Hybrid alpha-pseudovirus platform for riboviruses

Abstract

The present disclosure relates to a new system for generating and using a hybrid alpha pseudovirus for Riboviria viruses. The hybrid riboviria-alpha-pseudovirus (HRAP) present a new type of particle having an alphavirus-derived RNA genome and structural protein(s) of viruses in the riboviria realm. The instant HRAP particles assembled from structural proteins across diverse riboviria families may find use for vaccine development, antiviral drug screening, neutralization assays, initiating therapeutic or immune responses, and the like.

Description

HYBRID ALPHA-PSEUDOVIRUS PLATFORM FOR RIBOVIRUSESCROSS-REFERENCE TO RELATED APPLICACTIONS

[0001] This application claims priority benefit to United States Provisional Application no. 63 / 450,783 filed 08 March 2023 and United States Provisional Application no. 63 / 493,738 filed 01 April 2023, the entire contents of each of which are hereby incorporated herein in its entirety.FIELD

[0002] The present disclosure relates to a new system for generating and using a hybrid alpha- pseudovirus for Riboviria viruses. The hybrid riboviria-alpha-pseudovirus (HRAP) present a new type of particle having an alphavirus-derived RNA genome and structural protein(s) of viruses in the riboviria realm. The instant HRAP particles assembled from structural proteins across diverse riboviria families may find use for vaccine development, antiviral drug screening, neutralization assays, initiating therapeutic or immune responses, and the like.INTRODUCTION

[0003] RNA viruses are a diverse group of viruses that contain RNA as their genetic material instead of DNA They are responsible for a wide range of diseases in humans, animals, and plants, and are known to mutate rapidly, making them difficult to control. RNA viruses have been associated with some of the most significant global health challenges of recent times, including the COVID- 19 pandemic caused by the SARS-CoV-2 virus.

[0004] RNA viruses are classified into several families based on their genetic makeup, mode of replication, and other characteristics. Some of the most well-known RNA viruses include the influenza virus, hepatitis C virus, human immunodeficiency virus (HIV), and the Zika virus. These viruses have caused significant morbidity and mortality worldwide, leading to a range of public health challenges, including pandemics, chronic diseases, and increased healthcare costs.

[0005] One of the largest families of Riboviruses that cause human disease are the Paramyxoviruses. Paramyxovirus are negative-stranded RNA viruseses that include thehenipavirus genus which frequently causes outbreaks of severe illness in domestic animal and humans. For example, Hcnipaviruscs, including Hcndra and Nipah virus, cause respiratory and neurological diseases that have a high mortality rate in both humans and animals. Between the period of 1994 to 2015 over 40 horses had been killed or euthanized for Hendra virus infection. In a May 2018 outbreak, Nipah virus killed 17 people and during a previous outbreak in Malaysia, the government authorize killing over a million pigs to control the spread of the infection.

[0006] Several RNA viruses, such as Nipah and Marburg viruses, are classified as Biosafety Level 4 (BSL4) pathogens, which means that they are highly contagious and can cause severe or fatal disease in humans. Other RNA viruses, such as the SARS-CoV-2 virus responsible for the CO VID- 19 pandemic, are classified as Biosafety Level 3 (BSL3) pathogens, which require specialized containment facilities and strict safety protocols to prevent their spread.

[0007] Developing drugs and vaccines for BSL3 and BSL4 pathogens presents unique challenges due to the high level of risk associated with handling these pathogens, as well as the rapid mutation rate and genetic diversity of RNA viruses. The development of effective therapies and vaccines for these pathogens requires a thorough understanding of their biology, transmission, and pathogenesis, as well as the development of innovative technologies and platforms for drug and vaccine discovery.

[0008] Two of the most prevalent strategies for vaccines development are nucleotide encoded antigens, such as mRNA, or protein-based antigens, like virus-like particles (VLP or VLPs). A VLP is an empty particle which does not contain any nucleotide genome and cannot therefore express any gene in a target cell. Pseudo viruses are a single-cycle nonreplicating viral particle that contains a genome allowing for gene expression and utilizes a viral structure on its surface to facilitate entry into target cells.

[0009] One of the major challenges in developing drugs and vaccines for RNA viruses is the rapid evolution of these pathogens. RNA viruses have high mutation rates and can quickly develop resistance to antiviral drugs and vaccines, which makes it difficult to develop longterm solutions for disease prevention and control. Additionally, the development of vaccines and drugs for BSL3 and BSL4 pathogens is often hindered by the limited availability ofresearch funding, as well as the need for specialized containment facilities and trained personnel to conduct research on these pathogens safely. The high containment of BSL-3 and BSL4 restricts access to most researchers for vaccine and drug development. For some RNA viruses, lenti-pseudoviruses have been developed to allow for research in BSL2 conditions, but these particles contain proteins that are mostly HIV-derived, and only one or two of the structural proteins from the authentic virus. These pseudo virion particles are mostly comprised of proteins, such as the capsid and matrix proteins, that are not native to the live virus. Such systems may not truly reflect the infection processes of the virus. The previous lenti-pseudoviruses have considerably low titer and typically require 48-72 hours to generate reporter signal to quantify infectivity.SUMMARY

[0010] The present application provides a new system for generating and using a hybrid alpha- pseudovirus for Riboviria viruses. The hybrid riboviria-alpha-pseudovirus (HRAP) presents a new type of particle having an alphavirus-derived RNA genome and structural protein(s) of viruses in the riboviria realm. The instant HRAP particles assembled from structural proteins across diverse riboviria families may find use for vaccine development, antiviral drug screening, neutralization assays, initiating therapeutic or immune responses, and the like.

[0011] In one aspect, the application provides a hybrid riboviria-alpha-pseudovirus particle (HARP), comprising an alphavirus-derived RNA genome and one or more structural protein(s) from Riboviria family viruses.

[0012] In one embodiment, said alpha virus RNA encodes a gene of interest for delivery into target tissues and cells.

[0013] In one embodiment, said gene of interest is a reporter gene, vaccine antingen, or therapeutic target for delivery into target tissues and cells.

[0014] In one embodiment, said reporter gene encodes GFP or luciferase.

[0015] In one embodiment, said alpha virus-derived RNA genome derives from Venezuelan equine encephalitis virus, Sindbis virus, or Semliki forest virus.

[0016] In one embodiment, said one or more structural proteins is from any single or combination of virus in the Riboviria realm, including Filovirus, Flavivirus, Picornavirus, Calicivirus, Coronavirus, Paramyxovirus, Orthomyxovirus, Arenavirus, Hantavirus, Lentivirus, and DNA viruses with RNA intermediates like Hepadnaviruses.

[0017] In another aspect, the application provides a composition comprising a hybrid riboviria-alpha-pseudovirus particle (HARP), comprising an alphavirus-derived RNA genome and one or more structural protein(s) from Riboviria family viruses.

[0018] In one embodiment, said composition can stimulate an immune response by acting like a virus-like particle (VLP) and mRNA expression system simultaneously.

[0019] In another aspect, the application provides methodology for producing a hybrid riboviria-alpha-pseudovirus particle (HARP), comprising assembling by co-expression one or more Riboviria structural proteins and alphavirus RNA genome packaged into the HRAP particle.

[0020] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Filovirus structural proteins including VP40, GP and / or nucleoprotein wherein the particle also packages an alphavirus genome.

[0021] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Flavivirus structural proteins including Envelope, Membrane and / or Capsid wherein the particle also packages an alphavirus genome.

[0022] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Paramyxovirus structural proteins including attachment glycoprotein, Fusion, matrix and / or nucleocapsid wherein the particle also packages an alphavirus genome.

[0023] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Coronavirus structural proteins including spike, membrane, envelope and / or nucleocapsid wherein the particle also packages an alphavirus genome.

[0024] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Orthomyxovirus structural proteins including neuraminidase,hemagglutinin protein, Matrix 1 and / or Matrix 2 wherein the particle also packages an alphavirus genome.

[0025] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Picomavirus structural proteins including capsid subunits like VP1, VP2 and / or VP3 wherein the particle also packages an alphavirus genome.

[0026] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Arenavirus structural proteins including the glycoprotein and / or the matrix proteins wherein the particle also packages an alphavirus genome.

[0027] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Phlcbovirus structural proteins including the glycoproteins (Gn and Gc), and / or the nucleoprotein wherein the particle also packages an alphavirus genome.

[0028] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Hepadnavirus structural proteins including the surface antigen proteins and / or the core antigen proteins wherein the particle also packages an alphavirus genome.

[0029] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Rhabdovirus structural proteins including the matrix protein, the glycoprotein and / or nucleoprotein wherein the particle also also packages an alphavirus genome.

[0030] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Calicivirus structural proteins including capsid subunits VP1, and VP2 wherein the particle also packages an alphavirus genome.

[0031] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Pneumovirus structural proteins including attachment glycoprotein, Fusion, matrix, small hydrophobic and / or nucleocapsid wherein the particle also packages an alphavirus genome.

[0032] In one embodiment, the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Hantavirus structural proteins including the glycoproteins (Gn and Gc) and / or the nucleoprotein wherein the particle also packages an alphavirus genome.

[0033] In one embodiment, an HRAP particle can be assembled from structural proteins from any single or combination of virus in the Riboviria realm, including Filovirus, Flavivirus, Picomavirus, Calicivirus, Coronavirus, Paramyxovirus, Orthomyxovirus, Arenavirus, Hantavirus, Lentivirus, and DNA viruses with RNA intermediates like Hepadnaviruses.

[0034] In another aspect, the application provides a use of a hybrid riboviria-alpha- pseudovirus particle (HARP) for modeling RNA virus infections, quantifying neutralizing antibodies, viral entry inhibiting drugs, and / or cellular factors involved in viral replication.

[0035] In another aspect, the application provides use of a hybrid riboviria-alpha- pseudovirus particle (HARP), for delivering an alphaviral vector RNA to target tissues and cells for the purpose of gene therapy in a subject such as a mammal, including a human, cancer treatment or prevention in a subject such as a mammal, including a human, and / or to stimulate immune responses to antigens in a subject such as a mammal, including a human.

[0036] Additional objects and advantages of the embodiments will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosed embodiments. The objects and advantages may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0037] It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and thus not restrictive.

[0038] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles of the embodiment.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. Illustration of the HRAP particle and the alphavirus vector genome. (A) Model of the HRAP particle for paramyxovirus consisting of the paramyxoviral structural proteins (N, M, F, and G) and the SFV-based alphaviral vector. (B) SFV-based vector design with a paramyxoviral packaging signal ( IjJ) placed in the NSPI-4 region.

[0040] Figure 2. Illustration demonstrating the structural similarity of the HRAP particles and traditional pscudovirus with the authentic paramyxoviruses. The models of the HRAP particles show the structural similarities to the authentic paramyxoviruses while the models of traditional pseudoviruses have non-paramyxovirus IO structural proteins that effect the final particle structure.

[0001] Figure 3. Illustration for the production procedure of the HRAP particles. HEK293T cells were co-transfected with the plasmid vectors encoding the individual structural components of paramyxovirus (including N, M, F, and G) and a plasmid encoding the alphaviral vector genome. Supernatants with the HRAP particles are harvested 48hour post co-transfection, and can be purified and used to infect target cells.

[0042] Figure 4. Tracking Paramyxovirus Nipah HRAP particle infection with the GFP reporter. HEK293T cells were infected with a Nipah HRAP particle that carries a GFP reporter. GFP was observed 48hours post infection. (A) Brightfield microscopy image of infected cells (B) Florescent microscopy of infected cells for GFP expression (shown in black). (C) Overlay of the brightfield and florescent microscopy image.

[0043] Figure 5. Speed of the HRAP Particle to Deliver and Express the Alphaviral RNA genome. Vero cells were infected with a Nipah HRAP particle that carries an Alphaviral vector RNA genome that either expresses GFP or firefly luciferase (A) The GFP signal from the infection is visible within 6 hours post the infection and maximizes at 24 hours. The GFP positive cells are shown in black. (B) The infection with particle that expresses firefly luciferase has detectable signal above background 6 hours post infection.

[0044] Figure 6 illustrates various examples of HRAP particles for a diverse range of Riboviria families. HRAP particles can be assembled from the structural component of viruses in the Riboviria realm as shown in figure 3 and described previously. The illustration shows structural proteins that can be used to assemble HRAP particles in the most prevalent human and animal viral pathogens. Co-expressing these viral proteins with the alphaviral vector described in Figure IB will generate a HRAP particle.

[0045] Figure 7. Demonstrating different Riboviria structural proteins can be used to generate HRAP particles which express luciferase. HRAP particles were assembled with structural proteins from four major diverse Riboviria families, including Flavivirus withDengue serotypes 1-4 prME, Calicivirus with MNV-1 and HNV-GIT.4 VP1 and VP2, Hantavirus with Andes Gc and Gn and Phlcbovirus with Rift Valley Fever virus Gc and Gn. The particles all packaged an alphavirus genome derived from SFV that expresses a luciferase gene. The HRAP particles where then used to infect 293T cells and the luciferase was measured less than 24 hours post infection.

[0046] Figure 8. Demonstrating the diverse Riboviria families that can assemble HRAP particles and package an SFV derived RNA that expresses GFP HRAP particles were assembled with an SFV-derived replicon that expresses a GFP gene and the structural proteins from a diverse range of viral families including the Arenavirus, Rhabdo virus, Paramyxovirus, Filovirus, Orthomyxovirus, and Coronaviruses. These HRAP particles where then used to infect the appropriate target cell, including Vero, HEK293T and HEK293T(ACE2 / TMPRSS2) cells. There was detectible signal in all of the infections. This further demonstrates the HRAP can be used for modeling high-risk pathogens from diverse families of viruses including LASV, Nipah virus, MARV, Ebola virus, and SARS-CoV.

[0047] Figure 9. Demonstration of a separate HRAP RNA genome derived from a different alphavirus species with multiple viral structural proteins HRAP particles were assembled with an VEEV-derived replicon that expresses a GFP gene and the structural proteins from a diverse range of viral families including the Arenavirus, Rhabdovirus, Paramyxovirus, Filovirus, Orthomyxovirus, and Coronaviruses. There is detectable GFP signal from all of the HRAP particles which demonstrates that the RNA replicon can be derived from any alphavirus. The rate and efficiency at which a HRAP particle packages an alphavirus replicon will depend on the structural proteins making up the particles structure.

[0048] Figure 10. Utilization of the HRAP particles for antiviral drug screenings and neutralization assays. A neutralizing antibody to Nipah virus was serially diluted and was incubated with the Nipah PHAP particles. Following the incubation, HEK293T cells were infected with the particles and the luciferase signal was measured. (C) The antibody IC50 was calculated using the percent infection determined from the luciferase signal and the dilution of the antibody dosage. The final IC50 of the antibody was determined to be around the 1:687 dilution.DETAILED DESCRIPTION:

[0049] Timely development of vaccines and antiviral drugs is critical to control emerging infectious diseases to prevent future pandemics. RNA viruses represent the majority of major viral pathogens that cause human disease and include high-risk viral pathogens that can only be studied under strict BSL-3 or BSL-4 conditions. Previous pseudoviruses allow for the study of under BSL-2 conditions, but lack structural similarity to the live virus. Previous pseudoviral neutralization assays are time consuming and cannot be used in high throughput applications.

[0050] As explained below, the present application discloses methodology for making and using a new hybrid riboviria-alpha-pseudovirus (HRAP) particles for rapidly quantifying neutralization antibodies and antiviral drugs, and the HRAP particles can also be used as a gene delivery vehicle for vaccines, cancer and cell-based gene therapy.

[0051] HRAP particles are a non-replicating Ribovirus-like particle comprising one or more authentic ribo virus structural proteins. HRAP package a genome derived from an alphavirus-based vector, which can rapidly and robustly express genes of interest, like therapeutic or reporter genes, within a few hours (l-3hrs) after viral entry. Furthermore, and as detailed below, HRAP can be used as a robust platform for rapid quantification of neutralization antibodies and antiviral drugs.

[0052] Exemplary Riboviria viruses include Coronavirus, Picomavirus, Paramyxovirus, Orthomyxovirus, Pneumovirus, Hantavirus, Phlebovirus, Flavivirus, Filovirus, Arenavirus, Calicivirus, Rhabdovirus and DNA viruses with RNA intermediates like Hapadnavirus and others.

[0053] A HRAP is assembled using the structural proteins from RNA virus of interest that naturally form a virus-like particle (VLP) and package self-amplifying RNA genome derived from alphavirus replicon.

[0054] A virus-like particle (VLP) is an empty particle which does not contain any nucleotide genome and cannot therefore express any gene in a target cell. Pseudoviruses are a single-cycle non-replicating viral particle that contains a genome allowing for gene expression and utilizes a viral structure on its surface to facilitate entry into target cells.

[0055] For example, a HRAP particle assembly for Respiratory Syncytial Virus, would include the structural proteins that form a viral VLP including the nucleocapsid (N), matrix(M), fusion (F), and attachment glycoprotein (G) and this particle then packages an SFV- dcrivcd RNA rcplicon genome. The HRAP particles arc non-replicating particles. The RNA genome of these particles is derived from alphavirus vector (e.g. SFV, SINV, or VEEV), including the a) 5' untranslated region; b) an open-reading frame coding for a non- structural proteins (nsp) 1-4; c) an RNA virus packaging signal; d) the viral sub-genomic promoter; e) a gene of interest; f) 3' untranslated region; and g) a poly(A) tail.L0056J The RNA can encode any gene of interest, including reporter genes (e.g.GFP and Luciferase) or therapeutic targets, or vaccine antigens such as the receptor binding domain of a virus glycoprotein, downstream of the alphaviral subgenomic promoters. The alphavirus genome can rapidly express genes for quantifying viral infectivity, initiating immune responses, or for therapeutic purposes. Rapid reporter and gene expression facilitate rapid screening of anti-viral drugs and neutralizing antibodies. Additionally, the HRAP platform can be delivered through nasal or oral pathways to stimulate an immune response by acting like a virus-like particle (VLP) and mRNA expression system simultaneously. The structural elements of the particle can stimulate the immune response like traditional VLP and subunit vaccines. The viral structural proteins responsible for attachment and entry can facilitate the delivery of the alphavirus genome which can act like an mRNA vaccine to produce encoded antigens. Alphaviral vector vaccines can stimulate both the adaptive and the innate immune responses at the localized areas where infection occurs.

[0057] The figures below provide illustrative design of a HRAP particle, and the results demonstrating the use of the HRAPs in connection with a ribovirus for antibody and drug screenings. For example, and in no way limiting, the present application discloses as an example of the ability of the HRAPs to be used to model the entry of a ribovirus from the paramyxoviral family for antibody and drug screenings. It is understood that the instant HRAPs may be used to model the entry of any ribovirus.

[0058] Reference will now be made in detail to the present embodiments, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.1C

[0059] The conjunction “and” or “or” can be used in the list of members, but the “at least one of’ phrase is the controlling language. For example, at least one of A, B, and C is shorthand for A alone, B alone, C alone, A and B, B and C, A and C, or A and B and C.

[0060] The following examples are illustrative and non-limiting. One of ordinary skill in the art would readily understand that the present methodology, compositions, and constructs may be modified and / or optimized for particular embodiments, and retain the spirit and scope of the present application.Example 1; HRAP particle for th Paramyxoviridae family.

[0061] Fig. 1 describes the design of the HRAP particle for the Paramyxoviridae family. [A] the particles consist of the four major structural components of Paramyxoviridae including the viral attachment protein G, the viral fusion protein F, nucleocapsid protein N and the matrix protein M. Structural proteins self-assemble into a VLP that packages the RNA from the alphavirus vector. [B] The design of the alphavirus vector for the genome of the HRAP particles which express reporter or therapeutic genes of interest. The vector contains a common promoter, such as CMV, RSV, or any common promoter, at the 5' end. The promoter allows the transcription of full-length viral RNA genome to be packaged into the HRAP particles for gene expression in target cells. The genomic vector contains the 5' untranslated region and open-reading frames coding for nonstructural proteins (nsp) 1-4 from Semiliki Forest virus (SFV). The inclusion of nspl-4 allows for limited replication of viral RNA genomes in cells. An optional Paramyxoviridae packaging signal ('P) sequences can be inserted after a codon optimized NSP4 protein. The codon optimization destroys the original sub-genomic promoter, which is then replaced with the packaging signal downstream ofNSP4. Viral subgenomic promoters are utilized to drive the expression of reporter or therapeutic genes (such as GFP, Luc, lacZ, Amylase, or other transgenes). Downstream of the gene of interest is the 3' untranslated region from Semiliki Forest virus (SFV) and a poly(A) tail, which is required for mRNA synthesis and stability. The poly(A) tail is self-cleaved by the hepatitis delta virus ribozyme that is inserted just upstream of a mammalian ploy (A) signal. The expression of nspl-4 in particle-producer cells leads to the amplification of the HRAP particles mRNA genomes, likely producing abundant amounts of the genomic RNA that can be included into the budding Paramyxoviridae VLPs.

[0062] Fig. 2 is an illustration to demonstrate the structural similarity of the HRAP particles to authentic ribovirus from the paramyxovirus family. Traditional pscudoviruscs, like lentivirus and VSV (vascular stomatitis virus), have non-paramyxovirus structural proteins that may ultimately impact the structure of the pseudovirus particles. Additionally, antibodies may target and bind to structural proteins not present in traditional pseudovirus particles and these antibodies may ultimately impact the overall immune reaction and viral neutralization.

[0063] Fig. 3 describes the process that we currently used to assemble HRAP particles for Paramyxoviridae. Vectors expressing the structural proteins of paramyxovirus (F, G, M, and N) are co-transfected with the vector expressing the RNA genome of the HRAP particles (Fig IB) into human cells such as HEK293T cells. Virion particles are harvested at 48 hours post co- transfection, which can be further purified by various common methods such as sucrose gradient centrifugation. These particles are ready to be used for infection assays.

[0064] Example 2; HRAP particle for the Paramyxovirus family.

[0065] For viruses belonging to the Paramyxovirus family, including Mumps, Measles, HPIV, and Henipaviruses; Hendra and Nipah viruses, HRAP particles can be assembled from vectors expressing the viral matrix (M) and / or nucleocapsid (N) and / or the fusion protein (F) and / or the attachment glycoprotein or hemagglutinin-neuraminidase (G / HN) and / or the shall hydrophobic protein (SH) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0066] Example 3; HRAP particle for the Filovirus family.

[0067] For viruses belonging to the filovirus family, including Marburg and Ebola viruses, HRAP particles can be assembled from vectors expressing viral glycoprotein (GP) and / or the matrix protein (VP40) and / or the nucleocapsid (N) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0068] Example 4; HRAP particle for the Flavivirus family.

[0069] For viruses belonging to the flavivirus family, including Dengue, West Nile, and Zika viruses, HRAP particles can be assembled from vectors expressing viral capsid (C) and / or the membrane and / or the envelope polyprotein (prME) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0070] Example 5: HRAP particle for the Pneumovirus family.

[0071] For viruses belonging to the Pneumovirus family, including RSV-A and RSV-B viruses, HRAP particles can be assembled from vectors expressing the viral matrix (M) and / or nucleocapsid (N) and / or the fusion protein (F) and / or the attachment glycoprotein (G) and / or the shall hydrophobic protein (SH) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0072] Example 6: HRAP particle for the Rhabdovirus family.

[0073] For viruses belonging to the Rhabdovirus family, including vesiculovirus and rabies viruses, HRAP particles can be assembled from vectors expressing the viral matrix (M) and / or nucleoprotein (N) and / or the glycoprotein (G) and an alphavirus RNArcplicon with or without an optimized viral RNA packaging signal.

[0074] Example 7; HRAP particle for the Orthomyxoviridae family.

[0075] For viruses belonging to the Orthomyxoviridae family, including Influenza A and B viruses, HRAP particles can be assembled from vectors expressing viral the matrix and / or matrix 2 ion channel and / or nucleoprotein and / or the hemagglutinin (HA) protein and / or the neuraminidase (NA) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0076] Example 8; HRAP particle for the Phlebovirus family.

[0077] For viruses belonging to the Phlebovirus family, including Rift Valley Fever virus, HRAP particles can be assembled from vectors expressing viral glycoprotein (Gn and Gc) and / or the nucleoprotein (NP) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0078] Example 9: HRAP particle for the Calicivirus family.

[0079] For viruses belonging to the Calicivirus family, including human noroviruses, HRAP particles can be assembled from vectors expressing the major viral capsid (VP1) and / or the minor capsid protein (VP2) and an alphavirus RNA rcplicon with or without an optimized viral RNA packaging signal.

[0080] Example 10; HRAP particle for the Picornavirus family.

[0081] For viruses belonging to the Picomavirus family, including human rihnoviruses and poliovirus, HRAP particles can be assembled from vectors expressing the major viral capsid VP1 and / or the second capsid protein (VP2) and / or the third capsid protein (VP3) and / or the fourth capsid protein (VP4) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0082] Example 11: HRAP particle for the Arenavirus family.

[0083] For viruses belonging to the Arenavirus family, including Lymphocytic choriomeningitis virus and Lassa virus (LASV), HRAP particles can be assembled from vectors expressing viral glycoprotein (GP) and / or the matrix (Z) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0084] Example 12: HRAP particle for the Hantavirus family.

[0085] For viruses belonging to the Hantavirus family, including Andes and Hantaan viruses, HRAP particles can be assembled from vectors expressing viral glycoprotein (Gn and Gc) and / or the nucleoprotein (NP) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0086] Example 13: HRAP particle for the Coronavirus family.

[0087] For viruses belonging to the Coronavirus family, including Middle East Respiratory Syndrome (MERS) and the Severe Acute Respiratory Syndrome (SARS) viruses, HRAP particles can be assembled from vectors expressing the membrane (M) and / or nucleoprotein (N) and / or the envelope protein (E) and / or the spike protein (S) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0088] Example 14: HRAP particle for the Hepadnavirus family.

[0089] For viruses belonging to the Hepadnavirus family, including hepatitis B virus (HBV), HRAP particles can be assembled from vectors expressing the viral core protein (C) and / or the small, medium and large glycoprotein (S, M, L) and an alphavirus RNA replicon with or without an optimized viral RNA packaging signal.

[0090] It is commonly known that the co-transfection procedure can also be simplified by the use of stable cell lines that persistently express the viral structural proteins and the genomes.

[0091] Such cell lines can produce HRAP particles continuously with or without an inducer. Such an inducible system can be controlled with an inducible promoter to regulate the express of the structural proteins and viral genomes.

[0092] Example 15; HRAP particles infect and express report genes (GFP) in target cells.

[0093] Fig. 4 demonstrates the ability of the HRAP particles to infect and express report genes (GFP) in target cells. In this example, a construct designed for Paramyxoviridae HRAP particles were assembled and used to infect target HEK293T cells. GFP expression was observed at 24 hours post infection, demonstrating the ability of the HRAP particles to infect and express the GFP reporter gene in target cells.

[0094] Example 16: HRAP particles infect and express report genes (GFP) in target cells.

[0095] Figure 5 demonstrates the ability of the HRAP particles to deliver the alphaviral RNA genome to a target cell to express a gene of interest rapidly following infection. Figure 5A shows the GFP signal of a HRAP particle for Nipah virus is visible after 6 hours of infection on Vero cells. This signal is maximized after 24 hours post infection. In Figure 5B is a time course experiment demonstrating how firefly luciferase signal from a Nipah HRAP particle is detectable from the background after 6 hours post infection.

[0096] Example 17; HRAP particles assembled from a diverse range of riboviria orders and families.

[0097] Fig. 6 illustrates examples of various HRAP particles produced from the assembly method described in Fig.3 and how the HRAP particles can be assembled from a diverse range of riboviria orders and families. These particles represent the broad range of particles that can be assembled into a hybrid alpha-pseudovirus . To generate these particles the structural components of the virus particle are co-expressed with the alphavirus genome. Once co- expressed the particles will self-assemble.

[0098] Example 18: HRAP particles assembled from structural proteins across diverse riboviria families deliver and express the alphavirus RNA genome to target cells.

[0099] Fig. 7 demonstrates how HRAP particles assembled from structural proteins across diverse riboviria families can be used to deliver and express the alphavirus RNA genome to target cells. Positive signal after 6 to 18 hours post infection indicates the particles are infectious and can be used to model the viral entry mechanisms and deliver an RNA cargo.

[0100] In Fig. 7A the HRAP particles were prepared for Flaviviruses using the Dengue serotypes 1 through 4 prME structural polyprotein and the assembled particles were used to infect HEK293T. The luciferase signal was measured 12 hours posts infection and positive signal was detected 18 hours post infection.

[0101] In Fig. 7B HRAP particles were prepared for Calicivirses using the VP1 and VP2 of murine norovirus 1 (MNV-1) and human norovirus GII.4. Positive luciferase signal was detected in HEK293T cells infected with the Calicivirus HRAP particles.

[0102] In Fig. 7C a HRAP particle for Hantavirus using the Andes virus Gc and Gn and then used to infect HEK293T the resulting luciferase signal after 18 hours indicates positive infection signal.

[0103] In Fig. 7D a HRAP particle for Phlebovirus was made using the Gc and Gn of Rift Valley Fever virus then used to infect HEK293T the resulting luciferase signal after 18 hours indicates positive infection signal.

[0104] Example 19: HRAP particles can be assembled across a broad range of viral families and the particles are infectious and able to express reporter genes

[0105] Fig. 8 continues to demonstrate that HRAP particles can be assembled across a broad range of viral families and the particles are infectious and able to express reporter genes like GFP in target cells.

[0106] Briefly target cell including Vero, HEK293T and HEK293T(ACE2 / TEMPRSS2) cells were infected with a HRAP particles assembled with different viral structural proteins and packaged an SFV based RNA genome that expresses GFP. The GFP signal was measured by flowcytometry 48 hours post infection.

[0107] Fig. 8A, illustrating HRAP particles for Arenavirus with LASV GP infecting Vero cells; Fig. 8B, showcasing a HRAP particle for Rhabdovirus assembled with rabies virus structural proteins infecting HEK293T; Fig. 8C, presenting HRAP particles forParamyxovirus with Nipah virus structural proteins infecting Vero cells; Fig. 8D, displaying HRAP particles for filoviruses with Marburg and Ebola virus structural proteins infecting HEK293T cells; Fig. 8E, demonstrating Orthomyxovirus assembly with H5N1 structural proteins infecting HEK293T cells; Fig. 8F, depicting HEK293T(ACE2 / TMPRSS2) cells infected with HRAP particles for coronaviruses, assembled with MERS-CoV or SARS-CoV structural proteins; and Fig. 8G, featuring a HRAP particle for Pneumo virus with RSV structural proteins infecting HEK293T cells.

[0108] This data demonstrates that the HRAP particles can be generated for a broad range of viral families including the Coronavirus, Paramyxovirus, Orthomyxovirus, Pneumovirus, Hanatavirus, Phlebovirus, Flavivims, Filovirus, Arenavirus, Calicivirus, Rhabdovirus families.

[0109] Example 20: Packaging of the alphavirus RNA replicon into the HRAP particles.

[0110] Fig.9. demonstrates that the packaging of the alphavirus RNA replicon into the HRAP particles can be derived from VEEV. We have previously illustrated that an SFV RNA replicon can be optimized to more effectively package into the HRAP particle with addition of viral RNA packaging elements.

[0111] The present application demonstrates that the species of the alphaviral RNA genome can greatly impact the packaging of the replicon into the HRAP particles and the efficiency of the packaging is dependent on the viral structural proteins that are assembling the HRAP particles. The HRAP particles were assembled identically to the particles in Fig.8 exchanging the SFV-based GFP genome with a VEEV derived RNA replicon that expresses GFP. The particles were assembled and were used to infect the same target cells from Fig.8. The resulting infections show that the different HRAP particles have varying affinity for the SFV or the VEEV-based GFP genomes.

[0112] Example 21: HRAP particles for rapidly measuring neutralizing antibodies for clinical diagnostic, therapeutic discovery, and to facilitate rapid serological immunosurveillance of high risk pathogens

[0113] Fig. 10. demonstrates HRAP particles in efficiently measuring neutralizing antibodies for clinical diagnostic, therapeutic discovery and to facilitate rapid serological immuno-surveillance of high risk pathogens.

[0114] In Fig.10 a Nipah virus-specific HRAP was used to evaluate a commercially available Nipah virus-neutralizing antibody. The antibody underwent serial dilution, mixed with PHAP particles, and applied to HEK293T cells. Infection levels were quantified through luciferase expression (Fig 10A), and the 1C50 (half-maximal inhibitory concentration) of the antibody was determined based on the percentage of infection and loglO antibody dosage. The final IC50 for the neutralizing antibody was found to be around the 1 :687 dilution (Fig 10B).

[0115] The instant HRAP particles can be used to identify new host cell receptors, quantify viral neutralization for antibodies and antiviral drugs, measure viral variant infectivity, deliver therapeutic genes to specific target cells and activate immune responses as a prophylactic.

Claims

CLAIMS1. A hybrid riboviria- alpha-pseudovirus particle (HARP), comprising an alphavirus-derived RNA genome and one or more structural protein(s) from Riboviria family viruses.

2. The hybrid riboviria- alpha-pseudo vims particle (HARP) of claim 1, wherein said alphavirus RNA encodes a gene of interest for delivery into target tissues and cells.

3. The hybrid riboviria-alpha-pseudovirus particle (HARP) of claims 1-2, wherein said gene of interest is a reporter gene, vaccine antingen, or therapeutic target for delivery into target tissues and cells.

4. The hybrid riboviria-alpha-pseudovirus particle (HARP) of claims 1-3, wherein said reporter gene encodes GFP or luciferase.

5. The hybrid riboviria-alpha-pseudovirus particle (HARP) of claims 1-4, wherein said alphavirus-derived RNA genome derives from Venezuelan equine encephalitis virus, Sindbis vims, or Semliki forest virus.

6. The hybrid riboviria-alpha-pseudovirus particle (HARP) of claim 1-5, wherein said one or more structural proteins is from any single or combination of virus in the Riboviria realm, including Filovirus, Flavivirus, Picomavirus, Calicivirus, Coronavirus, Paramyxovirus, Orthomyxovirus, Arenavirus, Hantavirus, Lentivirus, and DNA viruses with RNA intermediates like Hepadnaviruses.

7. A composition comprising a hybrid riboviria-alpha-pseudovirus particle (HARP), comprising an alphavirus-derived RNA genome and one or more stmctural protein(s) from Riboviria family viruses.

8. The composition of claim 7, wherein said composition can stimulate an immune response by acting like a virus-like particle (VLP) and mRNA expression system simultaneously.

9. A method for producing a hybrid riboviria-alpha-pseudovirus particle (HARP), comprising assembling by co-expression one or more Riboviria structural proteins and alphavirus RNA genome packaged into the HRAP particle.

10. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Filovirus structural proteins including VP40, GP and / or nucleoprotein wherein the particle also packages an alphavirus genome.

11. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Flavivirus structural proteins including Envelope, Membrane and / or Capsid wherein the particle also packages an alphavirus genome.

12. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Paramyxovirus structural proteins including attachment glycoprotein, Fusion, matrix and / or nucleocapsid wherein the particle also packages an alphavirus genome.

13. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Coronavirus structural proteins including spike, membrane, envelope and / or nucleocapsid wherein the particle also packages an alphavirus genome.

14. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Orthomyxovirus structural proteins including neuraminidase, hemagglutinin protein, Matrix 1 and / or Matrix 2 wherein the particle also packages an alphavirus genome.

15. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Picomavirus structural proteins including capsid subunits like VP1, VP2 and / or VP3 wherein the particle also packages an alphavirus genome.

16. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Arenavirus structural proteins including the glycoprotein and / or the matrix proteins wherein the particle also packages an alphavirus genome.

17. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Phlebovirus structural proteins including theglycoproteins (Gn and Gc), and / or the nucleoprotein wherein the particle also packages an alphavirus genome.

18. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Hepadnavirus structural proteins including the surface antigen proteins and / or the core antigen proteins wherein the particle also packages an alphavirus genome.

19. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Rhabdovirus structural proteins including the matrix protein, the glycoprotein and / or nucleoprotein wherein the particle also also packages an alphavirus genome.

20. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Calicivirus structural proteins including capsid subunits VP1, and VP2 wherein the particle also packages an alphavirus genome.

21. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Pneumovirus structural proteins including attachment glycoprotein, Fusion, matrix, small hydrophobic and / or nucleocapsid wherein the particle also packages an alphavirus genome.

22. The method of claim 9, wherein the hybrid riboviria-alpha-pseudovirus particle (HARP) is assembled with one or more Hantavirus structural proteins including the glycoproteins (Gn and Gc) and / or the nucleoprotein wherein the particle also packages an alphavirus genome.

23. The method of claims 9-22, wherein an HRAP particle can be assembled from structural proteins from any single or combination of virus in the Riboviria realm, including Filovirus, Flavivirus, Picomavirus, Calicivirus, Coronavirus, Paramyxovirus, Orthomyxovirus, Arenavirus, Hantavirus, Lentivirus, and DNA viruses with RNA intermediates like Hepadnaviruses.

24. A use of a hybrid riboviria-alpha-pseudovirus particle (HARP) in any of the preceding claims for modeling RNA virus infections, quantifying neutralizing antibodies, viral entry inhibiting drugs, and / or cellular factors involved in viral replication.

25. A use of a hybrid riboviria-alpha-pseudovirus particle (HARP) in any of the preceding claims for delivering an alphaviral vector RNA to target tissues and cells for the purpose of gene therapy, cancer treatment or prevention, and / or to stimulate immune responses to antigens.

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