Mosaic alphavirus-pseudovirus for RNA gene delivery and multivalent antigen display

Abstract

An injectable, oral or nasal spray multivalent vaccine treats or prevents infection with a coronavirus, and / or influenza, and / or respiratory syncytial virus (RSV), or other viruses such as African swine fever virus (ASFV). The multivalent vaccine comprises a hybrid pseudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV p12 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA,M1,M2 and NP and / or RSV Matrix, G, and Nucleocapsid, and / or ASFV p35, p27, p14. These hybrid pseudoviruses package an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral, or nasal spray formulation.

Description

[0001] MOSAIC ALPHAVIRUS-PSEUDOVIRUS FOR RNA GENE DELIVERY AND MULTIVALENT ANTIGEN DISPLAY

[0002] Cross-Reference to Related Applications

[0003]

[0001] This application claims priority benefit to US application no. 63 / 661,089, filed 18 June 2025, the entire content of which is hereby incorporated herein by reference.

[0004] Field

[0005]

[0002] The present disclosure relates to a mosaic alphavirus-pseudovirus that can be used as a multivalent gene delivery and vaccine platform for treating or preventing infection with multiple viruses including but not limited to coronaviruses, influenza viruses, and respiratory syncytial virus (RSV), African swine fever virus et al. A vaccine platform comprises a new hybrid mosaic pseudovirus assembled as a virus-like particle (VLP) with the structural proteins of coronavirus, influenza, or RSV, or a combination of them. The particles carry multiple copies of viral attachment and entry proteins including the coronavirus spike, influenza hemagglutinin, and RSV fusion proteins. Additional viral structural proteins from coronaviruses, such as the nucleocapsid (N), membrane (M) and envelope (E) proteins, or Influenza or RSV can be incorporated to enhance particle production and delivery. The mosaic VLP pseudovirus packages an alphaviral vector that carries RNA genome encoding multiple viral antigens domains such as the receptor binding domain regions from the attachment or fusion proteins of these viruses. The receptor binding domains were selected to be conserved, consensus, or structure-based designer sequences from each family of viruses. The mosaic VLP pscudovirus and alphaviral vector can be assembled into a viral particle and formulated for injection, oral, or a nasal spray delivery, which allows for systematic and local immune activation in the blood, GI (gastro and intestinal tract), or nasal mucosa. The multivalent nature of the vaccine platform allows for the induction of immunity to multiple viruses such as coronaviruses, influenza viruses, and RSV, providing broader protection against emerging variants and multiple respiratory pathogens.

[0006]

[0003] As a gene delivery and vaccine platform, this new mosaic alphavirus-pseudovirus can be used to display antigens from multiple other viruses and administered as a vaccine through different routes.

[0007] Introduction

[0004] Coronaviruses are a family of pathogens that are known to cause both mild and severe respiratory illnesses. SARS-CoV-2, the virus that causes COVID-19, is a highly contagious and potentially deadly respiratory illness. Influenza and respiratory syncytial virus (RSV) are also highly contagious respiratory viruses that can cause severe illness, particularly in vulnerable populations such as young children and the elderly. The rapid spread of these viruses has led to significant morbidity and mortality, and the development of effective vaccines has been a priority for public health agencies worldwide.

[0008]

[0005] There are four FDA approved vaccines for SARS-CoV-2, the most commonly administered being Pfizer and Modema. These vaccines utilize an mRNA vaccine platform that delivers messenger RNA (mRNA) to stimulate an immune response against a pathogen specific protein. The mRNA vaccines can stimulate an immune response, but the strength and duration of this response can vary and require the mRNA vaccines need to be delivered directly to cells in the body in order to be effective. This can be challenging, as the mRNA needs to be protected from degradation and delivered to the correct cells in the body. This can affect the effectiveness of the vaccine and require booster doses to maintain immunity.

[0009]

[0006] The traditional flu vaccine, known as the inactivated influenza vaccine (11V), is made from heat- inactivated or killed flu viruses. The major challenge to the flu vaccine is that it must be updated each year- to match the strains of the flu virus that are expected to circulate during the upcoming flu season. The flu virus is constantly changing, and new strains can emerge at any time. As a result, the composition of the flu vaccine is reviewed and updated annually by the World Health Organization (WHO) based on data on the circulating flu strains.

[0010]

[0007] Nasal spray administration has been widely used in children for the live attenuated flu vaccine since 2007. Nasal spray vaccines are easy to administer and do not require a needle or injection. This can make them more appealing to people who are afraid of needles or who have a fear of injections. Many infections, including the flu, are transmitted through the respiratory system and enter the body through the nose. Nasal spray vaccines can stimulate an immune response at the site of infection, which can be more effective than an injection, particularly in young children. This may be due to the fact that the mucosa (the lining of the nose and lungs) is rich in immune cells, which can help to stimulate an immune response. Nasal spray vaccines may be more convenient for some people and may lead to better compliance with vaccination schedules.

[0011]

[0008] Virus-like particles (VLPs) have been frequently used as antigens for vaccines to stimulate immunity. VLPs are normally assembled in cells by expressing the self-assembly structural proteins of a virus. VLPs released from cells are empty shells that do not contain the viral genome for gene expression. Therefore, there is no new antigen synthesized from VLPs in target cells. VLPs are normally endocytosed into target cells and processed for antigen presentation. In contract, pseudoviruses are non-replicating viral particles that can enter cells through a normal infection process. Pseudoviruses contain a viral genome that can be used to express reporter or antigens in target cells. To assemble a pseudovirus in cells, producer cells are normally cotransfected with multiple vectors expressing viral structural proteins and with a vector expressing a viral genome (DNA or RNA). Viral structural proteins expressed will assemble into particles with the viral DNA / RNA genome packaged inside, but the vectors expressing the structural proteins are excluded from packaging into the particles. This selective packaging of only the viral genome limits the replication capacity of pseudoviruses to only a single-round in target cells. Pseudoviruses are normally superior than VLPs in stimulating immunity, as they resemble the real virus closer than VLPs, and can stimulate both MHC-class I- restricted and MHC-class Il-restricted immune responses. Nevertheless, assembly of pseudoviruses and maintenance of their infectivity are more complicated and difficult than the production of non-infectious VLPs. Frequently, it is even more difficult, if not impossible, to produce hybrid pseudoviruses bearing different combinations of structural proteins and viral genomes.

[0012] Summary

[0013]

[0009] The present application provides methodology, composition, construct, and formulation relating to a mosaic alphavirus-pseudovirus that can be used as a multivalent gene delivery and vaccine platform for treating or preventing infection with multiple viruses. In one aspect, the present application provides an injectable, oral or nasal spray multivalent vaccine for treating or preventing infection with a coronavirus, and / or influenza, and / or respiratory syncytial virus (RSV), or other viruses such as African swine fever virus (ASFV), comprising a hybrid pseudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV pl 2 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA, Ml, M2 and NP and / or RSV Matrix, G, and Nucleocapsid , and / or ASFV p35, p27, pl4.

[0014] These hybrid pseudoviruses package an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral, or nasal spray formulation.

[0015]

[0010] In one embodiment, in a multivalent vaccine, a hybrid pseudovirus can be assembled using the attachment proteins from other viruses.

[0016]

[0011] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, the hybrid pseudovirus is derived from mammalian cells, bacteriophage or another non-pathogenic virus expressing the primary and secondary proteins from coronaviruses, Influenza and / or RSV, and / or ASFV.

[0017]

[0012] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, the alphaviral vector is derived from an alphavirus, such as Venezuelan equine encephalitis vims, Sindbis vims, or Semliki forest vims.

[0018]

[0013] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, further comprising a pharmaceutically acceptable excipient.

[0019]

[0014] In another aspect, provided is methodology for treating or preventing infection with coronaviruses, influenza, and respiratory syncytial vims (RSV), or ASFV, or other viruses comprising administering to a subject in need thereof a nasal spray vaccine comprising a hybrid pseudovirus containing multiple copies of the coronavirus spike protein, influenza glycoprotein, and RSV glycoprotein on its surface, and an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the coronavims, influenza, and RSV viruses.

[0020]

[0015] In one embodiment, the is hybrid pscudovims derived from mammalian cells, a bacteriophage or another non-pathogenic virus.

[0016] In another embodiment, the alphaviral vector is derived from an alphavirus, such as Venezuelan equine encephalitis virus, Sindbis virus, or Scmliki Forest virus.

[0021]

[0017] In another embodiment, a vaccine further comprises a pharmaceutically acceptable excipient.

[0022]

[0018] In another embodiment, the alphaviral vector expresses multiple truncated or full- length attachment or fusion proteins of the coronaviruses, and / or influenza, and / or respiratory syncytial virus (RSV), and / or ASFV, or other viruses from either a single or multiple subgenomic RNAs.

[0023]

[0019] In another embodiment, the multivalent vaccine is an injectable, oral administration or spray, or nasal spray.

[0024]

[0020] In one aspect, the present application provides a method of treating or preventing infection with coronaviruses, influenza, and respiratory syncytial virus (RSV), or ASFV, or other viruses, comprising administering to a subject in need thereof a multivalent vaccine, wherein said multivalent vaccine comprising a hybrid pseudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV pl2 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA, Ml, M2 and NP and / or RSV Matrix, G, and Nucleocapsid , and / or ASFV p35, p27, pl4.

[0025]

[0021] These hybrid pseudoviruses package an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral, or nasal spray formulation.

[0026]

[0022] In one embodiment, in a multivalent vaccine, a hybrid pscudovirus can be assembled using the attachment proteins from other viruses.

[0027]

[0023] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, the hybrid pseudovirus is derived from mammalian cells, bacteriophage or another non-pathogenic virus expressing the primary and secondary proteins from coronaviruses, Influenza and / or RSV, and / or ASFV.

[0028]

[0024] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, the alphaviral vector is derived from an alphavirus, such as Venezuelan equine encephalitis virus, Sindbis virus, or Semliki forest virus.

[0029]

[0025] In another embodiment, in an injectable, oral formulation, or nasal spray multivalent vaccine, further comprising a pharmaceutically acceptable excipient.

[0030]

[0026] In another embodiment, provided are methodology for treating or preventing infection with African swine fever virus (ASFV). In a further embodiment, treating or preventing occurs by injectable, oral administration, or nasal spray.

[0031]

[0027] In another embodiment, provided are methodology for treating or preventing infection with coronavirus. In a further embodiment, treating or preventing occurs by injectable, oral administration, or nasal spray.

[0032]

[0028] In another embodiment, provided are methodology for treating or preventing infection with influenza. In a further embodiment, treating or preventing occurs by injectable, oral administration, or nasal spray.

[0033]

[0029] In another embodiment, provided are methodology for treating or preventing infection with respiratory syncytial virus (RSV). In a further embodiment, treating or preventing occurs by injectable, oral administration, or nasal spray.

[0034]

[0030] In one aspect, provided is a hybrid mosaic pseudovirus assembled as a virus-like particle (VLP) with the structural proteins of coronavirus, influenza, or RSV, or a combination of them. In one embodiment, a composition comprises the hybrid mosaic pseudovirus.

[0035]

[0031] In another aspect, provided is a virus-like particle (VLP) with the structural proteins of coronavirus, influenza, or RSV, or a combination of them.

[0036]

[0032] In one embodiment, said composition is an immunogenic composition administered to a subject in need thereof by injectable, oral administration, or nasal spray.

[0037]

[0033] In another aspect, provided is a hybrid pseudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV pl 2 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA, Ml, M2 and NP and / or RSV Matrix, G, and Nucleocapsid , and / or ASFV p35, p27, pl4, W wherein said hybrid pseudovirus packages an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral formulation, or nasal spray formulation.

[0038]

[0034] In one embodiment, an immunogenic composition comprises the hybrid pseudo virus.

[0039]

[0035] In another aspect, provided is a hybrid mosaic pseudovirus comprising multiple copies of universal coronavirus spike proteins, influenza hemagglutinins, and RSV fusion proteins, as well as an alphaviral vector that carries RNA encoding multiple universal receptor binding domain regions of the attachment and fusion proteins of these viruses, wherein the hybrid virus has structural similarity to a virus-like particle (VLP), but packages an alphaviral vector for gene expression.

[0040]

[0036] In one embodiment, a composition comprises the hybrid mosaic pseudovirus.

[0041]

[0037] In another embodiment, a vaccine comprises the the hybrid mosaic pseudovirus.

[0042]

[0038] In another embodiment, an immunogenic composition comprising the hybrid mosaic pseudovirus.

[0043]

[0039] In another embodiment, a formulation comprises the hybrid mosaic pseudovirus, wherein said formulation is injectable, oral administration, or nasal spray.

[0044] Brief Description of the drawings

[0045]

[0040] Figure 1A is an example illustration of the mosaic particle for the vaccine platform. Figure IB is an illustration of the alphavirus vector packaged by a mosaic VEP.

[0041] Figure 2 is an illustration of an example method of assembling a mosaic alpha- pscudoviral particles.

[0046]

[0042] Figure 2 is an illustration of an example method of assembling a mosaic alpha- pseudoviral particles.

[0047]

[0043] Each of Figures 3A-3B provides a specific example of a mosaic alphapseudovirus that can be used in a method to stimulate an immune response to a broad range of SARS-CoV-2 valiants.

[0048]

[0044] Figure 4 provides a specific example of a mosaic alpha-SARS-CoV-2 pseudoviruses that are assembled and used to infect a human target cell and to express a gene in the target cell.

[0049]

[0045] Each of Figures 5A-5B provides a specific example of an alpha-influenza pseudovirus (Ha-IAV).

[0050]

[0046] Each of Figures 6A-B provides a specific example of mosaic alpha-SARS-CoV-2 pseudoviruses are used as a vaccine in a K18-hACE2 mouse model.

[0051] Description

[0052]

[0047] To date limited multivalent vaccine has been developed to provide broad protection from multipipe respiratory viruses. One promising approach involves the use of viruslike particles (VLPs) that contain multiple copies of viral proteins. VLPs have been shown to be effective at inducing immunity to various viruses in preclinical studies. However, VLPs lack the ability to express genes in antigen presenting cells (APC) for MHC -class I-restricted antigen presentation. To address the need for broader protection against multiple respiratory pathogens, the present disclosure provides a new mosaic alphavirus-pseudovirus that can both express highly conserved antigen domains and display mosaic viral attachment proteins on the particles surface. This alphavirus-pseudovirus can be formulated as an oral or nasal spray multivalent vaccine platform for preventing infection with coronaviruses, influenzas, and RSV.

[0053]

[0048] Figure 1A is an example illustration of a mosaic particle for the vaccine platform. The vaccine platform comprises a hybrid mosaic pseudovirus that contains multiple copies of universal coronavirus spike proteins, influenza hemagglutinins, and RSV fusion proteins, as well as an alphaviral vector that carries RNA encoding multiple universal receptor binding domain regions of the attachment and fusion proteins of these viruses. Structurally, the hybrid virus is similar to a virus-like particle (VLP), but packages an alphaviral vector for gene expression. The VLP and alphaviral vector are assembled in a nasal spray formulation, which allows for easy administration and local immune activation in the nasal mucosa. The primary structural component of the pseudovirus are the three viral attachment and fusion proteins for the coronavirus, influenza and RSV viruses. The secondary structural proteins can either come from coronavirus (N, M and E), Influenza virus (NA, Ml, M2 and NP) or RSV (Matrix, G and Nucleocapsid) proteins.

[0054]

[0049] Figure IB is an illustration of the alphavirus vector packaged by a mosaic VLP. The alphavirus (e.g. SFV, SINV, or VEEV) vector carries the nonstructural proteins (NSP) 1 through 4 which allow for large amount of RNA production and protein expression form the downstream subgenomic promoters. The NSP4 protein can be codon optimized to disrupt the subgenomic promoter to allow for additional RNA sequences to be inserted such as RNA packaging signals from coronaviruses, influenza or RSV to enhance the alphaviral vector packaging. Downstream of the subgenomic promoter the vector encodes the RBDs (receptor binding domain) of the attachment or fusion proteins of the three viruses. Both the attachment, fusion protein and the RBDs can be selected by predictive protein modeling for identifying universal sequences that will allow for cross reactivity for multiple strains of each virus. The RBDs can be encoded on one subgenomic RNA by splitting the expression with self-cleaving peptides (e.g. viral 2A peptides) or with internal ribosomal entry sites (IRES). Alternatively, the RBDs can be expressed on separate subgenomic promoters.

[0055]

[0050] Figure 2 is an illustration of an example method of assembling a mosaic alpha- pseudoviral particles. The viral particles can be assembled by co-transfection of the viral primary and secondary proteins with the alphaviral vector in HEK293T cells or similar packaging cells lines. The particles are harvested and purified from the transfected cell supernatant. Larger scale particle production can also be performed with constitutive or inducible packaging cells that express the structural proteins.

[0056]

[0051] Figures 3A-3B provide a specific example of a mosaic alpha-pseudovirus that can be used in a method to stimulate an immune response to a broad range of SARS-CoV-2 variants. In Figure 3 A the particle is described as containing up to three SARS-CoV-2 spike variants on its surface in addition to the secondary structural protein’s membrane, nucleocapsid and the envelope proteins. Wuhan, Omicron and the Omicron sub-variant JN.l arc examples of variants that can be used. These proteins assemble and package an alphavirus genome described in Figure 3B. The genome expresses the nonstructural proteins of the alphavirus and a three -part RBD fusion protein from the 26S subgenomic promoter. The RBDs of the three spike proteins used on the surface of the particle are expressed and linked by a fusion peptide formed form a PanCoronavirus universal RBD peptide.

[0057]

[0052] Figure 4 provides a specific example of a mosaic alpha-SARS-CoV-2 pseudoviruses that are assembled and used to infect a human target cell and to express a gene in the target cell. In this example, mosaic alpha-pseudoviruses carrying multiple spike variants, including the Wuhan variant (WT) and the Omicron variant (WT+Omicron), the Wuhan variant and the JN.l variant (WT+JN.l), the Omicron variant and the JN1 variant (Omicron+JN.l), or the Wuhan(WT) and the Omicron variant and the JN.l variant (WT+Omicron+JN.l), are assembled by contrasfection of HEK293T cells with vectors expressing these spike variant proteins and the vectors expressing the SARS-CoV-2 N, M and E structural proteins. These mosaic alpha-pseudoviruses also package an alphavirus genome that express the luciferase reporter gene. These mosaic alpha-pseudoviruses are used to infect HEK293T(ACE2+TMPRSS2) target cell. Following infection for 18 hours, luciferase assay is performed to detect reporter gene expression. The mosaic pseodoviruses are shown to deliver genes for protein expressions in target cells.

[0058]

[0053] Each of Figures 5A-6B provides a specific example of an alpha-influenza pseudovirus (Ha-IAV). Figure 5 A illustrates the Ha-IAV particles that are pseudoviruses assembled from the structural proteins of the Influenza virus: Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), Matrix (Ml), and the Ion Channel (M2). They also encapsulate an alphaviral vector for reporter gene expression. The alpha-pseudoviruses are single-cycle viruses with self-amplifying RNA for rapid quantification of neutralizing antibodies, seroconversion, seroprotection, and entry -inhibiting drugs. These pseudoviruses are also used for stimulating immune responses and serves as vaccines. Figure 5B is an example of an alphainfluenza pseudovirus subtype H5N1 Avian Flu (Nevada) infection and target cells and the neutralization of the pseudoviruses by an H1 / H5 influenza neutralizing antibody.

[0054] Each of Figures 6A-6B provides a specific example of mosaic alpha-SARS-CoV- 2 pscudoviruscs arc used as a vaccine in a K18-hACE2 mouse model. Figure 6A is a schematic of the animal study, in which mice are vaccinated with a mosaic alpha-pseudovirus, Ha-CoV- 2(Tri-RBD), which carries an alphavirus genome that express the RBD domains from three SARS-CoV-2 spike protein variants (WT, Omicron, and JN.l). Following vaccination, mice are challenged with SARS-CoV-2 infection and clinical outcomes are scored. Vaccinated mice show improved clinical outcomes comparing with unvaccinated control mice. Figure 6B shows the quantification of the anti-SARS-CoV-2 neutralization activities in the serum of vaccinated mice.

[0059]

[0055] The purified particles can be loaded into an oral or nasal spray applicator to administer the vaccine. The particles will trigger host complement interactions if they have been exposed to any of the viruses previously which will activate the adaptive immune system. Any particles that escape the initial complement interactions will enter host cells using receptor mediated entry mechanisms. Upon entry the mosaic alpha-pseudovirus will begin to express the encoded RNA genome and activate factors of the innate immune system.

[0060]

[0056] The multivalent nature of the vaccine platform allows for the induction of immunity to multiple strains of coronaviruses, influenza, and RSV, providing broader protection against emerging variants and multiple respiratory pathogens. In addition, the nasal spray formulation allows for the induction of local immunity in the nasal mucosa, which may provide additional protection against respiratory infections.

[0061] Examples

[0062]

[0057] Figure 1A. An illustration of an example mosaic alpha-pseudovirus. The particle consists of the primary structural proteins, the coronavirus S, the influenza HA and the RSV F proteins and at least one set of viral secondary proteins such as the coronavirus N, M, and E proteins. Figure IB. An illustration of the alphaviral vector genome in the mosaic alphapseudovirus particle. The vector includes the 5’ untranslated region followed by the nonstructural proteins of the alphavirus replicase ,NSPl-4. An optional viral packaging signal. The viral subgenomic promoter over expresses the universal RBDs for coronavirus, influenza, and RSV separated by small self-cleaving picornavirus 2A peptides.

[0063]

[0058] Figure 2. An image of an example assembly procedure of the mosaic Alphapseudovirus particle nasal spray vaccine in HEK293T cells. Briefly HEK293T cells expressing the coronavirus structural proteins N, M and E are co-transfected with expression vectors for the coronavirus S proteins, influenza HA proteins, the RSV F proteins, and the alphaviral vector genome. The co-transfected cells produce a virus-like particle with an array of S, HA and F proteins on the surface and package the alphaviral vector vaccine encoding multiple receptor binding domains.

[0064]

[0059] Figure 3A. An illustration of an example mosaic alpha-pseudovirus particle specifically for multiple variants of SARS-CoV-2. The particle consists of up to three spike protein from various variants of SARS-CoV-2, such as the Wuhan, Omicron, and the JN.l variant and the secondary structural proteins of the coronavirus, including the membrane nucleocapsid and envelope proteins. Figure 3B. An illustration of the alphavirus vector for the multi-variant SARS-CoV-2 mosaic alpha-pseudovirus. The vector includes the 5’ untranslated region followed by the nonstructural proteins of the alphavirus replicase ,NSPl-4. An optional viral packaging signal. The viral subgenomic promoter over expresses three SARS-CoV-2 spike variant RBD regions that are linked by a pan-coronavirus RBD peptide.

[0065]

[0060] Figure 4. An example of mosaic alpha-SARS-CoV-2 pseudoviruses that are assembled and used to infect a human target cell and to express a luciferase reporter gene in the target cell. In this example, mosaic alpha-SARS-CoV-2 pseudoviruses carrying multiple spike protein variants, including the Wuhan variant (WT) and the Omicron variant (WT+Omicron), the Wuhan variant and the JN.l variant (WT+JN.1 ), the Omicron variant and the JN 1 variant (Omicron+JN.l), or the Wuhan(WT) and the Omicron variant and the JN.l variant (WT+Omicron+JN.l), are assembled by cotrasfection of HEK293T cells with vectors expressing these spike variant proteins and the vectors expressing the SARS-CoV-2 N, M and E structural proteins. These mosaic alpha-pseudoviruses also package an alphavirus genome that express the luciferase reporter gene. These mosaic alpha-pseudoviruses are used to infect HEK293T(ACE2+TMPRSS2) target cell. Following infection for 18 hours, luciferase assay is performed to detect reporter gene expression. The mosaic pseodoviruses are shown to deliver genes for reporter protein expressions in target cells, as quantified by luciferase assay of infected target cells.

[0066]

[0061] Figure 5A. Illustration of the Ha-IAV particles that are pseudoviruses assembled from the structural proteins of the Influenza virus: Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), Matrix (Ml), and the Ion Channel (M2). The particle also encapsulates an alphaviral vector for reporter gene expression. Figure 5B is an example of an alpha-influenza pseudovirus subtype H5N1 Avian Flu (Nevada) that infects target cells (IC, Infection control). The particle is also incubated with an H1 / H5 influenza neutralizing antibody that is serially diluted (from 40,000 ng / ml to 12.8 ng / ml). The antibody dose-dependent neutralization of the pseudoviruse is shown.

[0067] L062J Figure 6A. Schematic of the animal study, in which K18-hACE2 mice are intranasally vaccinated with a mosaic hybrid Ha-CoV-2(Tri-RBD) pseudovirus, which carries an alphavirus genome that express the RBD domains from three SARS-CoV-2 spike protein variants (WT, Omicron, and JN.l ). Following intranasal vaccination, mice are challenged with SARS-CoV-2 infection and clinical outcomes are scored. Figure 6B. Quantification of the anti- SARS-CoV-2 neutralization activities in the serum of vaccinated mice, as exampled in mouse 9B M3. Mice 2AM0 and 2BM0 are control mice treated with PBS buffer. Mouse serum samples collected at 120 days post vaccination were serially diluted and incubated with Ha-CoV-2(Luc) pseudovirus. The complex is used to infect HEK293T(ACE2+TMPRSS2) target cells. Following infection for 18 hours, luciferase assay is performed to detect reporter gene expression in infected cells. The neutralization cure is plotted, and 50% inhibition dosage (IC50) is calculated.

Claims

ClaimsWHAT IS CLAIMED IS:

1. An injectable, oral or nasal spray multivalent vaccine for treating or preventing infection with a coronavirus, and / or influenza, and / or respiratory syncytial virus (RSV), or other viruses such as African swine fever virus (ASFV), comprising a hybrid pscudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV pl2 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA, Ml, M2 and NP and / or RSV Matrix, G, and Nucleocapsid , and / or ASFV p35, p27, pl4.These hybrid pseudoviruses package an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral, or nasal spray formulation.

2. The multivalent vaccine of claim 1 , whereas the hybrid pseudovirus can be assembled using the attachment proteins from other viruses.

3. The injectable, oral, nasal spray multivalent vaccine of claim 1, wherein the hybrid pseudovirus is derived from mammalian cells, bacteriophage or another non-pathogenic virus expressing the primary and secondary proteins from coronaviruses, Influenza and / or RSV, and / or ASFV.

4. The injectable, oral, nasal spray multivalent vaccine of claim 1, wherein the alphaviral vector is derived from an alphavirus, such as Venezuelan equine encephalitis virus, Sindbis virus, or Semliki forest virus.

5. The injectable, oral, nasal spray multivalent vaccine of claim 1 , further comprising a pharmaceutically acceptable excipient.

6. A method of treating or preventing infection with coronaviruses, influenza, and respiratory syncytial virus (RSV), or ASFV, or other viruses comprising administering to a subject in need thereof a nasal spray vaccine comprising a hybrid pseudovirus containing multiple copies of the coronavirus spike protein, influenza glycoprotein, and RSV glycoprotein on its surface, and an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the coronavirus, influenza, and RSV viruses.

7. The method of claim 6, wherein the is hybrid pseudovirus derived from mammalian cells, a bacteriophage or another non-pathogenic virus.

8. The method of claim 6, wherein the alphaviral vector is derived from an alphavirus, such as Venezuelan equine encephalitis virus, Sindbis virus, or Semliki Forest virus.

9. The method of claim 6, further comprising a pharmaceutically acceptable excipient.

10. The method of claim 6, wherein the alphaviral vector expresses multiple truncated or full- length attachment or fusion proteins of the coronaviruses, and / or influenza, and / or respiratory syncytial virus (RSV), and / or ASFV, or other viruses from either a single or multiple subgenomic RNAs.

11. The multivalent vaccine of any one of claims 1-5, wherein the multivalent vaccine is an injectable.

12. The multivalent vaccine of any one of claims 1 -5, wherein the multivalent vaccine is a spray.

13. The multivalent vaccine of claim 12, wherein the spray is oral.

14. The multivalent vaccine of claim 12, wherein the spray is nasal.

15. A method of treating or preventing infection with coronaviruses, influenza, and respiratory syncytial virus (RSV), or ASFV, or other viruses, comprising administering to a subject in need thereof a multivalent vaccine of claims 1-5 or 11-14.

16. The method of claim 15, wherein the method is a method of treating or preventing infection with African swine fever virus (ASFV).

17. The method of claim 15, wherein the method is a method of treating or preventing infection with coronavirus.

18. The method of claim 15, wherein the method is a method of treating or preventing infection with influenza.

19. The method of claim 15, wherein the method is a method of treating or preventing infection with respiratory syncytial virus (RSV).

20. The method of any of claims 15-19, wherein treating or preventing occurs by injectable, oral administration, or nasal spray.21 . A hybrid mosaic pseudovirus assembled as a virus-like particle (VLP) with the structural proteins of coronavirus, influenza, or RSV, or a combination of them.

22. A virus-like particle (VLP) with the structural proteins of coronavirus, influenza, or RSV, or a combination of them.

23. A composition comprising the hybrid mosaic pseudovirus of claim 21.

24. The composition of claim 23, wherein said composition is an immunogenic composition administered to a subject in need thereof by injectable, oral administration, or nasal spray.

25. A hybrid pseudovirus assembled from any of the following combinations of primary proteins: coronavirus S, and / or influenza HA and / or RSV fusion protein, and / or ASFV pl2 plus any of the following secondary proteins from either: coronavirus N, M, and E, and / or Influenza NA, Ml, M2 and NP and / or RSV Matrix, G, and Nucleocapsid , and / or ASFV p35, p27, pl4, W wherein said hybrid pseudovirus packages an alphaviral vector carrying RNA encoding multiple receptor binding domain regions of the attachment or fusion proteins of these viruses, wherein the hybrid pseudovirus and alphaviral vector are combined in an injectable, oral formulation, or nasal spray formulation.

26. An immunogenic composition comprising the hybrid pseudovirus of claim 25.

27. A hybrid mosaic pseudovirus comprising multiple copies of universal coronavirus spike proteins, influenza hemagglutinins, and RSV fusion proteins, as well as an alphaviral vector that carries RNA encoding multiple universal receptor binding domain regions of the attachment and fusion proteins of these viruses, wherein the hybrid virus has structural similarity to a virus-like particle (VLP), but packages an alphaviral vector for gene expression.

28. A composition comprising the hybrid mosaic pseudovirus of claim 27.

29. A vaccine comprising the hybrid mosaic pseudovirus of claim 27.

30. An immunogenic composition comprising the hybrid mosaic pseudovirus of claim 27.

31. A formulation comprising the hybrid mosaic pseudovirus of claim 27, wherein said formulation is injectable, oral administration, or nasal spray.

32. Any product or use disclosed herein.

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