fluorometer; therefore, we used 5.4 .L of nasal wash RNA per reaction instead of lOOng. Triplicates were prepared for each sample, and samples were loaded into a MicroAmp Fast optical 96 well reaction plate (Applied Biosystems 4306737). Prepared reactions were run on the StepOnePlus Real-Time System machine using the parameters: Reverse transcription for 15 minutes at 48°C, activation of AmpliTaq Gold DNA polymerase for 10 minutes at 95°C, and 50 cycles of denaturing for 15 seconds at 95°C and annealing at 60°C for 1 minute. Example 10. Cytokine analysis.

[00137]R&D 5-plex mouse magnetic Luminex assay was used to quantify the cytokines CXCL13, TNFa, IL-6, IFN-y, and C reactive protein in the serum and lung supernatant. Manufacturer protocols were followed in preparing samples. 5 plex mouse cytokine plate was analyzed on the Luminex Magpix and pg/ mL were calculated based off standard curves generated for each cytokine in the assay.

Example 11. Histology.

[00138] Left lobes of lungs were fixed in lOmL of 10% neutral buffered formalin. Fixed lungs were paraffin embedded into 5 pm sections. Sections were stained with hematoxylin and eosin and sent to iHisto for pathological analysis. Lungs were scored for chronic and acute inflammation in the lung parenchyma, blood vessels, and airways by a blinded pathologist. Each mouse was scored individually using a standard qualitative toxicologic scoring criteria: 0-none; 1 -minimal; 2-mild; 3- moderate; 4-marked; 5-severe. Chronic inflammation was denoted by presence of lymphocytes and plasma cells and acute inflammation was scored by the presence of neutrophils and edema. Together, the chronic and acute inflammation scores were represented by the total inflammation score.

Statistical analyses.

[00139] All statistical analyses were performed using GraphPad Prism version 9. Statistical analyses were performed with n > 8 for all K18-ACE2 mice studies and n > 3 for the CD1 mice studies. Ordinary one-way ANOVA with Dunnetfs multiple comparisons test were used with single pooled variance for data sets following a normal distribution and Kruskal-Wallis for non-parametric distributed datasets. Kaplan-Meier survival curves were utilized, and Log-rank (Mantel-Cox) test were used to test significance of survival between sample groups.

Example 12. Formulation of RBD-VLP + BECC470 vaccine and K18-hACE2 mouse vaccination

[00140] The RBD-VLP vaccine was prepared using SARS-CoV-2 Wu RBD proteins. Viruslike particles (VLPs) were obtained by using a fusion between the bacteriophage AP205-derived capsid and a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag; SEQ ID NO: 7: AH1VMVDAYKPTK). To form the VLP, RED was conjugated to SpyTag to produce RBD-SpyTag antigens. SpyCatcher- VLPs were conjugated to HbsAg. RBD-SpyTag antigens were incubated overnight with HBsAg-SpyCatcher- VLP. Vaccines were prepared in batch by sonicating 25pg BECC 470 per dose in water for 15min, then adding RBD-HBsAg-VLP (lOpg per dose) and incubated at room temperature for 2hrs. Before administration, 10X PBS was added to bring the dose volume to 50pL.

[00141] Female 7 -week-old K18-hACE2 mice were intranasally vaccinated with 25pL per nare under anesthesia with intraperitoneal ketamine/xylazine. No-vaccine no-challenge control mice were administered 50pL IX PBS intramuscular in the hind flank. mRNA control mice were administered 50pL mRNA-1273 vaccine intramuscular in the hind flank as well. All vaccine groups received a second identical dose 4 weeks later.

[00142] Submandibular bleeds to collect serum were performed to assess immunogenicity 4- weeks after prime and boost doses. Serum was also collected at euthanasia via cardiac puncture. Anti- SARS-CoV-2 RED IgG levels were quantified using ELISA. High binding plates were coated overnight with RBD. The next day, plates were washed 3x and blocked with 3% nonfat milk in PBS- Tween20. After an hour incubation at room temperature, plates were washed 3x then prepared for sample:

5pL of serum in 95 pL 1% nonfat milk-PBS-Tween20 was added to the top row, and

50 pL of 1% nonfat milk-PBS-Tween20 was added to the remaining wells for dilution across two plates.

Samples were diluted 1:2 from row A of plate 1 to row G of plate 2, discarding before dilution into row H. Samples were incubated with shaking at room temperature for Ihr. Plates were washed 4x, then 100 pL goat anti-mouse IgG HRP as a secondary antibody was added to 1% nonfat milk-PBS- Tween20 solution. The resulting anti-mouse IgG HRP was added to all wells and incubated with shaking for an additional 1 hr at room temperature. Non-bound antibodies were washed five times, and lOOpL 5,5'-tetramethylbenzidine (TMB) substrate was added to all wells. After 15 min incubation in the dark, 25pL 2N sulfuric acid was added to stop development and plate absorbances were read at 450 nm. Serum antibody levels were quantified using Area Under the Curve analysis in GraphPad Prism V9.0.0. Example 13. In vitro SARS-CoV-2 RBD ACE2 binding assay

[00143] Neutralizing potential of serum antibodies collected from vaccinated K18-hACE2 mice pre- and post-challenge was analyzed using MSD’s V-PLEX SARS-CoV-2 Panel 22 Mouse IgG kit (K15563U-2). Binding to the following antigens was assessed:

COV-2 RBD,

Delta (B.1.617.2 variant) RBD,

Gamma (P.l variant) RBD, Beta (B.1.351) RBD, Alpha (B.l.1.7 variant) RBD, and Omicron (B.1.1.529 variant) RBD.

Serum from mice euthanized at 10 days post challenge or from 4-week post-boost submandibular bleeds was diluted at 1:5, 1:50, 1:500, and 1:5000 and analyzed following the manufacturer’s protocol. Binding (% neutralization) was measured via electrochemiluminescence.

[00144] Serum from intramuscular mRNA-immunized mice had high IgG levels post-prime, whereas IN RBD-VLP+BECC mice showed low early responses (FIG. 19A). Flowever, at post-boost the IN antibody levels rose to near mRNA levels, albeit mRNA remains significantly higher (FIG. 19B). At 10 days post challenge (DPC) with the Delta SARS-CoV-2 variant, IN RBD-VLP-BECC and mRNA RBD specific IgG levels (FIG. 19C) remained similar to post-boost levels. Immunization with RBD-VLP+BECC induced IgA antibodies that recognize RBD in both the nasal wash (FIG. 19D) and lung supernatants (FIG. 19E); levels of IgA antibodies in the nasal wash and lung supernatants were much lower in intramuscularly mRNA-immunized mice. Intranasal immunization with a BECC adjuvant thus induces greater levels of IgA antibodies than intramuscular vaccination with an mRNA vaccine.

[00145] To assess the neutralizing capabilities of intranasally raised anti-RBD IgG antibodies, an in vitro ACE2-RBD binding assay using the serum from intranasal and intramuscularly vaccinated K18-hACE2 mice was performed 4-weeks post-boost and 10 days post-challenge. In vitro, serum antibodies from intramuscularly mRNA-vaccinated mice prior to viral challenge bound RBD from WA-1, Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 and inhibited ACE2 binding at a rate of 82% or greater (FIG. 20). Ten days after viral challenge, IM antibody binding to all VOC RBD had no significant change. IN antibodies before challenge exhibited significantly less binding inhibition than IM antibodies (FIG. 20), but still inhibited ACE2 binding to WA-1 and Delta variants up to 97%. A trend was seen in inhibition capability 10 days after viral challenge where ACE2 inhibition increased against all VOC RBD except Omicron. Both IM mRNA-1273 and IN RBD-VLP were unable to elicit IgG antibodies that efficiently inhibited binding of Omicron RBD and ACE2 at either timepoint. This likely reflects the divergence of Omicron RBD epitopes from the ancestral RBD that both vaccines were formulated with.

[00146] Antibody neutralization was further evaluated in an authentic viral plaque reduction assay where the SARS-CoV-2 Delta variant was propagated in vitro with increasing concentrations of serum from vaccinated mice collected ten days after challenge. At low dilutions (1:10), IM vaccinated mouse serum fully prevented plaque formation (100% reduction) and at higher dilutions (1:100 and 1:1000) reduced plaque formation by 88% or more (FIG. 21). Serum from mice vaccinated intranasally was also highly effective at neutralizing virus to prevent plaque formation when added to culture media at a 1:10 dilution and reduced plaque formation more than 88% or 78% of plaques at lower dilutions (1:100 and 1:1000 respectively; FIG. 21). Although IgG antibodies elicited by the intranasal vaccine have reduced neutralizing activity compared to those from intramuscularly administered mRNA, total antibodies from these mice are still effective at limiting SARS-CoV-2 viral replication and plaque formation in vitro.

[00147] Referring now to FIGS. 25A to 25F, binding of serum antibodies collected from vaccinated and challenged K18-hACE2 mice was tested with COV-2 (WA-1) RBD, Delta RBD, Gamma RBD, Beta RBD, Alpha RBD, and Omicron RBD.

[00148] As seen in FIG. 25A, serum antibodies from vaccinated mice administered either an intramuscular mRNA vaccine or an intranasal BECC470+RBD-VLP vaccine showed strong binding to COV-2 RBD, either after administration of the boost (post-boost) or 10 days after being challenged with SARS-COV-2 Delta variant (10 DPC). Binding to COV-2 RBD post-boost was significantly lower for intranasal BECC470+RBD-VLP than for intramuscular mRNA; however, there was no significant difference between the intramuscular vaccine and the intranasal vaccine at 10 DPC. Similar results were observed for Alpha RBD (FIG. 25B) and Delta RBD (FIG. 25E).

[00149] Referring now to FIG. 25C, serum antibodies from vaccinated mice administered an intramuscular mRNA vaccine showed strong binding to Beta RBD, either post-boost or 10 DPC after being challenged with SARS-COV-2 Delta variant. However, serum antibodies from vaccinated mice administered an intranasal BECC470+RBD-VLP vaccine showed significantly reduced binding to Beta RBD when compared to serum from intramuscularly vaccinated mice, either post-boost (P<0.0001) or 10 DPC after being challenged with Beta RBD (P<0.0066). Similar results were observed for Gamma RBD (FIG. 25D). However, both the intramuscular and intranasal vaccine produced significant binding to either Beta RBD or Gamma RBD, relative to an NVC control (P<0.0001).

[00150] Referring now to FIG. 25F, serum antibodies from vaccinated mice administered an intramuscular mRNA vaccine showed relatively strong post-boost binding to Omicron RBD, relative to NVC control (P<0.0003). However, 10 DPC after being challenged with SARS-COV-2 Delta variant, serum antibody binding to Omicron RBD was reduced by about a factor of two. Serum antibodies from vaccinated mice administered an intranasal BECC470+RBD-VLP vaccine showed significantly reduced post-boost binding to Beta RBD when compared to serum from intramuscularly vaccinated mice. Moreover, 10 DPC after being challenged with Beta RBD (P<0.0066), intranasal BECC470+RBD-VLP vaccine showed very low binding to Omicron RBD.

[00151] Based on the above data, an intramuscular mRNA vaccine and an intranasal BECC470+RBD-VLP vaccine seem to have similar effectiveness against the RBD antigen from WA- 1 SARS-COV-2, as well as the RBD antigens from Alpha and Delta variants of SARS-COV-2. An intramuscular mRNA vaccine seems to be more effective than an intranasal BECC470+RBD-VLP vaccine against the RBD antigens from Beta and Gamma variants of SARS-COV-2; however, both intramuscular and intranasal vaccines are effective vaccines against the Beta and Gamma variants of SARS-COV-2 RBD. Both intramuscular and intranasal vaccines are relatively ineffective against the Omicron RBD antigen.

[00152] However, the serum antibodies were all generated against a SARS-CoV-2 Wu RBD antigen. Intranasal vaccination would likely be more effective against the Beta-, Gamma-, and Omicron-derived RBD antigens, if a corresponding antigen was used to prepare an intranasal BECC470+RBD-VLP vaccine.

Example 14. SARS-CoV-2 challenge of K18-hACE2 mice [00153] Stocks of the SARS-CoV-2 Delta vanant B.l.617.2 hCoV-19/USA/WV-VU- WV1 18685/2021 (GISAID Accession ID: EP I_ISL_1742834) were created from a patient sample at WVU that was propagated in Vero E6 cells (ATCC-CRL-1586). The stocks were sequenced to confirm there were no mutations. At the time of challenge, vaccinated and control K18-hACE2 mice were anesthetized with an IP injection of ketamine/xylazine, then 25ul . of a 10⁴ PFU solution of diluted virus was administered by pipette into each nare (50pL total dose).

Disease scoring of SARS-CoV-2 challenged mice.

[00154] K18-hACE2 mice were evaluated every day after challenge to track disease progression through in-person health checks and using the SwifTAG video monitoring system. Rectal temperatures and weight measurements were recorded each day in addition to scores related to weight loss, changes in activity, appearance, eye closure/ conjunctivitis, and respiration. Scores were awarded based on severity of disease phenotypes, as discussed in Example 4. On each day, scores in each category were combined and recorded as one overall numerical score. Mice that received a score of 5 or reached 20% weight loss before day 10 post-challenge were humanely euthanized.

[00155] Individuals receiving COVID-19 mRNA vaccines are administered two booster doses at four-week intervals after initial vaccination, and are considered fully protected after another four weeks. We followed this schedule to prime and boost K18-hACE2 mice with our candidate IN BECC470+RBD-VLP vaccine, then intranasally challenged them with a lethal dose (104 PFU) of the SARS-COV-2 Delta variant to measure protection. Intranasal and intramuscular vaccines were matched in their disease-limiting abilities.

[00156] Compared to PBS-vaccinated and challenged mice, authentically vaccinated mice, i.e., mice vaccinated with intramuscular mRNA or intranasal BECC470+RBD-VLP, maintained low disease scores over the course of the 10-day challenge window (FIG. 22A). Intranasal BECC470+RBD-VLP vaccinated mice did not experience dramatic weight loss or drops in temperature, similar to the intramuscular mRNA group (FIGS. 22B and 22C). Intranasal BECC470+RBD-VLP vaccine provided 100% survival to vaccinated mice over the 10-day challenge window, compared to non-vaccinated mice which reached total morbidity requiring humane euthanasia by day 6 post-challenge (FIG. 22D). This data together suggests that this intranasal BECC470+RBD-VLP vaccine is as effective as IM mRNA at conferring protection against morbidity and mortality in SARS-CoV-2 related disease. Mouse euthanasia and tissue collection

[00157] At day 2, 10, or due to humane endpoint criteria, IN VLP and IM mRNA vaccinated K18-hACE2 mice were euthanized with IP Euthasol (390mg/kg) (Pentobarbital) followed by cardiac puncture. Blood from cardiac puncture was centrifuged to collect the serum for downstream analysis. Lung and brain tissue were dissected out for downstream histology, serology, and quantification of viral burden. Homogenization of lung and brain was performed. A nasal wash was performed on each mouse by pushing PBS (ImL) by catheter through the nasal pharynx and collected for analysis. For RNA purification in BSL2, lung and brain homogenate as well as nasal wash was treated with TRIzol Reagent. qPCR quantification of SARS-CoV-2 viral copy number in mouse tissues

[00158] RNA from nasal wash, lung, and brain homogenates of virus -challenged mice was purified using the Direct-zol RNA miniprep kit (Zymo Research, R2053) according to the manufacturer’s protocol. qPCR of the SARS-CoV-2 nucleocapsid gene was then performed for each mouse and sample using the Applied Biosystems TaqMan RNA to CT One Step Kit (Ref: 4392938) to measure viral copy number via transcript number with specifications for each reaction that have been described previously.

[00159] Plaque assays using lung supernatant from the mice at day 2 showed that intranasal BECC470+RBD-VLP vaccination significantly limited viral replication in the lung compared to PBS- vaccination (FIG. 23A). qRT-PCR analysis of the mice’s nasal wash, lung and brain tissues further supported this finding. Viral nucleocapsid RNA burden was significantly lower in the nasal wash of IN VLP mice compared to PBS-vaccinated mice at day 2 and was further reduced at day 10 (FIG. 23B). In the lung, viral RNA burden was slightly lower in IM mRNA vaccinated mice than the PBS group, however there was no significant reduction in the viral RNA burden of IN VLP lungs at day 2 (FIG. 23C). At day 10, viral RNA burden in the lung in both vaccine groups had reduced significantly, although it was still slightly above the limit of detection (FIG. 23C). Both vaccines prevented dissemination to and detection of viral RNA in the brain at 10 days post-challenge, when it was detectable in the brain tissue of control mice (FIG. 23D).

Example 15. Histopathological evaluation of lung tissue inflammation [00160] The left lobe of mouse lungs were collected at euthanasia and stored in 10% neutral buffered formalin for one week to fix. Fixed tissues were sectioned and mounted on slides, then Hematoxylin and Eosin stained for analysis. H&E-stained slides were evaluated for acute and chronic inflammation. Acute inflammation was marked by the infiltration of neutrophils in the parenchyma, blood vessels, and airways. Chronic inflammation was marked by mononuclear infiltrates in the same areas of the tissue. Semiquantitative scores for each condition (0- none, 1- minimal, 2- mild, 3- moderate, 4- marked, 5- severe) were awarded for tissue from each mouse.

[00161] Delta challenge alone resulted in scorable acute and chronic inflammation in the lungs of all mice, characterized by the respective identification of mononuclear cells as well as neutrophils. Intramuscular mRNA vaccinated mice euthanized 10 days post-challenge were given low scores for acute inflammation, while intranasal RBD-VLP mice received no scores (FIG. 24B). At 2 days postchallenge, intramuscularly vaccinated mice were given higher acute inflammation scores than the intranasal group (FIG. 24A), suggesting that the intranasal vaccine better protects mice from early development of lung infection than IM mRNA, perhaps due to the route of administration and localized immune responses. Chronic inflammation scores were similar between vaccine groups, albeit slightly lower on average in the intramuscular group. Overall, histopathological analysis shows that IN RBD-VLP protects mice against SARS-CoV-2 related lung inflammation to a degree similar to IM mRNA.

[00162] Although the various exemplary embodiments have been described in detail with particular reference to certain exemplary aspects thereof, it should be understood that the invention is capable of other embodiments and its details are capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purposes only and do not in any way limit the invention, which is defined only by the claims.

Claims

What is claimed is: A method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method comprising administering a coronavirus antigen to the mammalian patient, wherein the coronavirus antigen is least one of a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; wherein the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC)- derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof. The method of claim 1, wherein the viral pathogen is selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Canine coronavirus HuPn-2018 (CCoV-HuPn-2018), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKUl (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), and Human coronavirus NL63 (HCoV-NL63). The method of claim 2, wherein the viral pathogen is selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The method of claim 1, wherein the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC).

- 45 - The method of claim 1, wherein the coronavirus antigen is: the recombinant coronavirus RBD, wherein the recombinant coronavirus RBD has at least 80% sequence identity to an RBD from the viral pathogen; or the recombinant coronavirus spike protein, wherein the recombinant coronavirus spike protein has at least 80% sequence identity to a spike protein from the viral pathogen. The method of claim 1, wherein the coronavirus antigen is administered intranasally, intravenously, intramuscularly, subcutaneously, intradermally, or intraperitoneally. The method of claim 6, wherein the coronavirus antigen is administered intranasally or intramuscularly. A vaccine composition, comprising: a coronavirus antigen conjugated to a carrier protein, wherein the coronavirus antigen comprises a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; and wherein the carrier protein comprises detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); the outer membrane protein complex of serogroup B meningococcus (OMPC) , or a combination thereof; an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier. A vaccine composition, comprising: a coronavirus antigen,

- 46 - wherein the coronavirus antigen comprises a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier. The vaccine composition of claim 9, wherein: the coronavirus antigen is conjugated to a carrier protein; and the carrier protein is a detoxified diphtheria toxin produced in E. coli. A method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method comprising intranasally administering a coronavirus antigen to the mammalian patient, wherein the coronavirus antigen is least one of a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; wherein the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus, an inactivated virus, and a viruslike particle. The method of claim 11, wherein the coronavirus antigen is conjugated to a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (TT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (PD); and the outer membrane protein complex of serogroup B meningococcus (OMPC). The method of claim 11, wherein the coronavirus antigen is administered in combination with an adjuvant selected from the group consisting of a live attenuated virus and an inactivated

- 47 - virus, and the coronavirus antigen is conjugated to: a carrier protein selected from the group consisting of detoxified diphtheria toxoid protein (DT); detoxified tetanus toxoid protein (IT); a detoxified diphtheria toxin produced in E. coli; Haemophilus protein D (ED); and the outer membrane protein complex of serogroup B meningococcus (OMPC); or a virus-like particle. The method of claim 11, wherein the adjuvant is a live attenuated virus, wherein the live attenuated virus is obtained from at least one of a live attenuated influenza vaccine; a live attenuated Japanese encephalitis vaccine; a live attenuated measles vaccine; a live attenuated mumps vaccine; a live attenuated measles and rubella (MR) vaccine; a live attenuated measles, mumps, and rubella (MMR) vaccine; a live attenuated measles, mumps, rubella and varicella (MMRV) vaccine; a live attenuated polio vaccine; a live attenuated rotavirus vaccine; a live attenuated rubella vaccine; a live attenuated smallpox vaccine; a live attenuated varicella vaccine; a live attenuated yellow fever vaccine; and a live attenuated zoster/ shingles vaccine. The method of claim 14, wherein the adjuvant is a live attenuated virus, wherein the live attenuated virus is obtained from a live attenuated influenza vaccine. The method of claim 11, wherein the adjuvant is an inactivated virus, wherein the inactivated virus is obtained from at least one of an inactivated polio vaccine, an inactivated hepatitis A vaccine, an inactivated rabies vaccine, an inactivated influenza vaccine, and an inactivated tick- borne encephalitis vaccine. The method of claim 16, wherein the adjuvant is an inactivated virus, wherein the inactivated virus is obtained from an inactivated influenza vaccine. The method of claim 11, wherein the adjuvant is a virus-like particle. A vaccine composition, comprising: a coronavirus antigen, wherein: the coronavirus antigen comprises a coronavirus Receptor Binding Domain (RBD), a recombinant coronavirus RBD, a coronavirus spike protein, a recombinant coronavirus spike protein, or a combination thereof; and the coronavirus antigen conjugated to a virus-like particle; an adjuvant selected from the group consisting of bacterial enzymatic combinatorial chemistry (BECC) -derived Lipid A analogs, monophosphoryl lipid A, and combinations thereof; and an aqueous carrier. A method of immunizing a mammalian patient against infection by a viral pathogen, wherein the viral pathogen is a coronavirus, the method comprising intranasally administering the vaccine composition of claim 19 to the mammalian patient. The method of claim 20, wherein the viral pathogen is selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome— related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Canine coronavirus HuPn-2018 (CCoV-HuPn-2018), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKUl (HCoV-HKUl), Human coronavirus 229E (HCoV-229E), and Human coronavirus NL63 (HCoV-NL63). The method of claim 20, wherein the coronavirus antigen is: the recombinant coronavirus RBD, wherein the recombinant coronavirus RBD has at least 80% sequence identity to an RBD from the viral pathogen; or the recombinant coronavirus spike protein, wherein the recombinant coronavirus spike protein has at least 80% sequence identity to a spike protein from the viral pathogen.