WO2020010474A1

WO2020010474A1 - The ebola virus glycoprotein as a tool to stimulate an immune response

Info

Publication number: WO2020010474A1
Application number: PCT/CA2019/050975
Authority: WO
Inventors: Gary Kobinger; Marc-Antoine DE LA VEGA; Hiva AZIZI; Xiaojian Yao
Original assignee: UNIVERSITé LAVAL; The University Of Manitoba
Priority date: 2018-07-13
Filing date: 2019-07-15
Publication date: 2020-01-16

Abstract

Pseudotyped vesicular stomatitis viruses comprising the Ebola virus glycoprotein and antigens, as well as viral preparations and pharmaceutical composition for generating an immune response in a host or for vaccination are provided herewith. VSV-based vectors for preparing pseudotyped vesicular stomatitis viruses comprising the Ebola virus glycoprotein and antigens are also provided.

Description

THE EBOLA VIRUS GLYCOPROTEIN AS A TOOL TO STIMULATE AN IMMUNE RESPONSE

FIELD

Pseudotyped vesicular stomatitis viruses comprising the Ebola virus glycoprotein and antigens, as well as viral preparations and pharmaceutical composition for generating an immune response in a host or for vaccination are provided herewith. Vesicular stomatitis virus (VSV)-based vectors for preparing pseudotyped VSVs comprising the Ebola virus glycoprotein and antigens are also provided.

BACKGROUND

Vesicular stomatitis virus (VSV) is an enveloped, negative -stranded RNA virus of the Rhabdoviridae family that encodes five viral proteins: the nucleocapsid (N), the phosphoprotein (P, a.k.a., the polymerase P), the matrix protein (M), the surface glycoprotein (G), which assembles in trimers, and the polymerase L (L).

Recombinant VSV DNA vectors forming viral particles capable of self-replication have been generated, thereby allowing manipulation of the VSV genome (Lawson, N.D. et al., Proc. Natl. Acad. Sci., 92, p.4477-4481, 1995). Heterogeneous viral antigens including antigens from HIV, influenza virus, Marburg virus, Lassa virus and Ebola virus have thus been cloned into the VSV genome and the resulting pseudotyped viral particles tested as putative vaccines in animal models.

In fact, VSV makes an excellent vaccine vector as the general population lacks antibodies against VSV, most infections are asymptomatic, the virus does not integrate into the host genome and it can accommodate large antigenic transgenes (Tober, R. et al., J. Virol., Vol. 88(9), p.4897-4907, 2014). Recombinant VSV can be produced in high-titer and in large quantities in vitro, which facilitates its production to commercial-scale.

Although, recombinant VSVs can be generated to accommodate large foreign gene inserts or multiple genes into their genomes, VSV exhibits a strong 3’ to 5’ gradient of gene expression due to discontinuous messenger RNA transcription across intergenic regions resulting in a modulation of gene expression (including foreign genes) as a function of gene distance from the 3’ transcription promoter.

The 2013-2016 Ebola virus outbreak mobilized the scientific community to test experimental VSV- based vaccines against Ebola virus. The rVSV-ZEBOV Ebola vaccine is a recombinant live-attenuated, replication-competent VSV-based vaccine expressing a surface glycoprotein of Zaire Ebola virus in replacement of the VSV glycoprotein (G). This vaccine was previously shown to confer protection in several challenge models of animal species including in healthy and immunocompromised non-human primates (NHP) (Jones SM et al., Nat Med 2005; 11:786-90; Geisbert TW et al .; 2008 PloS Pathog. el000225; Geisbert TW et al., J. Infect. Dis. 2011; 204(Suppl. 3):S 1075-S 1081)). This vaccine was since found to be highly protective against infection in an open-label, cluster-randomized ring vaccination clinical trial (Henao-Restrepo AM et al., Lancet 2017; 389:505-518). Limited protection was observed in post exposure models (Marzi AJ et al., Infect. Dis. 2016; 2l4(Suppl. 3):S360-S366).

Pseudotyped VSV carrying the Ebola virus glycoproteins have been tested in animal models. For example, Mire et al., generated a trivalent viral vaccine expressing the glycoproteins of the Marburg marburgvirus (MARV), the Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) using the VSVAG platform (J. Infect. Dis. 2015, vol. 2012 (Suppl. 2): S384-S388). Twenty-eight days post-vaccination, all animals had detectable levels of circulating anti -MARV, anti -EBOV and anti-SUDV glycoprotein IgGs. However, the level of circulating IgGs against the SUDV glycoprotein was the lowest. All animals survived the challenge with either the guinea pig-adapted-(GPA-) MARV, GPA-EBOV or GPA-SUDV although the authors underlined the limitations of the GPA-SUDV model.

Wong et al, generated a bivalent VSV-based vaccine expressing both the avian Hanoi/30408/2005 H5N 1 hemagglutinin (HA) influenza virus and the glycoprotein from the Zaire species (ZGP) of Ebolavirus (Wong G etal., J. Infect. Dis. 20l5;2l2(Suppl. 2): S435-S442). The HA and ZGP proteins are both located downstream of the VSV matrix gene (VSVAG-HA-ZGP). Wong et al., showed that the vaccine conferred 100% protection in mice when administered 28 days before challenge with homologous influenza virus or mouse-adapted Ebola virus. However, even an increased dose of the vaccine was insufficient to confer complete post-exposure protection (lxlO⁷ PFU in this study vs 2xl0⁵ PFU in previous studies). The authors hypothesize that this may be due to competition between HA and ZGP antigens, the VSVAG-HA-ZGP vaccine may be attenuated due to the addition of 2 antigens instead of one, the replication kinetics of the VSVAG-HA-ZGP virus may be slower thus impairing the ability of the bivalent vaccine to mount a quick and robust immune response or that the placement of the ZGP gene downstream of HA may result in a lower expression of ZGP.

Tsuda Y. et al, generated a bivalent viral vaccine expressing the Zaire Ebola virus (EBOV) glycoprotein and the Andes virus glycoprotein (ANVD) using the VSVAG platform (J. Infect. Dis. 2011; 204 (Suppl. 3): S1090-S 1097). The Andes virus is a hantavirus that is the major cause of hantavirus pulmonary syndrome (HPS) in South America. The Ebola virus and Andes virus share a common lethal small animal disease model, the Syrian hamster. The authors showed that the vaccine conferred complete and sterile protection following a single immunization against a lethal challenge with both MA-EBOV and ANVD. Partial protection was also observed when animals were immunized with the vaccine after infection with EBOV.

International patent application No. PCT/US2009/001821 by Geisbert el al., published on October 1, 2009 under No. W02009/120306A1 describes a recombinant vesicular stomatitis virus (rVSV) vector lacking the glycoprotein (G) gene and having a multiple cloning site (MCS) for foreign genes positioned between the matrix protein gene and the polymerase gene, as well as an additional MCS upstream of the nucleocapsid gene. This vector called pATX VSVAG4 accommodates the insertion of four additional genes into the rVSV vector. The inventors generated viral vaccines expressing the glycoproteins of different Ebola virus species with or without the glycoprotein of the Marburg virus.

International patent application No . PCT/CA2016/05 1374 by Yao el al.. published on June I^st, 2017 under No. W02017/088053 describes HIV-based vectors expressing the Ebola virus glycoprotein in replacement of the HIV envelope and generated replicating and non-replicating GP/HIV chimeric virus like particles to trigger an immune response against HIV and Ebola virus.

Racine, T. el al., reports that the only HIV vaccine trials showing promising results to date is the RV144 trial conducted in Thailand where the individuals were primed with a canarypox vector expressing HIV gag/pol/nef and boosted with a recombinant HIV gpl20. The authors suggest generating an HIV vaccine based on the VSVAG platform but reports several challenges associated with this approach (Racine et al, AIDS Res Ther 14:55, 2017).

Viral particles carrying the Ebola virus glycoprotein and an antigen, as well as viral vectors expressing the Ebola virus glycoprotein and the antigen are provided herewith.

SUMMARY

The Applicant came to the unexpected discovery that the Ebolavirus glycoprotein (EBOV GP) may be used to redirect an antigen to a sub-population of immune cells thereby modulating or increasing the host’s immune response towards said antigen.

The present disclosure therefore relates to the use of the Ebola virus glycoprotein to increase a host’s immune response towards an antigen.

The present disclosure thus provides in a first aspect, particles (including without limitations, viral particles, liposomes and the like) carrying the Ebola glycoprotein at its surface and comprising an antigen or antigens. The antigen or antigens may be located inside the viral particles or at its surface. More particularly, the viral particles of the present disclosure may comprise vesicular stomatitis virus (V SV) nucleocapsid, phosphoprotein, matrix protein, and polymerase L, an Ebola virus glycoprotein and an antigen or antigens. In accordance with the present disclosure, the antigen or antigens may be from a pathogen, from a tumor (a tumor specific antigen), from an allergen, etc. Viral particles of the present disclosure include recombinant viral particles. Viral particles of the present disclosure may also include viral-like particles.

In an additional aspect, the present disclosure provides viral preparations and pharmaceutical compositions comprising the viral particles disclosed herein. The viral preparations and pharmaceutical compositions may be used for in vitro or in vivo applications including for increasing an immune response against an antigen or antigens in a host or for vaccination.

In yet an additional aspect, the present disclosure provides pharmaceutical compositions comprising the viral particles or the viral preparation disclosed herein. The pharmaceutical composition may come in a vial or vials or in a pre -filled syringe and may comprise an adjuvant.

In a further aspect, the present disclosure provides methods of increasing an immune response towards an antigen or antigens by administering the pharmaceutical composition of the present disclosure to a host in need.

The present disclosure also relates to a nucleic acid or a set of nucleic acids that may be suitable for expressing a VSV genome, an Ebola glycoprotein and an antigen or antigens.

In accordance with the present disclosure the nucleic acid or set of nucleic acids may express a VSV genome that lacks VSV glycoprotein. Exemplary embodiments of VSV genome include the VSVAG genome.

In an additional aspect, the nucleic acid expressing the VSV genome and the Ebola glycoprotein may be on a single vector and the nucleic acid expressing the antigen or antigens may be on a separate vector or vectors.

In a further aspect, the nucleic acids expressing the VSV genome and the antigen or antigens may be on a single vector and the nucleic acid expressing the Ebola glycoprotein may be on a separate vector.

In yet a further aspect, the nucleic acids expressing the antigen or antigens and the Ebola glycoprotein may be on a single vector and the nucleic acid expressing the VSV genome may be on a separate vector. In another aspect the nucleic acids expressing the VSV genome, the Ebola glycoprotein and the antigen or antigens may be on a single vector and may be operably linked.

In accordance with the present disclosure the Ebola glycoprotein may be expressed from the VSV genome.

In accordance with the present disclosure the antigen or antigens may be expressed from the VSV genome.

In yet a further aspect, the present disclosure provides a vector or a set of vectors for recombinant expression of a VSV genome encoding vesicular stomatitis virus (VSV) proteins, an Ebola virus glycoprotein and an antigen or antigens.

In accordance with the present disclosure, the vector or set of vectors may comprise the nucleic acid or set of nucleic acids disclosed herein.

In accordance with the present disclosure, the vector may comprise elements and sequences allowing for transcription of the genome and/or for expression the genes.

The present disclosure provides in another aspect, an isolated cell comprising the vector or vectors of the present disclosure. In accordance with the present disclosure, the isolated cell may further comprise helper plasmids expressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV polymerase L or combination thereof.

When one or more vectors are used they may be co-transfected in cells to allow incorporation of the proteins into the viral particles.

In an additional aspect, the present disclosure provides a kit which may comprise a vector or a set of vectors for recombinant expression of VSVs pseudotyped with an Ebola virus glycoprotein and the antigen or antigens. In accordance with the present disclosure, the kit may comprise a vector encoding a VSV genome encoding VSV proteins, an Ebola virus glycoprotein and an antigen or antigens. The kit may further comprise a vector or vectors encoding additional proteins such as the VSV nucleocapsid, the VSV phosphoprotein, the VSV polymerase L and/or a bacteriophage RNA polymerase. The vectors may be provided in separate vials.

In an additional aspect, the present disclosure provides a kit which may comprise the nucleic acid or set of nucleic acids disclosed herein. In yet an additional aspect, the present disclosure provides a kit which may comprise the vector or set of vectors disclosed herein.

In a further aspect the present disclosure provides a method of making pseudotyped VSV particles. The method may comprise allowing expression in a cell of a) a VSV positive-sense genome comprising genes encoding VSV proteins, b) an Ebola glycoprotein and c) an antigen or antigens. The Ebola glycoprotein and/or antigen or antigens may be expressed, for example, from the VSV genome. In accordance with the present disclosure, the method may further comprise overexpressing the VSV nucleocapsid, the VSV phosphoprotein, and/or the VSV polymerase L. The method may further involve a purification step.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a schematic representing the VSV-based viral vector expressing the Ebola glycoprotein (pATX-VSV-Ebo-GP).

Figure IB is a schematic representing the VSV-based viral vector expressing the Ebola glycoprotein and the HIV env (pATX-VSV-Ebo-HIV/T7l3).

Figure 1C a schematic representing the VSV-based viral vector expressing the VSV glycoprotein and the HIV env (pATX-VSVG-HIV/T7l3).

Figure ID a schematic representing the VSV-based viral vector expressing the VSV glycoprotein and the HIV/SIVtm env (pATX-VSVG-HIV/SIVtm).

Figure IE a schematic representing the VSV-based viral vector expressing the Ebola glycoprotein and the HIV/SIVtm env (pATX-VSV-Ebo-HIV/SIVtm).

Figure IF a schematic representing the VSV-based viral vector expressing the modified Ebola glycoprotein (Ebo/V3) and the HIV/SIVtm env (pATX-VSV-Ebo/V3-HIV/SIVtm).

Figures 2A-D are schematics representing the helper plasmids encoding the polymerase L (A), the nucleocapsid (B), the phosphoprotein (C) and the T7 Polymerase (D).

Figure 3A and Figure 3B represent Western blots performed on viral preparations and shows expression of HIV envelope in VSV-G-HIV/T713 (A) and of HIV envelope and Ebola glycoprotein in VSV-Ebo-HIV/T7l3 (B). Figure 3C is a schematic representation of vectors VSV-EBO (expressing the wild type Ebola glycoprotein), VSVG-HIV/T713 (expressing the VSV glycoprotein and atruncated HIV envelope), VSVG- HlV/SIVtm (expressing the VSV glycoprotein and a chimeric HIV/SIVtm envelope), VSV-EBO- HIV/T713 (expressing the wild type Ebola glycoprotein and a truncated HIV envelope) and VSV-EBO- HlV/SIVtm (expressing the wild type Ebola glycoprotein and a chimeric HIV/SIVtm envelope).

Figure 4 is a histogram of the optical density measured following an ELISA assay of the HIV env antibodies in sera of mice vaccinated with the viral preparations expressing only the Ebola glycoprotein (mock= VSVEbo), the Ebola glycoprotein and the HIV envelope (VSV-Ebo-HIV/T7l3) or the VSV glycoprotein and the HIV envelope (VSV-G/HIV/T713).

Figure 5A and Figure 5B represent Western blots performed on viral preparations and shows expression of HIV envelope and VSV glycoprotein in VSVG-HIV/T713 and VSVG-HIV/SIVtm (A) and of HIV envelope and Ebola glycoprotein in VSV-EBO- HIV/T713 and VSV-EBO-HIV/SIVtm (B).

Figure 6 is a histogram of the optical density measured following an ELISA assay of the HIV env antibodies in sera of mice vaccinated with the viral preparations expressing only the Ebola glycoprotein (VSV-EBO), the Ebola glycoprotein and the HIV envelope truncation (VSV-EBO-HIV/T713) the Ebola glycoprotein and the HIV/SIVtm envelope chimera (VSV-EBO-HIV/SIVtm), the VSV glycoprotein and the HIV envelope truncation (VSVG-HIV/T713) or the VSV glycoprotein and the HIV/SIVtm envelope chimera (VSVG-HIV/SIVtm).

Figure 7A is a schematic representation of vectors rVSVG-HIV/SIVtm (expressing the VSV glycoprotein and the HIV/SIVtm chimera), rVSV-EBO-HIV/SIVtm (expressing the wild type Ebola glycoprotein and the HIV/SIVtm chimera) and rVSV-EboAM/V3 -HIV/SIVtm (expressing the chimeric Ebola glycoprotein (EboGPAM/HIV-V3) and the HIV/SIVtm chimera).

Figure 7B is a picture of a Western blot showing the expression of of HIV envelop glycoprotein and Ebola glycoproteins as detected by anti-EboGP antibodies (ID6, 5F7 monoclonal antibodies) or and anti-HIV-GP antibodies in VSV infected MDCK cells (lane 1 : VSVG-HIV/SIVtm, lane 2: VSV-EBO- HIV/SIVtm, lane 3: VSV-EBOAM-V3-HIV/SIVtm, lane 4: Mock).

Figure 8 is a graph of gpl20 IgG titer after immunization of mice with different rVSV viral particles. Briefly, the BALB/c mice were injected subcutaneously with lxlO⁶ TCID50 of each of rVSV as indicated. At 35 days post-immunization, mice were sacrificed, and sera were collected. The anti -HIVgp 120 antibody levels in the sera of immunized B ALB/c mice with different rVSVs were measured by anti- HIVgpl20 ELISAs. The values shown were the average levels of each group from 4 mice.

Figure 9 is a representative sequence of Ebola virus glycoprotein comprising a deletion in the mucin-like domain.

Figure 10 is a representative sequence of HIV envelope V3 region.

Figure 11A is a Western Blot performed on VSV-EBO-HIV/EboTM and VSVG-EBO- HIV/EboTM and hybridized with various antibodies against HIV env, EBOGP, VSVG or VSVN.

Figure 11B is an ELISA assay performed on sera collected from mice immunized with the described vaccines, 4 weeks post-immunization. T-test statistical analysis was done using Prism 7 and considered positive when p value falls under 0.05.

Figure 12A is a schematic representing the VSV-based viral vector expressing the Ebola glycoprotein and an HIV envelope carrying the Ebola transmembrane domain (pATX-EBO-HIV/EboTM).

Figure 12B is a schematic representing the VSV-based viral vector expressing the VSV glycoprotein) and an HIV envelope carrying the Ebola transmembrane domain (pATX-VSVG- HIV/EboTM).

DETAILED DESCRIPTION

In a first aspect, the present disclosure relates to particles carrying an Ebola virus glycoprotein (GP) and an antigen or antigens.

In another aspect, the present disclosure relates to viral particles carrying an Ebola virus glycoprotein (GP) and an antigen or antigens. These viral particles may be used to generate an immune response against the antigen or antigens. The antigen or antigens may be anchored at the surface of the viral particles or located within the viral particles.

In accordance with an embodiment of the disclosure, the viral particles and the viral vectors used to generate them may be from the Rhabdoviridae family. More particularly, the viral vectors or viral particles may be based on the vesicular stomatitis virus (VSV). Any VSV serotype may be used. In an exemplary embodiment the VSV genome is from serotype Indiana

VSV-based viral nucleic acids and vectors The present disclosure relates to a nucleic acid or a set of nucleic acids encoding VSV proteins, the Ebola glycoprotein and the antigen or antigens.

The present disclosure also relates to a nucleic acid or a set of nucleic acids encoding operably linked sequences encoding the VSV proteins and the Ebola glycoprotein and optionally the antigen or antigens. In a particular embodiment of the disclosure, the nucleic acid encodes the VSV proteins, the Ebola glycoprotein and the antigen or antigens. The nucleic acids may serve as templates for transcription of a VSV antigenome (positive -sense).

In an embodiment of the disclosure, the VSV proteins encoded by the nucleic acids of the present disclosure includes the nucleocapsid, the phosphoprotein, the matrix protein, the VSV surface glycoprotein (or a portion of the glycoprotein such as the Gstem (US2009/0175900)) and the polymerase L. In particular embodiments of the disclosure, the nucleic acids do not encode a functional VSV surface glycoprotein.

In accordance with an embodiment of the disclosure, the nucleic acid may be a DNA or RNA molecule encoding in a 5’ to 3’ fashion; the nucleocapsid, the phosphoprotein, the matrix protein, the antigen or antigens, the Ebola virus glycoprotein, and the polymerase L. The nucleic acid encodes the antigenomic sense (positive sense) RNA of VSV.

In accordance with another embodiment of the disclosure, the nucleic acid may be a DNA or RNA molecule encoding in a 5’ to 3’ fashion; the nucleocapsid, the phosphoprotein, the matrix protein, the Ebola virus glycoprotein, the antigen or antigens, and the polymerase L.

The nucleic acid may comprise sequences necessary for the transcription and/or replication of the VSV genome such as the leader sequence and the intergenic sequences. The nucleic acids may be“codon- optimized” to enhance protein expression. An exemplary embodiment of a“codon-optimized” nucleic acid sequence encoding the Ebola glycoprotein is provided in US Pat. No. 8,663,981.

In another aspect, the present disclosure relates to viral vectors for expressing viral particles carrying the Ebola virus glycoprotein and the antigen or antigens. The viral vector may be a plasmid comprising the necessary elements for its replication and maintenance in bacteria such as an origin of replication and antibiotic resistance gene(s). The plasmid may also comprise a suitable promoter for in vitro transcription and/or translation.

In accordance with an embodiment of the disclosure, the vector is a VSV-based viral vector engineered so that it does not contain or does not express the gene encoding the VSV glycoprotein. An exemplary embodiment of the VSV-based viral vector is VSVAG. In accordance with another embodiment of the disclosure, the VSV glycoprotein may be mutated so that its expression is attenuated or silenced. Other non-limitative exemplary embodiments of suitable VSV-based viral vectors are disclosed in patent publication Nos. US2009/0175900 from Parks et al., US2007/0218078 from Clarke et al., US2009/0169580 from Whelan et al.

It is to be understood herein that the gene encoding the antigen or antigens may be cloned into the VSV-based vector thereby being part of the VSV genome. Alternatively, the gene encoding the antigen or antigens may be provided in a separate vector.

In yet another aspect, the present disclosure relates to a kit comprising a vector or a set of vectors for making the viral particles of the present disclosure.

The kit may comprise, for example, vials comprising a vector for producing the VSV-based viral genome and helper plasmids. The helper plasmids may be selected from the group consisting of plasmids encoding VSV nucleocapsid, VSV polymerase L, VSV phosphoprotein or combination thereof. In the case where the gene encoding the antigen or antigens is not part of the VSV genome, it may be provided by a separate vector. The kit may thus further comprise a plasmid encoding the antigen or antigens.

In an additional aspect thereof, the present disclosure therefore relates to isolated cells comprising vectors for expressing and/or making viral particles carrying the Ebola virus glycoprotein and an antigen.

The Ebola virus glycoprotein

The viral particle of the present disclosure comprises, in an aspect thereof, an Ebola virus glycoprotein which may allow the viral particle to be targeted to a subpopulation of immune cells for modulating or increasing the host’s immune response towards the antigen comprised inside or at the surface of the viral particles.

Exemplary embodiments of Ebola virus glycoprotein include for example and without limitation; the Zaire Ebola virus glycoprotein, Sudan Ebola virus glycoprotein, Taϊ Forest Ebola virus glycoprotein, Bundibugyo Ebola virus glycoprotein and Reston Ebola virus glycoprotein. In a particular aspect of the disclosure, the Ebola virus glycoprotein may be the Zaire Ebola virus glycoprotein (EBOV) and more particularly, the Mayinga strain, the Makona strain or the Kikwit strain. The Ebola virus glycoprotein may be obtained from any Ebola isolates.

The Ebola virus glycoprotein sequence used in the present disclosure may originate from a naturally occurring Ebola virus including those obtained from clinical isolates, reference sequences such as those published in sequence databases, recombinant and/or chimeric Ebola virus glycoproteins and mutated Ebola virus glycoprotein.

Exemplary embodiments of Ebola virus glycoprotein sequence can be obtained, for example, from GenBank Accession numbers NC_002549 ( Zaire Ebola virus), NC_006432 ( Sudan Ebola virus), NC_0l4372 (Taϊ Forest Ebola virus), NC_0l4373 ( Bundibugyo Ebola virus) and NC_004l6l ( Reston Ebola virus). In other exemplary embodiments of the disclosure, the Ebola virus glycoprotein may be modified and may include for example, variants having at least 70%, at least 80%, at least 85%, at least 90% sequence identity, at least 95% sequence identity or at least 98% sequence identity with those of the Zaire Ebola virus, Sudan Ebola virus, Taϊ Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus. Ebola virus glycoprotein variants may remain immunogenic or not.

In yet other embodiments the Ebola virus glycoprotein may be encoded by the sequence comprising SEQ ID NO: 1 or by a sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO: 1 or as set forth in SEQ ID NO: 1.

In a further embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:2 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In yet a further embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:3 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In an additional embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:4 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In another embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:5 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In yet another embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:6 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto. In a further embodiment, the Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:7 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In certain embodiments, the Ebola virus glycoprotein may be modified by rendering the mucin -like domain non-functional. Such modification may occur for example, by deletion, by amino acid substitutions and the like.

As such, the Ebola virus glycoprotein may comprise the amino acid sequence set forth in any of SEQ ID NO:2 to 7 wherein the mucin-like domain is deleted or mutated.

Exemplary embodiments of Ebola virus glycoprotein may comprise a sequence as set forth in SEQ ID NO:9 or a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

Alternatively, the viral glycoprotein may originate from Marburg virus (e.g., Accession number YP_001531156.1) and may include Marburg virus glycoprotein variants having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the sequence published under Accession number YP_001531156.1.

The list or sequence of Ebola virus glycoproteins provided in this section or disclosed elsewhere in the text is not intended to be construed as limiting the scope of the disclosure. As such, other Ebola virus glycoproteins than those listed may be used.

Antigens

The antigen or antigens selected for expression in the viral vector may be from a pathogen, from a tumor (a tumor specific antigen), from an allergen, etc.

The antigen may be monovalent or multivalent (e.g., a multi-chain protein composed of several antigens from a single pathogen, from multiple pathogens, from different strains, isolates, or serotypes of a given pathogen). The antigen may also be a consensus sequence derived from the amino acid sequence of different strains, isolates, or serotypes of a given pathogen.

The antigen may also be a mosaic of different sequences derived from different proteins or epitopes of one or more pathogens, tumor or allergen. Generally, the specific strain(s), isolate(s) or serotype(s) of a pathogen used for generating the vaccine of the present disclosure may be selected from the strain(s), isolate(s) or serotype(s) that is(are) prevalent in a given population. In the case of new outbreaks, the gene expressing the antigen or antigens may be sequenced and cloned into the vector of the present disclosure using methods known in the art involving for example, amplification by polymerase chain reaction, use of restriction enzymes, ligation, transformation of bacteria, sequencing, etc.

Exemplary embodiments of antigens include for example and without limitation, viral antigens from Retroviridae (HIV, HTLV), Flaviviridae (e.g., Zika, Hepatitis C, West Nile, Dengue, Yellow fever, Japanese encephalitis, tick-bome encephalitis, Saint Louis encephalitis, Alkhurma hemorrhagic fever virus, Kyasanur Forest Disease virus, Omsk hemorrhagic fever virus etc.), Togaviridae (e.g., Chikungunya, Rubella virus), Picomaviridae (Hepatitis A, Polio virus, Enterovirus (EV71)), Caliciviridae (Norwalk virus, Sapporo virus), Astroviridae, Coronaviridae (e.g., Middle East Respiratory syndrome coronavirus (MERS- CoV), Severe acute Respiratory Syndrome coronavirus (SARS-CoV, etc.), Rhabdoviridae (rabies), Filoviridae (Ebola virus, Marburg virus), Paramixoviridae (Nipah virus, Hendra virus, Measles virus, Mumps virus, Respiratory syncytial virus), Orthomixoviridae (Influenza virus H1N1, H3N2, H5N1, H7N9), Bunyaviridae (Rift Valley Fever Disease virus, Crimean-Congo hemorrhagic fever, Hantaan, Dobrava, Saarema, Seoul and Puumala viruses, Hanta virus), Arenaviridae (Lassa virus, Junin virus, Guanarito virus, Lujo virus, Sbia virus, Machupo virus, Whitewater Arroyo virus, Chapare virus, Lymphocytic choriomeningitis virus), Reoviridae (rotavirus), Papovaviridae (human papilloma viruses), Adenoviridae, Parvoviridae, Herpesviridae (Herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus), Poxviridae (smallpox virus, vaccinia virus), Hepadnaviridae (Hepatitis B).

Exemplary embodiments of antigens include for example and without limitation, bacterial antigens from Salmonella Typhi, Salmonella Parathyphi, Yersinia pestis, Vibrio cholera, Corynebacterium diphtheria, Haemophilus influenza type B, Neisseria meningitidis, Bordetella pertussis, Streptococcus pneumoniae, Clostridium tetani, Clostridium difficile, Mycobacterium tuberculosis, Campylobacter jejuni, enterotoxigenic Escherichia coli, Streptococcus agalactiae (group B), Streptococcus pneumoniae, Streptococcus pyrogenes, Salmonella enterica, Shigella, Staphylococcus aureus.

Exemplary embodiments of antigen also include for example and without limitations, parasite antigens from Plasmodium ( Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium Knowlesi), Trypanosome ( Trypanosoma cruzi), Necator americanus, Leishmania, Schistosoma haematobium, Schistosoma mansoni, H. anatolicumanatolicum, H. dromedarii, Rhipicephalus sanguineus, etc. For veterinary purposes, the pathogen may be selected amongst animal-specific pathogens or amongst pathogens causing zoonotic diseases. Examples of veterinary vaccines are provided for example, in Roth, J.A., 2011 (Procedia in Vaccinology 5: 127-136, 2011) and Redding L. and Weiner D.B., 2009 (Expert Rev. Vaccines 8(9), 1251-1276, 2009). Licensed products for animal vaccination include preventative vaccines for West Nile virus in horses and infectious haematopoietic necrosis virus in fish, a therapeutic cancer vaccine for dogs, and a growth hormone gene therapy to increase litter survival in breeding pig sows.

Exemplary embodiments of tumor antigens include without limitation; 707 alanine proline-AFP (707-AP), alpha (a)-fetoprotein (AFP), adenocarcinoma antigen recognized by T cells 4 (ART-4), B antigen; b-catenin/mutated (BAGE), breakpoint cluster region-Abelson (Bcr-abl), CTL-recognized antigen on melanoma (CAMEL), carcinoembryonic antigen peptide- 1 (CAP-l), caspase-8 (CASP-8), cell-division- cycle 27 mutated (CDC27m), cycline-dependent kinase 4 mutated (CDK4/m), carcino-embryonic antigen (CEA), cancer testis antigen (CT), cyclophilin B (Cyp-B), differentiation antigen melanoma (DAM), elongation factor 2 mutated (ELF2M), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETV6- AML1), glycoprotein 250 (G250), G antigen (GAGE), N-acetylglucosaminyltransferase V (GnT-V), glycoprotein 100 kDa (GplOO), helicose antigen (HAGE), human epidermal receptor-2/ neurological (HER-2/neu), arginine (R) to isoleucine (I) exchange at residue 170 of the a-helix of the a2 -domain in the HLA-A2 gene (HLA-A * 0201-R1701), human papilloma virus E7 (HPV-E7), heat shock protein 702 mutated (HSP70-2M), human signet ring tumor-2 (HST-2), human telomerase reverse transcriptase (hTERT or hTRT), intestinal carboxyl esterase (iCE), KIAA0205, L antigen (LAGE), low-density lipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-L-fucosyltransferase (LDLR/FUT), melanoma antigen (MAGE), melanoma antigen recognized by T cells- l/melanoma antigen A (MART-l/Melan-A), melanocortin 1 receptor (MC1R), myosin mutated (Myosin/m), mucin 1 (MUC1), melanoma ubiquitous mutated 1, 2, 3 (MUM-l, -2, -3), NA cDNA clone of patient M88 (NA88-A), New York-esophagus 1 (NY- ESO-l), protein 15 (P15), protein of 190 kDa ber-abl (pl90 minor bcr-abl), promyelocytic leukaemia/retinoic acid receptor a (Pml/RARa), preferentially expressed antigen of melanoma (PRAME), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), renal antigen (RAGE), renal ubiquitous 1 or 2 (RU 1 or RU2), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1 or 3 (SART- 1 or SART-3), translocation Ets-family leukemia/acute myeloid leukemia 1 (TEL/ AML1), triosephosphate isomerase mutated (TPI/m), tyrosinase related protein 1 or gp75 (TRP-l), tyrosinase related protein 2 (TRP-2), TRP-2/intron 2 (TRP-2/ INT2), Wilms' tumor gene (WTl).

In instances where the antigen does not have a transmembrane domain, it may be desirable to add an anchoring domain so that the antigen can be incorporated into the VSV external envelope. VSV glycoprotein amino acid residues may be used for such purposes (Rabinovich S. et al., PloS one, 9(9):el06597, 2014), including for example, the VSV glycoprotein transmembrane domain, cytoplasmic tail and amino acids of the ectodomain or combination thereof. In some instances, the SIV envelope short cytoplasmic tail may be used for anchoring an antigen. In other instances, the transmembrane domain and a short cytoplasmic tail of Ebola virus glycoprotein may be used for anchoring an antigen.

In accordance with the present disclosure, antigen or antigens are other than the Ebola virus glycoprotein or the Marburg virus glycoprotein.

In accordance with the present disclosure, desired antigens include for example and without limitations, antigens from the Lassa fever virus, antigens from human immunodeficiency virus (HIV), antigens from the Nipah virus, antigens from the Zika virus, antigens from MERS CoV, antigens from CCHF, etc.

In order generate a stronger immune response in a host, it may be desirable to select a surface antigen of a pathogen, such as glycoproteins of viruses or suitable fragments thereof (e.g., HIV gpl60, gpl40 or gpl20, Ebola virus glycoprotein (e.g., from the Zaire species), Nipah virus glycoprotein, Zika virus envelope and/or pre-membrane M (prM), Lassa fever virus glycoprotein, Crimean Congo Hemorrhagic Fever virus glycoprotein, MERS -CoV S protein (envelope)). However, a vaccine for a given pathogen may include other types of antigens. For example, structural proteins such as the viral capsid, nucleocapsid, matrix, including HIV gag, CCHF nucleocapsid, etc. These antigens may be located inside the VSV-based viral particles or at their surface.

In certain embodiments, the antigen may be from HIV and may include for example HIV envelope sequences (gpl20, gpl40) or portion thereof of naturally occurring HIV including those obtained from clinical specimens, recombinant and/or chimeric envelopes (e.g., HIV/SIVtm chimeras and the like), mutated HIV envelopes (such as those disclosed in US20190127466, etc).

Exemplary embodiments of HIV antigens that may be used in the present disclosure include for example and without limitations, "mosaic" HIV-l Gag, Pol and Env antigens, derived from HIV Group Antigen (Gag), Polymerase (Pol), and Envelope (Env) proteins, such as those described in Barouch et al, Nat Med 2010, 16: 319-323, WO 2010/059732), trimeric HIV envelope proteins such as for example, clade C gpl40 proteins or mosaic envelope trimer protein (WO 2014/042942, WO 2014/107744, Nkolola et al 2014 J. Virol. (2014) 88(17), 9538-9552). The list or sequence of antigens provided in this section or disclosed elsewhere in the text is not intended to be construed as limiting the scope of the disclosure. As such, other antigens than those listed may be used.

Variants

In accordance with an aspect of the present disclosure, the Ebola glycoprotein and/or the antigens used in the present disclosure includes variants of naturally occurring proteins.

Variants encompassed by the present disclosure include those which may comprise an insertion, a deletion or an amino acid substitution (conservative or non-conservative). These variants may have at least one amino acid residue in its amino acid sequence removed and a different residue inserted in its place.

Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

Other exemplary embodiments of conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in an undesired property, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.

It is known in the art that variants may be generated by substitutional mutagenesis and retain the biological activity of the polypeptides of the present disclosure. These variants have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its place. Examples of substitutions identified as“conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated“exemplary substitutions” in Table 1 A, or as further described herein in reference to amino acid classes, are introduced and the products screened.

Naturally occurring residues are divided into groups based on common side chain properties:

(group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (He)

(group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln),

(group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)

(group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg) (group 5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and

(group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

Table 1. Exemplary amino acid substitutions

Generally, the degree of similarity and identity between two sequences is determined using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) using default settings, i.e., meagablast program (see NCBI Handout Series | BLAST homepage & search pages | Last Update September 8, 2016).

Percent identity will therefore be indicative of amino acids which are identical in comparison with the original peptide and which may occupy the same or similar position.

Percent similarity will be indicative of amino acids which are identical and those which are replaced with conservative amino acid substitution in comparison with the original peptide at the same or similar position.

Variants of the present disclosure therefore comprise those which may have at least 70%, 75%,

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99% or 100% sequence identity with an original sequence or a portion of an original sequence.

Exemplary embodiments of variants are those having at least 80% sequence identity to a sequence described herein. Other exemplary embodiments of variants are those having at least 85% sequence identity to a sequence described herein. Further exemplary embodiments of variants are those having at least 90% sequence identity to a sequence described. Other exemplary embodiments of variants are those having at least 95% sequence identity to a sequence described herein. Additional exemplary embodiments of variants are those having at least 99% sequence identity to a sequence described herein.

Due to the inherent degeneracy of the genetic code, DNA sequences that encode the same, substantially the same or a functionally equivalent amino acid sequence may be produced and used. The nucleotide sequences of the present disclosure may be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

Method of making VSV-based viral particles

The viral particles of the present disclosure comprise the Ebola virus glycoprotein at their surface and one or more antigens for which an immune response is sought. The viral particle may thus carry a protein or polypeptide originating from a pathogen (e.g., a virus, a bacterium, a parasite, etc) or from a tumor (a tumor-specific antigen).

The present disclosure thus provides a method of making pseudotyped vesicular stomatitis viral particles. The method may comprise allowing expression in a cell of a) a VSV positive-sense genome comprising genes encoding VSV proteins, b) an Ebola glycoprotein and c) an antigen or antigens.

The viral particles are produced in accordance with the methods described in Lawson et al., (PNAS, USA, Vol. 92, p. 4477-4481, 1995), Stillman et al, (J. Virol., 69:2946-2953, 1995) or Whitt M.A, (J Virol Methods, l69(2):365-374, 2010).

In an exemplary embodiment of the disclosure, both the Ebola glycoprotein and antigen or antigens are expressed from the VSV positive-sense genome.

However, the viral particles of the disclosure may be made by expressing the Ebola glycoprotein from the VSV positive-sense genome and providing the antigen or antigens by other means (i.e., constitutive/inducible expression by the cells, infection with vaccinia virus-expressing the antigen or antigens, etc.).

The viral particles of the disclosure may also be made by expressing the antigen or antigens from the VSV positive-sense genome and providing the Ebola glycoprotein by other means (i.e., constitutive/inducible expression by the cells, infection with vaccinia virus-expressing the antigen or antigens, etc.).

In instances where expression of the Ebola glycoprotein and antigen or antigens from the VSV positive-sense genome is not sufficient, the method may also involve overexpressing the Ebola glycoprotein and antigen or antigens using plasmids or other techniques.

In a particular embodiment, the gene encoding the Ebola virus glycoprotein and the gene of interest are cloned in the VSV-based vector which encodes the VSV antigenomic RNA under the control of a T7 polymerase promoter. The VSV-based vector is transfected together with three helper plasmids for overexpression of the nucleocapsid (pBS-N), the phosphoprotein (pBS-P) and the polymerase L (pBS-L).

In some instances, the T7 polymerase is provided by infecting cells with vaccinia virus-expressing the T7 polymerase. However, the T7 polymerase may be provided by transfecting cells with an expression plasmid encoding the T7 polymerase or by constitutive/inducible expression.

Cells (e.g., BHK, a mixture of HEK293T/VERO-E6 cells, etc.) are infected with the T7 polymerase-expressing vaccinia viruses and transfected with the VSV-based vector and the helper plasmids. The cell supernatant containing the viral particles is recovered once cytopathic effects are observed. In instances where no cytopathic effects are observed, the supernatant is nevertheless recovered for blind passages.

The viral particles are plaque purified and titered. The cell supernatant is diluted by 10-fold serial dilutions which are used to infect cells. After an incubation period sufficient to allow cells to become infected, an agar gel is overlaid on the infected cells. The plaques are isolated with a sterile Pasteur pipette and the viral particles contained in the agar plug recovered by elution. The infectivity titer may be determined by plaque assay (plaque forming units (PFUs)) or by calculating the TCID50.

Viral stocks are prepared by repeating an infection cycle using a low dilution of the plaque purified viral preparation. Viral particles of the disclosure may be purified by any methods known in the art, including through sucrose gradient. The infectivity of viral stocks may be determined by titration assays.

The viral particles thus generated are replicative. However, the present disclosure encompasses attenuated, replicative or partially replicative (i.e., one cycle) viral particles.

The method may also comprise a step of purifying the pseudotyped vesicular stomatitis viral particles.

The exemplary methods provided in this section or disclosed elsewhere in the text are not intended to be construed as limiting the scope of the disclosure. As such, other methods may be used.

Viral Preparations and Pharmaceutical Compositions

Viral preparations may be obtained by purifying viruses from the supernatant of virus-producing cells. The viral preparations of the present disclosure may be substantially free of cell debris (e.g. substantially pure). The viral preparation may be used for research purposes (including in vitro testing) as well as in pre-clinical or clinical settings.

In order to generate the pharmaceutical compositions of the present disclosure, substantially pure viral particles or viral preparations may be diluted in pharmaceutically acceptable carriers or excipients.

As such in a further aspect, the present disclosure relates to pharmaceutical compositions comprising the viral particles of the present disclosure and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be formulated for delivery by injection (e.g., intramuscular, intradermal, subcutaneously) or for mucosal administration (oral, intranasal). The present disclosure also encompasses immunogenic compositions. The pharmaceutical compositions of the disclosure may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used. To prepare such a composition, the viral preparation of the disclosure may be mixed with one or more pharmaceutically acceptable carriers and/or excipients. The carriers and excipients may be“acceptable” in the sense of being compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

A pharmaceutical composition may also be formulated in the form of an oil-in-water emulsion. The oil-in-water emulsion may be based, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ or tetratetracontane; oil resulting from the oligomerization of alkene(s), e.g., isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, such as plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, e.g., isostearic acid esters. The oil advantageously is used in combination with emulsifiers to form the emulsion. The emulsifiers may be nonionic surfactants, such as esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, such as the Pluronic® products, e.g., L121. The adjuvant may be a mixture of emulsifier(s), micelle-forming agent, and oil such as that which is commercially available under the name Provax® (DEC Pharmaceuticals, San Diego, Calif.). The pharmaceutical compositions of the disclosure may contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).

Adjuvants include, but are not limited to, mineral salts (e.g., AlK(SC>4)2, AlNa(SC>4)2, AlNH(S04)2, silica, alum, Al(OH)3, Ca3(PC>4)2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs), CpG oligonucleotides, poly IC or poly AU acids, saponins such as QS21, QS 17, and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, such as 3- de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod, etc.

The disclosure also relates to a kit comprising a vial including the viral particles, viral preparation or pharmaceutical composition of the present disclosure and instructions for their use or administration.

The type of viruses, vectors, viral preparations and pharmaceutical compositions provided in this section or disclosed elsewhere in the text is not intended to be construed as limiting the scope of the disclosure. As such, other viruses, vectors, viral preparations and pharmaceutical compositions than those listed may be used.

Method of administration

Pharmaceutical compositions comprising the viral particles of the present disclosure may be administered to a host including for example mammals and non-mammals, (e.g., humans and animals such as non-human primates, cattle, rabbits, mice, rats, sheep, goats, horses, birds, poultry, fish, etc.).

The pharmaceutical composition may be administered alone or co-administered with other therapeutics aimed at increasing the host’s immune response such as for example, adjuvants, immunomodulators (cytokine, chemokines, checkpoint inhibitors, etc.), etc. In accordance with the present disclosure, the pharmaceutical composition may be administered in multiple rounds.

The pharmaceutical composition may be administered as a single dose or in multiple doses. In certain aspects of the disclosure, the method of generating an immune response includes administering a single dose of the pharmaceutical composition. In other aspects of the disclosure, the method of generating an immune response includes administering multiple doses of the pharmaceutical composition (e.g., two or three times in a lifespan, three times a year, twice a year, every 2 months, every month etc.).

The pharmaceutical compositions may comprise a dose of viral particles ranging from about 10⁵ to about 10¹⁰ PFUs. In an exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁹ PFUs. In another exemplary embodiment to dose of viral particles may range from about 10⁶ to about 10⁹ PFUs. In yet another exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁸ PFUs. In a further exemplary embodiment to dose of viral particles may range from about 10⁶ to about 10⁸ PFUs. In yet a further exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁸ PFUs. In an additional exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁷ PFUs. In another exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁶ PFUs.

The pharmaceutical compositions may comprise a dose of viral particles ranging from about 10⁵ to about 10¹⁰ TCID50. In an exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁹ TCID50. In another exemplary embodiment to dose of viral particles may range from about 10⁶ to about 10⁹ TCID50. In yet another exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁸ TCID50. In a further exemplary embodiment to dose of viral particles may range from about 10⁶ to about 10⁸ TCID50. In yet a further exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁸ TCID50. In an additional exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁷ TCID50. In another exemplary embodiment to dose of viral particles may range from about 10⁵ to about 10⁶ TCID50.

The pharmaceutical composition may be administered by injection, intramuscularly, intradermally, transdermally, subcutaneously, to the mucosa (oral, intranasal), etc.

The present disclosure therefore relates to a method of generating an immune response towards an antigen or antigens by administering viral particles carrying the Ebola virus glycoprotein and the antigen or antigens to a host.

The method may include administering a first pharmaceutical composition that may act as a “prime” and a second pharmaceutical composition that may act as a“boost”. One or more of the antigens used in the prime or boost may be provided in the form of the viral particles disclosed herein. In an exemplary embodiment, the boost step may be performed with the same VSV serotype or with another VSV serotype.

In the case of HIV vaccination, the first pharmaceutical composition may comprise for example, one or more of HIV gag, pol or nef or portions or mosaic thereof and the second pharmaceutical composition may comprise for example, an HIV envelope. In some instances, an immune response may be generated by administering viral particles expressing Ebola GP and only the HIV envelope. Following administration, the host’s immune response towards the antigen may be assessed using methods known in the art. In some instances, the level of serum antibodies against the antigen may be measured by ELISA assays or by other techniques known to a person skilled in the art. The cellular immune response towards the antigen may be assessed by ELISPOT or by other techniques known to a person skilled in the art.

In the case of pre-clinical studies in animals, the level of protection against the pathogen may be determined by challenge experiments where the pathogen is administered to pre -vaccinated or post- vaccinated animals and the animals’ health or survival is assessed. The level of protection conferred by the vaccine expressing a tumor-specific antigen may be determined by tumor shrinkage or inhibition of tumor growth in animal models carrying the tumor.

In some instances, protective immunity may sometimes be evaluated by determining the presence of neutralizing antibodies.

Challenge models for Nipah virus include hamsters, cats, ferrets, African green monkey and pigs (Satterfield, B. A et al, Vaccine 34:2971-2975, 2016).

The primary HIV animal challenge models include Rhesus macaques challenged with either SIV or a chimeric construct of SIV and HIV Env (SHIV). It is presumed that the best vaccine candidates would elicit neutralizing antibodies and/or non-neutralizing antibodies that mediate viral clearance through other mechanisms (such as antibody-dependent cell-mediated cytotoxicity (ADCC) or virus inhibition, (ADCVI)), and/or T cells (primarily CD8+) that suppress virus growth or kill virus-infected cells (Safrit, J. T. et al, Vaccine 34:2921-2925, 2016).

Animal models for Lassa Fever challenge or infection include guinea pigs, but non-human primates (e.g., rhesus monkeys, cynomolgus monkeys) are preferred.

Animal models for MERS challenge or infection include, for example, non-human primates (e.g., rhesus and common marmoset), dromedary camels, transgenic mice expressing hDPP4 (human receptor for MERS), lamas, etc.

Animal models for CCHF challenge or infection include, for example, mice (e.g., new bom), rats, rabbits, goats, guinea pigs, cattle, non-human primates (e.g. cynomologus macaques), IFNAR and STAT- 1 mice, etc. Animal models for Zika challenge or infection include, for example, IFNAR mice, non-human primates, etc.

Definitions

The use of the terms "a" and "an" and "the" and similar referents in the context of describing embodiments (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Unless specifically stated or obvious from context, as used herein the term“or” is understood to be inclusive and covers both“or” and“and”.

The term“and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other.

The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. The term“consisting of’ is to be construed as close-ended.

As used herein the term “VSV-based viral vectors” or “VSV-based vectors” are used interchangeably. The term“VSV-based viral vectors” encompasses vectors expressing all VSV proteins except the VSV glycoprotein (either though total or partial deletion of the VSV gene or through silencing mutations). An exemplary embodiment of a“VSV-based viral vector” is“VSV AG”. Another exemplary embodiment of a“VSV-based viral vector” is the Gstem vector disclosed in US2009/0175900. The term “VSV-based viral vectors” also encompasses vectors capable of expressing all VSV proteins including the VSV glycoprotein.

As used herein the term“particles” refers to a vehicle in which proteins can be attached or anchored and which comprises a membrane such as a lipidic membrane (e.g., lipid bilayer including artificial or derived from cell membrane).

As used herein the term“VSV-based viral particles” means recombinant viral particles comprising VSV proteins (with or without VSV glycoprotein). As used herein the term“pseudotyped vesicular stomatitis virus” refers to VSV -based viral particles carrying non-VSV proteins (i.e., foreign proteins) such as the Ebola glycoprotein and the antigen or antigens.

As used herein the term“VSV-based vaccine” means a pharmaceutical composition comprising “VSV-based viral particles” or“pseudotyped vesicular stomatitis virus”.

As used herein the term“host in need” refers to a host for which delivery of an antigen or antigens to the host’s cell is desired. A“host in need” includes a host having a disease or susceptible of having a disease. For example, a“host in need” may include hosts infected or susceptible or being infected with pathogen, hosts having or susceptible of having a tumor, hosts having or susceptible or having allergies, etc.

As used herein the term “90% sequence identity”, includes all values contained within and including 90% to 100%, such as 91%, 92%, 92,5%, 95%, 96.8%, 99%, 100%. Likely, the term“at least 75% identical” includes all values contained within and including 75% to 100%.

It is to be understood herein that the nucleic acid sequences encoding protein(s) or peptide(s) of interest may be codon-optimized. The term“codon-optimized” refers to a sequence for which a codon has been changed for another codon encoding the same amino acid but that is preferred or that performs better in a given organism (increases expression, minimize secondary structures in RNA, etc.). “Codon- optimized” sequences may be obtained, using publicly available softwares or via service providers including GenScript (OptimumGene™, US Pat. No. 8,326,547).

As used herein,“pharmaceutical composition” means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically effective amount" as used herein refers to that amount that provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), etc.

As used herein the term“immunogenic composition” refers to a composition or pharmaceutical composition that induces a humoral and/or cellular immune response in a subject. The term“treatment” for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object slows down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

In addition to the embodiments described and provided in this disclosure, the following non-limiting embodiments are particularly contemplated.

1. Particles comprising an Ebola virus glycoprotein and an antigen or antigens.

2. The particles of any one of the preceding embodiments, wherein the particles are viral particles, liposomes and the like.

3. Viral particles comprising an Ebola virus glycoprotein and an antigen or antigens.

4. Viral particles comprising vesicular stomatitis virus (VSV) nucleocapsid, phosphoprotein, matrix protein, and polymerase L, an Ebola virus glycoprotein and an antigen or antigens.

5. The viral particles of any one of the preceding embodiments lacking the VSV glycoprotein.

6. The viral particles of any of the preceding embodiments, wherein the antigen or antigens are from one or more pathogens.

7. The viral particles of any of the preceding embodiments, wherein the pathogen is selected from the group consisting of viruses, bacteria and parasites.

8. The viral particles of any of the preceding embodiments, wherein the antigen or antigens are tumor- specific antigen or antigens.

9. The viral particles of any of the preceding embodiments, wherein the antigen or antigens comprise a sequence allowing anchoring at the surface of vesicular stomatitis viruses.

10. The viral particles of any of the preceding embodiments, wherein the vesicular stomatitis virus is replicative.

11. The viral particles of any of the preceding embodiments, wherein the viral particles are recombinant viral particles.

12. A viral preparation comprising the viral particles of any of the preceding embodiments.

13. The viral preparation of any of the preceding embodiments, for increasing an immune response against an antigen or antigens in a host, wherein the viral preparation comprises substantially purified viral particles. A pharmaceutical composition comprising the viral particles or the viral preparation of any of the preceding embodiments.

The pharmaceutical composition of any of the preceding embodiments, further comprising an adjuvant. An immunogenic composition comprising the viral particles or the viral preparation of any of the preceding embodiments.

A vial or vials comprising the pharmaceutical composition of any of the preceding embodiments. A kit comprising the vial or vials of any of the preceding embodiments and instructions for the use of the pharmaceutical composition.

A method of increasing an immune response towards an antigen or antigens, the method comprising administering the pharmaceutical composition of any of the preceding embodiments to a host in need. The method of any of the preceding embodiments, wherein the pharmaceutical composition is administered by injection.

The method of any of the preceding embodiments, wherein the injection is performed intramuscularly, subcutaneously intradermally.

The method of any of the preceding embodiments, wherein the pharmaceutical composition is delivered transdermally.

The method of any of the preceding embodiments, wherein the pharmaceutical composition is delivered to a mucosa.

The method of any of the preceding embodiments, wherein the pharmaceutical composition is administered in a single-dose regimen.

The method of any of the preceding embodiments, comprising prime and boost steps.

A nucleic acid or a set of nucleic acids suitable for expressing a VSV genome, an Ebola glycoprotein and an antigen or antigens.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the VSV genome does not encode a functional VSV glycoprotein.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the VSV genome is a VSV AG genome. The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the nucleic acids expressing the VSV genome and the Ebola glycoprotein are on a single vector and wherein the nucleic acid expressing the antigen or antigens is on a separate vector or vectors.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the nucleic acids expressing the VSV genome and the antigen or antigens are on a single vector and wherein the nucleic acid expressing the Ebola glycoprotein is on a separate vector.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the nucleic acids expressing the antigen or antigens and the Ebola glycoprotein are on a single vector and wherein the nucleic acid expressing the VSV genome is on a separate vector.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the nucleic acids expressing the VSV genome, the Ebola glycoprotein and the antigen or antigens are on a single vector. The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the Ebola glycoprotein is expressed from the VSV genome.

The nucleic acid or set of nucleic acids of any of the preceding embodiments, wherein the antigen or antigens are expressed from the VSV genome.

A vector or set of vectors comprising the nucleic acid or set of nucleic acids of any of the preceding embodiments.

A nucleic acid, set of nucleic acids, vector or set of vectors for recombinant expression of a VSV positive-sense genome (antigenome) comprising genes encoding vesicular stomatitis virus (VSV) proteins, an Ebola virus glycoprotein and an antigen or antigens, wherein the genes are operably linked. The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, comprising sequences allowing for transcription of the genome and/or for expression of the genes. The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the vector is a VSV-based vector expressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, and the VSV polymerase L.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the vector is a VSV-based vector expressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, the VSV surface glycoprotein and the VSV polymerase L.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, the antigen or antigens, the Ebola vims glycoprotein, and the polymerase L are arranged sequentially in a 5’to 3’ fashion.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the Ebola vims glycoprotein is the Zaire Ebola vims glycoprotein.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the Zaire Ebola vims glycoprotein is from the Makona strain, the Mayinga strain or the Kikwit strain.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the antigen or antigens are from one or more pathogens.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the pathogen is selected from the group consisting of vimses, bacteria and parasites.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the antigen or antigens are tumor-specific antigen or antigens.

The nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments, wherein the antigen or antigens comprise a sequence allowing anchoring at the surface of VSV-based viral particles.

An isolated cell comprising the nucleic acid, set of nucleic acids, vector or set of vectors of any of the preceding embodiments.

The isolated cell of any of the preceding embodiments, further comprising helper plasmids for overexpressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV polymerase L or combination thereof.

The isolated cell of any of the preceding embodiments, wherein the cell comprises a plasmid for expressing a bacteriophage RNA polymerase under the control of a bacteriophage RNA polymerase promoter.

A kit comprising the nucleic acid, set of nucleic acid, the vector or set of vectors of any of the preceding embodiments.

The kit of any of the preceding embodiments, wherein the kit is for recombinant expression of vesicular stomatitis viruses pseudotyped with an Ebola virus glycoprotein and the antigen or antigens. The kit of any of the preceding embodiments, wherein the kit comprises a vial containing a vector comprising gene encoding a vesicular stomatitis virus (VSV) positive-sense genome (antigenome) including genes encoding VSV proteins, an Ebola virus glycoprotein and an antigen or antigens. The kit of any of the preceding embodiments, further comprising one or more vial comprising a vector or vectors encoding the VSV nucleocapsid, the VSV phosphoprotein, and/or the VSV polymerase L. A method of making pseudotyped vesicular stomatitis viral particles, the method comprising allowing expression in a cell of a) a VSV positive-sense genome comprising genes encoding VSV proteins, b) an Ebola glycoprotein and c) an antigen or antigens.

The method of any of the preceding embodiments, wherein the Ebola glycoprotein is expressed from the VSV positive-sense genome.

The method of any of the preceding embodiments, wherein the antigen or antigens are expressed from the VSV positive-sense genome.

The method of any of the preceding embodiments, further comprising overexpressing the VSV nucleocapsid, the VSV phosphoprotein, and/or the VSV polymerase L.

The method of any of the preceding embodiments, further comprising overexpressing the Ebola glycoprotein.

The method of any of the preceding embodiments, further comprising overexpressing the antigen or antigens.

The method of any of the preceding embodiments, further comprising the purification of viral particles. Use of Ebola glycoprotein for increasing an immune response towards an antigen or antigens.

Use of a viral particle comprising vesicular stomatitis virus (VSV) nucleocapsid, phosphoprotein, matrix protein, and polymerase L, an Ebola virus glycoprotein and an antigen or antigens for treating or preventing a disease or condition associated with an antigen or antigens.

The use of any of the preceding embodiments, wherein the disease or condition is cancer.

The use of any of the preceding embodiments, wherein the disease or condition is an infection or is related to an infection.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein is as described herein. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein is a natural Ebola virus glycoprotein, a mutated Ebola virus glycoprotein, a chimeric Ebola virus glycoprotein or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein does not contain a functional mucin-like domain.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein is at least 80% identical to the Zaire Ebola virus glycoprotein.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Zaire Ebola virus glycoprotein is from the Makona strain, the Mayinga strain or the Kikwit strain.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the mucin-like domain is deleted or mutated. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are as described herein. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from the Human Immunodeficiency Virus (HIV).

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise the HIV envelope or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise HIV-l Gag, Pol and Env antigens, a portion thereof or a mosaic thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from the Lassa virus. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise Lassa virus glycoproteins or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from the Nipah virus. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise Nipah virus glycoproteins, Nipah fusion proteins or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from the Crimean Congo Hemorrhagic Fever CCHF) virus.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise CCHF glycoprotein or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from the Middle East Respiratory coronavirus (MERS-CoV).

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprise MERS-CoV S protein or a portion thereof.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from a tick.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from Retroviridae (HIV, HTLV), Flaviviridae (e.g., Zika, Hepatitis C, West Nile, Dengue, Yellow fever, Japanese encephalitis, tick-borne encephalitis, Saint Louis encephalitis, Alkhurma hemorrhagic fever virus, Kyasanur Forest Disease virus, Omsk hemorrhagic fever virus etc.), Togaviridae (e.g., Chikungunya, Rubella virus), Picomaviridae (Hepatitis A, Polio virus, Enterovirus (EV71)), Caliciviridae (Norwalk virus, Sapporo virus), Astroviridae, Coronaviridae (e.g., Middle East Respiratory syndrome coronavirus (MERS-CoV), Severe acute Respiratory Syndrome coronavirus (SARS-CoV, etc.), Rhabdoviridae (rabies), Filoviridae (Ebola virus, Marburg virus), Paramixoviridae (Nipah virus, Hendra virus, Measles virus, Mumps virus, Respiratory syncytial virus), Orthomixoviridae (Influenza virus H1N1, H3N2, H5N1, H7N9), Bunyaviridae (Rift Valley Fever Disease virus, Crimean-Congo hemorrhagic fever, Hantaan, Dobrava, Saarema, Seoul and Puumala viruses, Hantavirus), Arenaviridae (Lassa virus, Junin virus, Guanarito virus, Lujo virus, Sbia virus, Machupo virus, Whitewater Arroyo virus, Chapare virus, Lymphocytic choriomeningitis virus), Reoviridae (rotavirus), Papovaviridae (human papilloma viruses), Adenoviridae, Parvoviridae, Herpesviridae (Herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus), Poxviridae (smallpox virus, vaccinia virus), Hepadnaviridae (Hepatitis B).

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens are from Salmonella Typhi, Salmonella Parathyphi, Yersinia pestis, Vibrio cholera, Corynebacterium diphtheria, Haemophilus influenza type B, Neisseria meningitidis, Bordetella pertussis, Streptococcus pneumoniae, Clostridium tetani, Clostridium difficile, Mycobacterium tuberculosis, Campylobacter jejuni, enterotoxigenic Escherichia coli, Streptococcus agalactiae (group B), Streptococcus pneumoniae, Streptococcus pyrogenes, Salmonella enterica, Shigella, Staphylococcus aureus.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens Plasmodium ( Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium Knowlesi), Trypanosome ( Trypanosoma cruzi), Necator americanus, Leishmania, Schistosoma haematobium, Schistosoma mansoni, H. anatolicumanatolicum, H. dromedarii, Rhipicephalus sanguineus, etc.

The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens is selected amongst 707 alanine proline-AFP (707-AP), alpha (a)-fetoprotein (AFP), adenocarcinoma antigen recognized by T cells 4 (ART-4), B antigen; b-catenin/mutated (BAGE), breakpoint cluster region-Abelson (Bcr-abl), CTF-recognized antigen on melanoma (CAMEF), carcinoembryonic antigen peptide-l (CAP-l), caspase-8 (CASP-8), cell-division-cycle 27 mutated (CDC27m), cy cline -dependent kinase 4 mutated (CDK4/m), carcino-embryonic antigen (CEA), cancer testis antigen (CT), cyclophilin B (Cyp-B), differentiation antigen melanoma (DAM), elongation factor 2 mutated (ELF2M), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETV6-AML1), glycoprotein 250 (G250), G antigen (GAGE), N-acetylglucosaminyltransferase V (GnT-V), glycoprotein 100 kDa (GplOO), helicose antigen (HAGE), human epidermal receptor-2/ neurological (HER-2/neu), arginine (R) to isoleucine (I) exchange at residue 170 of the a-helix of the a2-domain in the HLA-A2 gene (HLA-A * 0201-R1701), human papilloma virus E7 (HPV-E7), heat shock protein 702 mutated (HSP70-2M), human signet ring tumor-2 (HST-2), human telomerase reverse transcriptase (hTERT or hTRT), intestinal carboxyl esterase (iCE), KIAA0205, L antigen (LAGE), low-density lipid receptor/GDP-L-fucose: b-D- galactosidase 2-a-L-fucosyltransferase (LDLR/FUT), melanoma antigen (MAGE), melanoma antigen recognized by T cells- l/melanoma antigen A (MART-l/Melan-A), melanocortin 1 receptor (MC1R), myosin mutated (Myosin/m), mucin 1 (MUC1), melanoma ubiquitous mutated 1, 2, 3 (MUM-l, -2, - 3), NA cDNA clone of patient M88 (NA88-A), New York-esophagus 1 (NY -ESO-l), protein 15 (P15), protein of 190 kDa ber-abl (pl90 minor bcr-abl), promyelocytic leukaemia/retinoic acid receptor a (Pml/RARa), preferentially expressed antigen of melanoma (PRAME), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), renal antigen (RAGE), renal ubiquitous 1 or 2 (RU1 or RU2), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1 or 3 (SART-l or SART- 3), translocation Ets-family leukemia/acute myeloid leukemia 1 (TEL/ AML1), triosephosphate isomerase mutated (TPI/m), tyrosinase related protein 1 or gp75 (TRP-l), tyrosinase related protein 2 (TRP-2), TRP-2/intron 2 (TRP-2/ INT2), Wilms' tumor gene (WT1).

95. The particles, viral particles, nucleic acids, vectors, cells, pharmaceutical compositions, kits, uses or methods of any of the preceding embodiments, wherein the antigen or antigens comprises an anchoring domain from Ebola virus glycoprotein.

Other aspects and embodiments of the present disclosure will become apparent to those skilled in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating exemplary or preferred embodiments of the disclosure, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed disclosure will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

All patents, patent applications, and publications referred to herein are incorporated by reference in their entirety.

It is to be understood herein that due to the degeneracy of the genetic code, the nucleic acid used in the present experiments may be different than those used. For example, any nucleic acid sequences encoding an Ebola glycoprotein may be used including for example, codon-optimized sequences.

EXAMPLE 1

V SV-based vectors expressing Ebo GP and an HIV antisen

The genomic plasmid pATX, encoding the VSV genome of serotype Indiana, excluding VSVG, was synthesized by Genscript. The Ebola glycoprotein gene (Ebo GP) was amplified from a plasmid containing Ebola Makona, variant B6 (SEQ ID NO: 1), using primers containing 15 base pair (bp) homology arms complementary to the pATX plasmid after it was linearized by Xhol-Nhel sites. The amplified gene was then ligated to the linear plasmid using In-Fusion™ cloning kit from Takara to form the plasmid pATX- VSV-Ebo GP (Figure 1A). To generate the pATX-VSV-Ebo-HIV/T7l3 plasmid (Figure IB), HIV envelope (from the NL4.3 isolate) was amplified from a plasmid using a primer pair with 15 bp homology to the pATX-Ebo GP plasmid, linearized by MluI-AvrII. The resulting amplicon, which contains the HIV transmembrane domain and short cytoplasmic tail, also called truncation 713 (SEQ ID NO: 14), was ligated into the pATX-VSV- Ebo GP vector using In-Fusion™ cloning kit. To construct the pATX-VSVG-HIV/T7l3 (Figure 1C), the VSVG gene was amplified from pMD2.G plasmid and cloned in the pATX plasmid. Ectodomain of HIV gene was then cloned via MluI-AvrII sites as described above.

Cloning of pATX-VSVG-HIV/SIVtm (Figure ID) was accomplished by inserting HIV/SIVtm (SEQ ID NO: 12) gene into the pATX-VSVG plasmid linearized with MluI-AvrII.

Construction of the pATX-VSV-Ebo-HIV/SIVtm plasmid (Figure IE) is similar to pATX-VSV- Ebo-HIV/T7l3, except that a distinct reverse primer containing SIV transmembrane and short cytoplasmic tail sequence was used to amplify the chimeric gene.

Construction of pATX-EBO-HIV/EboTM and pATX-VSVG-HIV/EboTM is similar to pATX- EBO-HIV/SIVtm and pATX-VSVG-HIV/SIVtm, except that a specific primer harbouring EboTM+CT was used to amplify the gene (Figure 12A and Figure 12B).

The pATX-VSV-Ebo/V3 -HIV/SIVtm (Figure IF) was constructed in a similar fashion.

To generate helper plasmids, pBS-N, pBS-P and pBS-L (Figures 2A-C), the VSV N, VSV P and VSV L genes were all amplified from the pATX plasmid and cloned into plasmid pBS via Ecorl-Hindlll sites. Finally, pCAGGs-T7 (Figure 2D) was constructed by cloning T7 polymerase gene in the pCAGGs plasmid via BglII-EcoRI sites.

Similar plasmids containing the Ebo GP and the HIV envelope from the BG505 or A74 isolates of HIV have been generated.

VSV-based viral particles

The recombinant VSV vaccine platform is based on the reverse genetic system used for an attenuated strain of VSV serotype Indiana (Lawson et al., PNAS, USA, Vol. 92, p. 4477-4481, 1995). Marzi, A et al., indicates that the entire VSV genome was cloned into a plasmid under the control of the bacteriophage T7 polymerase promoter. Subsequently, the open reading frame for the VSV glycoprotein (G) gene was excised and unique restriction enzyme sites for cloning were introduced. The resulting vector can be used to insert foreign virus glycoproteins in place of VSV-G. Transfection of the VSV genomic plasmid together with expression plasmids for the viral replication complex (VSV nucleocapsid, phosphoprotein and polymerase L) and the T7 polymerase in a co-culture of Vero and 293T cells results in viral transcription, protein expression, genome replication and production of recombinant VSV particles that bear the foreign glycoprotein on their surface (Marzi et al., J. Bioterr Biodef 2011, Sep 25; Suppl 1(4), 2157-2526-S1-004).

More particularly, viral particles of the present disclosure were generated as follow. Genomic plasmid corresponding to each vector along with the helper plasmids pCAGGs-T7, pBS-L, pBS-N and pBS-P were transfected into a mixture of HEK293T/VERO-E6 cells in a 6-well plate format, using lipofectamine 3000 (Thermo Scientific) as per manufacturer’s instructions. Blind passages were carried out at 72 and 96 hours post-transfection on VERO-E6 cells. The presence of virus was confirmed when cytopathic effect (CPE) was observed in VERO-E6 cells. The cell supernatant was collected and viral particles were then titrated using tissue culture infectious dose (TCID50) method on VERO-E6 cells. Briefly, serial dilution of each virus was prepared in DMEM medium supplemented with 2% FBS and incubated with VERO-E6 cells for 1 hour in an incubator at 37°C, 5% CO2. Cell culture medium containing unattached virus particles was removed from the wells and fresh DMEM with 2% FBS was added back to the wells. Cultures were monitored for up to 5 days following initial infection, until CPE was observed. Then the TCID50 dose was calculated.

In order to generate virus stocks for mouse studies, VERO-E6 cells that were cultured in four 175 cm² flasks were infected at a multiplicity of infection (MOI) of 0.1 for 1 hour. Cell culture medium containing unattached viruses was removed and fresh DMEM medium containing 10% FBS was added to each well. Supernatant from the cultures was harvested when CPE was observed in approximately 80% of the cells. Viruses were then purified by ultracentrifugation at 28000 g on a 20% sucrose cushion. Pellets were resuspended in phosphate saline buffer (PBS). Viruses were then directly titrated or stored at -80°C.

The different viral preparations were analyzed by Western blot to confirm expression of HIV gpl40, Ebola glycoprotein and of VSV’s structural proteins. Briefly, VSV viruses carrying VSV glycoprotein, Ebola glycoprotein either alone or in combination with HIV Env were purified using sucrose cushion, then lysed in laemmli buffer and subjected to Western blotting using anti-HIV gpl20 monoclonal antibody (ID6) or anti -VSV glycoprotein antibody or anti-VSV nucleocapsid antibody. The signal obtained for the nucleocapsid serves as a loading control of virus input. Recombinant VSV (control) and VSV- Ebo/T7l3 viruses were similarly purified using sucrose cushion.

In vivo studies 1 x l0⁶TCID50 dose of each purified virus was injected intramuscularly in three groups of BALB/c mice, each comprising 7 animals. Blood samples were collected by saphenous bleed on days -1, 7, 14, 21 and 28 post-immunization and sera was then isolated and tested for the presence of anti-HIV env antibodies by ELISA assays.

An ELISA assay was performed on the sera of mice vaccinated with the VSV-Ebo vaccine (Mock), the VSV-Ebo-HIV/T7l3 vaccine, the VSVG-HIV/T713 vaccine, the VSV-Ebo-HIV/SIVtm vaccine or the VSVG-HIV/SIVtm vaccine and collected four weeks after immunization.

A recombinant HIV-l gpl20 LAV protein (GenBank accession number Ml 9921) was used to coat the ELISA plates at a final concentration of 1 pg/ml. ELISA plates were then blocked with blocking reagent for an hour at 37°C and 1/250 diluted sera was subsequently incubated with antigen for two hours at room temperature. Plates were then washed 3 times with PBS-Tween, 0.05 % and incubated for an hour with a secondary, HRP -conjugated anti-mouse antibody. Linally, after three washing steps, substrate was added and ELISA plates were incubated for 30 minutes at 37°C, then absorbance was measured at 405 nm.

In this study we showed that co-expression of Ebola glycoprotein with HIV envelope on the surface of VSV significantly increases the antibody titers against HIV envelope (Figures 4 and 6). As a control we included a VSV vector containing its native glycoprotein (VSV G) instead of Ebo GP. We show that the antibody titer against HIV envelop in this group is almost comparable to mock group (see Figures 4 and 6), indicating that Ebola GP enables this strong humoral immune response against HIV. In addition, by replacing transmembrane domain and short cytoplasmic tail of native HIV with that of SIVmac239, we generated chimeric HIV envelope (HIV/SIVtm) capable of high expression levels. Analysis of HIV envelope protein expression of these particles demonstrate that high levels of gpl20 is incorporated on the surface of these VSV particles, compared to the wild type HIV (Figures 5A and 5B) and this is independent of Ebo GP or VSVG. Our ELISA results show that presence of SIVtm further increases antibody titers directed against HIV envelope when co-expressed with Ebo GP (Figure 6). We hypothesize that Ebo GP confers this ability to the VSV-Ebo/T7l3 vector through widening cell tropism in the host, possibly outside T-cells, which results in a stronger humoral immune response. Our expression analysis also indicates that both VSV-Ebo/T7l3 and VSV-G/T713 vectors express gpl40 and the processed form, gpl20 on the surface of purified viruses (Figures 3A, 3B, 5A and 5B).

We generated a construct in which the mucin-like domain of Ebola GP was replaced with the V3 domain of HIV envelope (EboGPAM/V3 or EboAM/V3: SEQ ID NO: 8). We then tested whether the co expression of the EboAM/V3 protein (SEQ ID NO:9) and HIV Env glycoprotein in recombinant vesicular stomatitis virus (rVSV) vector can also induce robust host immune responses in vivo. Different VSV vectors, including rVSV co-expressing HIV envelop glycoprotein and VSV glycoprotein (rVSVG- HlV/SIVtm), rVSV co-expressing HIV envelop glycoprotein and the wild type Ebola glycoprotein (rVSV- EBO-HIV/SIVtm), or rVSV co-expressing HIV envelop glycoprotein and EboGPAM/V3 (rVSV- EboAM/V3-HIV/SIVtm) (Figure 7A) were used to immunize the BALB/c mice. Briefly, each rVSV stock was produced in VeroE6 cells and virus titers were titrated in Vero E6 cells, and expression of various proteins were detected by anti-EboGP antibodies or and anti-HIV-GP antibodies (Figure 7B). Then, the BALB/c mice were injected subcutaneously with lxlO⁶ TCID50 of each of VSVG/HIV/SIVtm, VSV-EBO- HlV/SIVtm and VSV-EBOAM/V3-HIV/SIVtm, as indicated. At 35 days post-immunization, the sera from immunized mice were collected and measured for the anti-HIVgpl20 antibody levels with anti-HIVgpl20 ELIS As.

Results have shown that the inoculation of rV SV-EboAM/V3-HIV/SIVtm elicited more robust anti- HIV antibody responses than other rVSV vectors co-expressing either HIV envelop glycoprotein and the wild type Ebola glycoprotein (rVSV-EBO-HIV/SIVtm) or HIV envelop glycoprotein and VSV glycoprotein (rVSVG-HIV/SIVtm) (Figure 8).

We further engineered the HIV antigen by fusing its ectodomain with transmembrane domain and a short cytoplasmic tail of Ebola glycoprotein (EBOGP). Given the high surface expression of EBOGP on Ebola virus, we anticipated that by replacing the native transmembrane domain of HIV env with EBOGP, we might increase surface expression of HIV envelope on the VSV particles. Our results show that the transmembrane domain of EBOGP increases the expression level of HIV env, regardless of co-expressing antigen (Figure 11A).

To test the in vivo efficacy of these new vaccines in animal model, we immunized groups of ten mice with either lxE06 TCID50 dose of VSV-EBO-HIV/EboTM or VSVG-HIV/EboTM. ELISA results from sera of these mice show that, A) EboTM greatly increases overall antibody response against HIV env, B) EBOGP induces significantly higher HIV antibody titers (Figure 11B). Since expression level of HIV/EboTM is quite comparable in both vaccines, high antibody titers could be attributed to EBOGP and its role in stimulating the immune response in a way that results in greater humoral immune response.

In order to perform challenge studies, non-human primates are bleed, after an acclimatization period, on Day -45 and -15 (in relation to vaccination) to establish baseline immunity levels. On Day 0, the animals are vaccinated via intramuscular (IM) injection, in the deltoid muscle, with 2xl0⁸ PFU in 0.5 mL. Control animals are vaccinated with 0.5 mL of saline via IM injection. Blood samples are collected on days 14, 28, 60 and 90 post-vaccination to follow the immune response. On day 90, animals are challenged intrarectally with lxlO⁴ TCID50 SHIV. Animals are subsequently challenged once every week for a total of 10 weeks. Blood samples are collected prior to each infection in order to evaluate the T and B cell response, as well as to determine the animals viral load. The animals are followed for an additional two months with blood samples taken each week for immune analysis and viral load determination.

EXAMPLE 2

VSV-based vectors expressing Ebo GP and CCHF glycoprotein

To generate the pATX-VSV-Ebo GP-CCHF GP plasmids, the gene encoding the CCHF glycoprotein from the“Turkey kelkit 06” strain is amplified using a forward primer with 15 bp homology to the pATX-Ebo GP plasmid, linearized by MluI-AvrII and a reverse primer containing a 15 bp homology with the 3’-end of the CCHF glycoprotein. The resulting amplicon is ligated into the pATX-VSV-Ebo-GP vector using In-Fusion™ cloning kit.

Viral particles expressing the Ebola glycoprotein and the CCFH glycoprotein are obtained and tested using methods described in Example 1.

SEQUENCE TABLES

SEQ ID NO: 1 Ebola Glycoprotein Gene Sequence of Makona B6 variant

ATGGGTGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACATCATTCTTTCTTTGGGTAA

TTATCCTTTTCCAAAGAACATTTTCCATCCCGCTTGGAGTTATCCACAATAGTACATTACAGGTTAGTGA

TGTCGACAAACTAGTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTGAATCTC

GAGGGGAATGGAGTGGCAACTGACGTGCCATCTGTGACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCAC

CAAAGGTGGTCAATTATGAAGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGA

CGGGAGTGAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAA

GTATCAGGAACGGGACCATGTGCCGGAGACTTTGCCTTCCACAAAGAGGGTGCTTTCTTCCTGTATGATC

GACTTGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCC

CCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCTGGTCAATGCAACGGAGGACCCGTCG

AGTGGCTATTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACTAATGAGACAGAGTACTTGT

TCGAGGTTGACAATTCGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAGCTGAA

TGAGACAATATATGCAAGTGGGAAGAGGAGCAACACCACGGGAAAACTAATTTGGAAGGTCAACCCCGAA

ATTGATACAACAATCGGGGAGTGGGCCTTCAGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTG

AAGAGTTGTCTTTCACAGCTGTATCAAACGGACCCAAAAACATCAGTGGTCAGAGTCCGGCGCGAACTTC

TTCCGACCCAGAGACCAACACAACAAATGAAGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATG

GTTCAAGTGCACAGTCAAGGAAGGAAAGCTGCAGTGTCGCATCTGACAACCCTTGCCACAATCTCCACGA

GTCCTCAACCTCCCACAACCAAAACAGGTCCGGACAACAGCACCCATAATACACCCGTGTATAAACTTGA

CATCTCTGAGGCAACTCAAGTTGGACAACATCACCGTAGAGCAGACAACGACAGCACAGCCTCCGACACT

CCCCCCGCCACGACCGCAGCCGGACCCTTAAAAGCAGAGAACACCAACACGAGTAAGAGCGCTGACTCCC

TGGACCTCGCCACCACGACAAGCCCCCAAAACTACAGCGAGACTGCTGGCAACAACAACACTCATCACCA

AGATACCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTAGCA

GGACTGATCACAGGCGGGAGAAGGACTCGAAGAGAAGTAATTGTCAATGCTCAACCCAAATGCAACCCCA

ATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGATTGGCCTGGATACCATATTTCGGGCC

AGCAGCCGAAGGAATTTACATAGAGGGGCTAATGCACAACCAAGATGGTTTAATCTGTGGGTTGAGGCAG

CTGGCCAACGAAACGACTCAAGCTCTCCAACTGTTCCTGAGAGCCACAACTGAGCTGCGAACCTTTTCAA

TCCTCAACCGTAAGGCAATTGACTTCCTGCTGCAGCGATGGGGTGGCACATGCCACATTTTGGGACCGGA

CTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGATTTT

GTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGG

CAGGTATTGGAGTTACAGGTGTTATAATTGCAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTTA

G

SEQ ID NO:2 Ebola Glycoprotein protein sequence of Makona B6 variant MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNL

EGNGVATDVPSVTKRWGFRSGVPPKWNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHK

VSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGWAFLILPQAKKDFFSSHPLRELVNATEDPS

SGYYSTTIRYQATGFGTNETEYLFEVDNSTYVQLESRFTPQFLLQLNETIYASGKRSNTTGKLIWKVNPE

IDTTIGEWAFRETKKNLTRKIRSEELSFTAVSNGPKNISGQSPARTSSDPETNTTNEDHKIMASENSSAM

VQVHSQGRKAAVSHLTTLATISTSPQPPTTKTGPDNSTHNTPVYKLDISEATQVGQHHRRADNDSTASDT

PPATTAAGPLKAENTNTSKSADSLDLATTTSPQNYSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVA

GLITGGRRTRREVIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQ

LANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDF

VDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF

SEQ ID NO:3 >NP_066246.1 spike glycoprotein [Zaire ebolavirus ]

MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNL

EGNGVATDVPSATKRWGFRSGVPPKWNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHK

VSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGWAFLILPQAKKDFFSSHPLREPVNATEDPS

SGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEELSFTWSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAM

VQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDT

PSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVA

GLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQ

LANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDF

VDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF

SEQ ID NO: 4 >YP_138523.1 spike glycoprotein [Sudan ebolavirus]

MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQLVCKDHLASTDQLKSVGLNL

EGSGVSTDIPSATKRWGFRSGVPPKWSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHK

AQGTGPCPGDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIREAVNYTENTS

SYYATSYLEYEIENFGAQHSTTLFKIDNNTFVRLDRPHTPQFLFQLNDTIHLHQQLSNTTGRLIWTLDAN

INADIGEWAFWENKKNLSEQLRGEELSFEALSLNETEDDDAASSRITKGRISDRATRKYSDLVPKNSPGM

VPLHIPEGETTLPSQNSTEGRRVGVNTQETITETAATIIGTNGNHMQISTIGIRPSSSQIPSSSPTTAPS

PEAQTPTTHTSGPSVMATEEPTTPPGSSPGPTTEAPTLTTPENITTAVKTVLPQESTSNGLITSTVTGIL

GSLGLRKRSRRQTNTKATGKCNPNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQ

LANETTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTCRILGPDCCIEPHDWTKNITDKINQIIHDF

IDNPLPNQDNDDNWWTGWRQWIPAGIGITGIIIAIIALLCVCKLLC SEQ ID NO :5 >YP_003815426.1 spike glycoprotein [Tai Forest ebolavirus ]

MGASGILQLPRERFRKTSFFVWVIILFHKVFSIPLGVVHNNTLQVSDIDKFVCRDKLSSTSQLKSVGLNL

EGNGVATDVPTATKRWGFRAGVPPKWNCEAGEWAENCYNLAIKKVDGSECLPEAPEGVRDFPRCRYVHK

VSGTGPCPGGLAFHKEGAFFLYDRLASTIIYRGTTFAEGVIAFLILPKARKDFFQSPPLHEPANMTTDPS

SYYHTTTINYVVDNFGTNTTEFLFQVDHLTYVQLEARFTPQFLVLLNETIYSDNRRSNTTGKLIWKINPT

VDTSMGEWAFWENKKNFTKTLSSEELSFVPVPETQNQVLDTTATVSPPISAHNHAAEDHKELVSEDSTPV

VQMQNIKGKDTMPTTVTGVPTTTPSPFPINARNTDHTKSFIGLEGPQEDHSTTQPAKTTSQPTNSTESTT

LNPTSEPSSRGTGPSSPTVPNTTESHAELGKTTPTTLPEQHTAASAIPRAVHPDELSGPGFLTNTIRGVT

NLLTGSRRKRRDVTPNTQPKCNPNLHYWTALDEGAAIGLAWIPYFGPAAEGIYTEGIMENQNGLICGLRQ

LANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPQDWTKNITDKIDQIIHDF

VDNNLPNQNDGSNWWTGWKQWVPAGIGITGVIIAIIALLCICKFML

SEQ ID NO :6 >YP_003815435.1 spike glycoprotein [Bundibugyo

ebolavirus ]

MVTSGILQLPRERFRKTSFFVWVIILFHKVFPIPLGVVHNNTLQVSDIDKLVCRDKLSSTSQLKSVGLNL

EGNGVATDVPTATKRWGFRAGVPPKWNYEAGEWAENCYNLDIKKADGSECLPEAPEGVRGFPRCRYVHK

VSGTGPCPEGYAFHKEGAFFLYDRLASTIIYRSTTFSEGWAFLILPETKKDFFQSPPLHEPANMTTDPS

SYYHTVTLNYVADNFGTNMTNFLFQVDHLTYVQLEPRFTPQFLVQLNETIYTNGRRSNTTGTLIWKVNPT

VDTGVGEWAFWENKKNFTKTLSSEELSVIFVPRAQDPGSNQKTKVTPTSFANNQTSKNHEDLVPEDPASV

VQVRDLQRENTVPTPPPDTVPTTLIPDTMEEQTTSHYEPPNISRNHQERNNTAHPETLANNPPDNTTPST

PPQDGERTSSHTTPSPRPVPTSTIHPTTRETHIPTTMTTSHDTDSNRPNPIDISESTEPGPLTNTTRGAA

NLLTGSRRTRREITLRTQAKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGIMHNQNGLICGLRQ

LANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDF

IDKPLPDQTDNDNWWTGWRQWVPAGIGITGVIIAVIALLCICKFLL

SEQ ID NO :7 >NP_690583.1 spike glycoprotein [Reston ebolavirus]

MGSGYQLLQLPRERFRKTSFLVWVIILFQRAISMPLGIVTNSTLKATEIDQLVCRDKLSSTSQLKSVGLN

LEGNGIATDVPSATKRWGFRSGVPPKVVSYEAGEWAENCYNLEIKKSDGSECLPLPPDGVRGFPRCRYVH

KVQGTGPCPGDLAFHKNGAFFLYDRLASTVIYRGTTFAEGVVAFLILSEPKKHFWKATPAHEPVNTTDDS

TSYYMTLTLSYEMSNFGGNESNTLFKVDNHTYVQLDRPHTPQFLVQLNETLRRNNRLSNSTGRLTWTLDP

KIEPDVGEWAFWETKKNFSQQLHGENLHFQIPSTHTNNSSDQSPAGTVQGKISYHPPANNSELVPTDSPP

VVSVLTAGRTEEMSTQGLTNGETITGFTANPMTTTIAPSPTMTSEVDNNVPSEQPNNTASIEDSPPSASN

ETIYHSEMDPIQGSNNSAQSPQTKTTPAPTTSPMTQDPQETANSSKPGTSPGSAAGPSQPGLTINTVSKV

ADSLSPTRKQKRSVRQNTANKCNPDLYYWTAVDEGAAVGLAWIPYFGPAAEGIYIEGVMHNQNGLICGLR QLANETTQALQLFLRATTELRTYSLLNRKAIDFLLQRWGGTCRILGPSCCIEPHDWTKNITDEINQIKHD

FIDNPLPDHGDDLNLWTGWRQWIPAGIGIIGVIIAIIALLCICKILC

SEQ ID NO:8 Nucleic acid sequence of EboGPAM/V3 (a.k.a., EboAM/V3 or EboAM-V3 )

ATGGGTGTGACCGGTATCCTGCAGCTGCCGCGTGATCGCTTCAAACGTACCTCTTTCTTTCTGTGGGTTA

TCATCCTGTTCCAGCGTACCTTTTCTATCCCGCTGGGTGTTATTCATAACTCCACCCTGCAGGTGAGCGA

CGTTGATAAACTGGTTTGCCGTGACAAACTGTCTTCTACCAACCAGCTGCGCTCCGTGGGCCTGAACCTG

GAAGGTAACGGTGTTGCAACCGACGTGCCGTCTGCGACCAAACGCTGGGGTTTCCGCTCCGGTGTTCCGC

CGAAAGTTGTTAACTACGAAGCGGGCGAATGGGCTGAAAACTGTTATAACCTGGAAATCAAGAAACCGGA

CGGCTCCGAGTGCCTGCCGGCAGCTCCGGACGGTATTCGCGGCTTTCCGCGCTGTCGTTACGTTCATAAA

GTTAGCGGTACTGGTCCGTGCGCAGGTGACTTTGCTTTCCACAAAGAGGGCGCGTTTTTCCTGTATGACC

GCCTGGCATCCACCGTTATTTACCGTGGCACCACCTTCGCGGAAGGCGTTGTGGCGTTCCTGATCCTGCC

GCAGGCTAAGAAAGATTTCTTTAGCAGCCACCCGCTGCGCGAGCCGGTTAACGCGACTGAGGATCCGTCT

TCTGGTTATTACTCCACCACTATCCGTTACCAGGCAACTGGTTTCGGTACCAACGAAACTGAATACCTGT

TCGAAGTTGATAACCTGACCTACGTTCAGCTGGAAAGCCGCTTCACTCCGCAGTTCCTGCTGCAGCTGAA

CGAAACCATCTACACCAGCGGTAAACGTTCCAACACCACCGGCAAACTGATCTGGAAAGTTAACCCGGAG

ATCGATACCACTATTGGTGAGTGGGCGTTTTGGGAAACCAAGAAAAACCTGACCCGCAAAATCCGTTCCG

AGGAAGGATTCAATGGAACAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATCAGGCC

AGTAGTATCAACTCAACTGCTGTTAAATGGCAGTCTAGCAGAAGAAGATGTAGTAATTAGATCTGCCAAT

TTCACAGACAATGCTAAAACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCA

ACAACAATACAAGAAAAAGTATCCGTATCCAGAGGGGACCAGGGAGAGCATTTGTTACAATAGGAAAAAT

AGGAAATATGAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAGATAGCT

AGCAAATTAAGAGAACAATTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAAACACCATCG

CAGGTGTTGCTGGTCTGATCACCGGCGGTCGTCGTACCCGCCGTGAAGCTATTGTTAACGCACAGCCGAA

ATGTAACCCGAACCTGCACTACTGGACCACTCAGGATGAAGGCGCTGCTATCGGCCTGGCATGGATCCCG

TACTTCGGTCCGGCGGCTGAAGGTATCTATATCGAAGGTCTGATGCACAACCAGGATGGTCTGATTTGCG

GTCTGCGTCAGCTGGCGAACGAAACCACTCAGGCGCTGCAGCTGTTCCTGCGCGCAACCACCGAGCTGCG

TACCTTCTCTATCCTGAACCGTAAGGCGATCGACTTTCTGCTGCAGCGTTGGGGTGGTACCTGCCATATC

CTGGGTCCGGACTGCTGTATCGAGCCGCATGATTGGACTAAAAACATCACTGACAAAATCGACCAGATCA

TTCACGACTTCGTTGACAAAACCCTGCCGGACCAGGGCGATAACGACAACTGGTGGACCGGCTGGCGTCA

GTGGATTCCGGCAGGCATCGGCGTTACCGGTGTTATTATTGCTGTGATTGCACTGTTTTGCATTTGCAAG

TTCGTTTTCTGA

SEQ ID NO: 9 Amino acid sequence of EboGPAM/V3 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNL

EGNGVATDVPSATKRWGFRSGVPPKWNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHK

VSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGWAFLILPQAKKDFFSSHPLREPVNATEDPS

SGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPE

IDTTIGEWAFWETKKNLTRKIRSEEGFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEDWIRSAN

FTDNAKTIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIA

SKLREQFGNNKTIIFKQSSGNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIP

YFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHI

LGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICK

FVF

SEQ ID NO: 10 Nucleotide sequence HIVenv of pNL4.3

ATGAGAGTGAAGGAGAAGTATCAGCACTTGTGGAGATGGGGGTGGAAATGGGGCACCATGCTCCTTGGGA

TATTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGA

AGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCC

ACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTA

ACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAA

GCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACC

AATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAA

GCATAAGAGATAAGGTGCAGAAAGAATATGCATTCTTTTATAAACTTGATATAGTACCAATAGATAATAC

CAGCTATAGGTTGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCA

ATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAA

CAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATCAGGCCAGTAGTATCAACTCAACT

GCTGTTAAATGGCAGTCTAGCAGAAGAAGATGTAGTAATTAGATCTGCCAATTTCACAGACAATGCTAAA

ACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAA

GTATCCGTATCCAGAGGGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGC

ACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAA

TTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTT

TTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTAC

TTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAA

TTTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTT

CATCAAATATTACTGGGCTGCTATTAACAAGAGATGGTGGTAATAACAACAATGGGTCCGAGATCTTCAG

ACCTGGAGGAGGCGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAA

CCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAG

GAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGT

ACAGGCCAGACAATTATTGTCTGATATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAA CAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT

ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCC

TTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATAACATGACCTGGATGGAGTGGGAC

AGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGA

ATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCT

GTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT

TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAATCCCGAGGG

GACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGT

GAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGA

GACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATT

GGTGGAATCTCCTACAGTATTGGAGTCAGGAACTAAAGAATAGTGCTGTTAACTTGCTCAATGCCACAGC

CATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTATTACAAGCAGCTTATAGAGCTATTCGCCAC

ATACCTAGAAGAATAAGACAGGGCTTGGAAAGGATTTTGCTATAA

SEQ ID NO: 11 amino acid sequence of HIVenv of pNL4.3

MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWA

THACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNT

NSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEP

IPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEDWIRSANFTDNAK

TIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQ

FGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQ

FINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKWKIE

PLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAIEAQ

QHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWD

REINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVL

SIVNRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLR

DLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVNLLNATAIAVAEGTDRVIEVLQAAYRAIRH

IPRRIRQGLERILL

SEQ ID NO: 12 HIVenv/SIVtm nucleic acid sequence based on pNL4.3 (underlined sequence represents the SIV transmembrane (nucleotides 2044- 2121) and twice underlined sequence (nucleotides 2122-2130) represents the short SIV cytoplasmic tail of the construct with stop codon (2131- 2133) ) ATGAGAGTGAAGGAGAAGTATCAGCACTTGTGGAGATGGGGGTGGAAATGGGGCACCATGCTCCTTGGGA

TATTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGA

AGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCC

ACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTA

ACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAA

GCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACC

AATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAA

GCATAAGAGATAAGGTGCAGAAAGAATATGCATTCTTTTATAAACTTGATATAGTACCAATAGATAATAC

CAGCTATAGGTTGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCA

ATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAA

CAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATCAGGCCAGTAGTATCAACTCAACT

GCTGTTAAATGGCAGTCTAGCAGAAGAAGATGTAGTAATTAGATCTGCCAATTTCACAGACAATGCTAAA

ACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAA

GTATCCGTATCCAGAGGGGACCCGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGC

ACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAA

TTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTT

TTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTAC

TTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAA

TTTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTT

CATCAAATATTACTGGGCTGCTATTAACAAGAGATGGTGGTAATAACAACAATGAGTCCGAGATCTTCAG

ACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAA

CCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAG

GAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGT

ACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAA

CAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT

ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCC

TTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGAC

AGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGA

ATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCT

GTGGTATATAAAGTACATCCAGTACGGAGTCTACATCGTCGTAGGAGTCATCCTGTTGAGGATCGTGATC

TACATCGTCCAGATGCTGGCTAAGTTGCTCTAG

SEQ ID NO: 13 HIVenv/SIVtm amino acid sequence

MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWA

THACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNT

NSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEP IPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEDWIRSANFTDNAK

TIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQ

FGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQ

FINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNESEIFRPGGGDMRDNWRSELYKYKWKIE

PLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQ

QHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWD

REINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKYIQYGVYIWGVILLRIVI

YIVQMLAKLL

SEQ ID NO: 14 HIVenv/truncation713 nucleic acid sequence based on pNL4.3 (underlined sequence represents the transmembrane (nucleotides 2053- 2133) and twice underlined sequence (nucleotides 2134-2148) represents the short cytoplasmic tail of the construct with stop codon (nucleotides 2149-2151)

ATGAGAGTGAAGGAGAAGTATCAGCACTTGTGGAGATGGGGGTGGAAATGGGGCACCATGCTCCTTGGGA

TATTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGA

AGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCC

ACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTA

ACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAA

GCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACC

AATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAA

GCATAAGAGATAAGGTGCAGAAAGAATATGCATTCTTTTATAAACTTGATATAGTACCAATAGATAATAC

CAGCTATAGGTTGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCA

ATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAA

CAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATCAGGCCAGTAGTATCAACTCAACT

GCTGTTAAATGGCAGTCTAGCAGAAGAAGATGTAGTAATTAGATCTGCCAATTTCACAGACAATGCTAAA

ACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAA

GTATCCGTATCCAGAGGGGACCCGGGAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACA

AGCACATTGCAACATTAGTAGAACAAAATGGAATAACACTTTAAATCAAATAGCTACAAAATTAAAAGAA

CAATTTGGGAATAATAAAACAATAGTCTTTAATCAATCCTCAGGAGGGGACCCAGAAATTGTAATGCACA

GTTTTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATTTTAA

TGGTACTTGGAATTTAACACAATCGAATGGTACTGAAGGAAATGACACTATCACACTCCCATGTAGAATA

AAACAAATTATAAATATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGAGGACAAATTA

GATGCTCATCAAATATTACAGGGCTAATATTAACAAGAGATGGTGGAACTAACAGTAGTGGGTCCGAGAT

CTTCAGACCTGGGGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAA ATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAAAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGG

GAACGATAGGAGCTATGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATAAC

GCTGACGGTACAGGCCAGACTATTATTGTCTGGTATAGTGCAACAGCAGAACAATTTGCTGAGGGCTATT

GAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAGTCCTGGCTG

TGGAAAGATACCTAAGGGATCAACAGCTCCTAGGGATTTGGGGTTGCTCTGGAAAACTCATCTGCACCAC

TGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATG

GAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGC

AAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAAC

AAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTT

GCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTCTAG

SEQ ID NO: 15 HIVenv/truncation713 amino acid sequence based on pNL4.3

MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWA

THACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNT

NSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEP

IPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEDWIRSANFTDNAK

TIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFYTTGEIIGDIRQAHCNISRTKWNNTLNQIATKLKE

QFGNNKTIVFNQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNFNGTWNLTQSNGTEGNDTITLPCRI

KQIINMWQEVGKAMYAPPIRGQIRCSSNITGLILTRDGGTNSSGSEIFRPGGGDMRDNWRSELYKYKWK

IEPLGVAPTKAKRRVVQREKRAVGTIGAMFLGFLGAAGSTMGAASITLTVQARLLLSGIVQQQNNLLRAI

EAQQHLLQLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWM

EWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVF

AVLSIVNRVRQGYSPF

SEQ ID NO : 16 DNA sequence upstream ofHIV envelop gene (between VSVM and HIVenv; Mlul cut site underlined)

GCTAGTCTAACTTCTAGCTTCTGAACAATCCCCGGTTTACTCAGTCTCCCCTAATTCCAGCCTCTCGAAC

AACTAATATCCTGTCTTTTCTATCCCTATGAAAAAAACTAACAGAGATCGATCTGTTTACGCGT

SEQ ID NO : 17 DNA sequence upstream of Ebola GP (between HIVenv and Ebo GP; AvrII and Xhol cut site underlined)

CCTAGGAAAGTAACTCAAATCCTGCTAGGTATGAAAAAAACTAACAGATATCACGCTCGAG

SEQ ID NO: 18 DNA sequence upstream of VSV-L (between Ebo GP and VSVL: Nhel cute site underlined)

GCTAGCCAGATTCTTCATGTTTGGACCAAATCAACTTGTGATACCATGCTCAAAGAGGCCTCAATTATAT

TTGAGTTTTTAATTTTTATGAAAAAAACTAACAGCAATC SEQ ID NO: 19 pATX-VSV genomic plasmid (VSVN; nucleotide position: 261- 1529, VSVP : 1593-2390, VSVM: 2447-3136, VSVL: 3558-9887)

ACGCGACTAGTAATACGACTCACTATAGGACGAAGACAAACAAACCATTATTATCATTAAAAGGCTCAGG

AGAAACTTTAACAGTAATCAAAGGTACCAATTAATTCCCGGGCGCATAATATCAGAACGAAGCAAATATT

TATTAGCTAGGTATGAAAAAAACTAACAGATATCAGCGTACGTAATTATAGGTTACCACGAAGACAAACA

AACCATTATTATCATTAAAAGGCTCAGGAGAAACTTTAACAGTAATCAAAATGTCTGTTACAGTCAAGAG

AATCATTGACAACACAGTCGTAGTTCCAAAACTTCCTGCAAATGAGGATCCAGTGGAATACCCGGCAGAT

TACTTCAGAAAATCAAAGGAGATTCCTCTTTACATCAATACTACAAAAAGTTTGTCAGATCTAAGAGGAT

ATGTCTACCAAGGCCTCAAATCCGGAAATGTATCAATCATACATGTCAACAGCTACTTGTATGGAGCATT

AAAGGACATCCGGGGTAAGTTGGATAAAGATTGGTCAAGTTTCGGAATAAACATCGGGAAAGCAGGGGAT

ACAATCGGAATATTTGACCTTGTATCCTTGAAAGCCCTGGACGGCGTACTTCCAGATGGAGTATCGGATG

CTTCCAGAACCAGCGCAGATGACAAATGGTTGCCTTTGTATCTACTTGGCTTATACAGAGTGGGCAGAAC

ACAAATGCCTGAATACAGAAAAAAGCTCATGGATGGGCTGACAAATCAATGCAAAATGATCAATGAACAG

TTTGAACCTCTTGTGCCAGAAGGTCGTGACATTTTTGATGTGTGGGGAAATGACAGTAATTACACAAAAA

TTGTCGCTGCAGTGGACATGTTCTTCCACATGTTCAAAAAACATGAATGTGCCTCGTTCAGATACGGAAC

TATTGTTTCCAGATTCAAAGATTGTGCTGCATTGGCAACATTTGGACACCTCTGCAAAATAACCGGAATG

TCTACAGAAGATGTAACGACCTGGATCTTGAACCGAGAAGTTGCAGATGAAATGGTCCAAATGATGCTTC

CAGGCCAAGAAATTGACAAGGCCGATTCATACATGCCTTATTTGATCGACTTTGGATTGTCTTCTAAGTC

TCCATATTCTTCCGTCAAAAACCCTGCCTTCCACTTCTGGGGGCAATTGACAGCTCTTCTGCTCAGATCC

ACCAGAGCAAGGAATGCCCGACAGCCTGATGACATTGAGTATACATCTCTTACTACAGCAGGTTTGTTGT

ACGCTTATGCAGTAGGATCCTCTGCCGACTTGGCACAACAGTTTTGTGTTGGAGATAACAAATACACTCC

AGATGATAGTACCGGAGGATTGACGACTAATGCACCGCCACAAGGCAGAGATGTGGTCGAATGGCTCGGA

TGGTTTGAAGATCAAAACAGAAAACCGACTCCTGATATGATGCAGTATGCGAAAAGAGCAGTCATGTCAC

TGCAAGGCCTAAGAGAGAAGACAATTGGCAAGTATGCTAAGTCAGAATTTGACAAATGACCCTATAATTC

TCAGATCACCTATTATATATTATGCTACATATGAAAAAAACTAACAGATATCATGGATAATCTCACAAAA

GTTCGTGAGTATCTCAAGTCCTATTCTCGTCTGGATCAGGCGGTAGGAGAGATAGATGAGATCGAAGCAC

AACGAGCTGAAAAGTCCAATTATGAGTTGTTCCAAGAGGATGGAGTGGAAGAGCATACTAAGCCCTCTTA

TTTTCAGGCAGCAGATGATTCTGACACAGAATCTGAACCAGAAATTGAAGACAATCAAGGCTTGTATGCA

CCAGATCCAGAAGCTGAGCAAGTTGAAGGCTTTATACAGGGGCCTTTAGATGACTATGCAGATGAGGAAG

TGGATGTTGTATTTACTTCGGACTGGAAACAGCCTGAGCTTGAATCTGACGAGCATGGAAAGACCTTACG

GTTGACATCGCCAGAGGGTTTAAGTGGAGAGCAGAAATCCCAGTGGCTTTCGACGATTAAAGCAGTCGTG

CAAAGTGCCAAATACTGGAATCTGGCAGAGTGCACATTTGAAGCATCGGGAGAAGGGGTCATTATGAAGG

AGCGCCAGATAACTCCGGATGTATATAAGGTCACTCCAGTGATGAACACACATCCGTCCCAATCAGAAGC

AGTATCAGATGTTTGGTCTCTCTCAAAGACATCCATGACTTTCCAACCCAAGAAAGCAAGTCTTCAGCCT

CTCACCATATCCTTGGATGAATTGTTCTCATCTAGAGGAGAGTTCATCTCTGTCGGAGGTGACGGACGAA TGTCTCATAAAGAGGCCATCCTGCTCGGCCTGAGATACAAAAAGTTGTACAATCAGGCGAGAGTCAAATA

TTCTCTGTAGACTATGAAAAAAAGTAACAGATATCACGATCTAAGTGTTATCCCAATCCATTCATCATGA

GTTCCTTAAAGAAGATTCTCGGTCTGAAGGGGAAAGGTAAGAAATCTAAGAAATTAGGGATCGCACCACC

CCCTTATGAAGAGGACACTAGCATGGAGTATGCTCCGAGCGCTCCAATTGACAAATCCTATTTTGGAGTT

GACGAGATGGACACCTATGATCCGAATCAATTAAGATATGAGAAATTCTTCTTTACAGTGAAAATGACGG

TTAGATCTAATCGTCCGTTCAGAACATACTCAGATGTGGCAGCCGCTGTATCCCATTGGGATCACATGTA

CATCGGAATGGCAGGGAAACGTCCCTTCTACAAAATCTTGGCTTTTTTGGGTTCTTCTAATCTAAAGGCC

ACTCCAGCGGTATTGGCAGATCAAGGTCAACCAGAGTATCACGCTCACTGCGAAGGCAGGGCTTATTTGC

CACATAGGATGGGGAAGACCCCTCCCATGCTCAATGTACCAGAGCACTTCAGAAGACCATTCAATATAGG

TCTTTACAAGGGAACGATTGAGCTCACAATGACCATCTACGATGATGAGTCACTGGAAGCAGCTCCTATG

ATCTGGGATCATTTCAATTCTTCCAAATTTTCTGATTTCAGAGAGAAGGCCTTAATGTTTGGCCTGATTG

TCGAGAAAAAGGCATCTGGAGCGTGGGTCCTGGACTCTATCGGCCACTTCAAATGAGCTAGTCTAACTTC

TAGCTTCTGAACAATCCCCGGTTTACTCAGTCTCCCCTAATTCCAGCCTCTCGAACAACTAATATCCTGT

CTTTTCTATCCCTATGAAAAAAACTAACAGAGATCGATCTGTTTACGCGTCACTATCAAGTGCCTTTTGT

ACTTAGCCTTTTTATGCATGCGGTACGACATAGAGATGAACCGCCTAGGAAAGTAACTCAAATCCTGCTA

GGTATGAAAAAAACTAACAGATATCACGCTCGAGAATTAATTGCTAGGTATGAAAAAAACTAACAGATAT

CACGCTCGAGAATTAATTGCTAGCCAGATTCTTCATGTTTGGACCAAATCAACTTGTGATACCATGCTCA

AAGAGGCCTCAATTATATTTGAGTTTTTAATTTTTATGAAAAAAACTAACAGCAATCATGGAAGTCCACG

ATTTTGAGACCGACGAGTTCAATGATTTCAATGAAGATGACTATGCCACAAGAGAATTCCTGAATCCCGA

TGAGCGCATGACGTACTTGAATCATGCTGATTACAACCTGAATTCTCCTCTAATTAGTGATGATATTGAC

AATTTAATCAGGAAATTCAATTCTCTTCCAATTCCCTCGATGTGGGATAGTAAGAACTGGGATGGAGTTC

TTGAGATGTTAACGTCATGTCAAGCCAATCCCATCCCAACATCTCAGATGCATAAATGGATGGGAAGTTG

GTTAATGTCTGATAATCATGATGCCAGTCAAGGGTATAGTTTTTTACATGAAGTGGACAAAGAGGCAGAA

ATAACATTTGACGTGGTGGAGACCTTCATCCGCGGCTGGGGCAACAAACCAATTGAATACATCAAAAAGG

AAAGATGGACTGACTCATTCAAAATTCTCGCTTATTTGTGTCAAAAGTTTTTGGACTTACACAAGTTGAC

ATTAATCTTAAATGCTGTCTCTGAGGTGGAATTGCTCAACTTGGCGAGGACTTTCAAAGGCAAAGTCAGA

AGAAGTTCTCATGGAACGAACATATGCAGGATTAGGGTTCCCAGCTTGGGTCCTACTTTTATTTCAGAAG

GATGGGCTTACTTCAAGAAACTTGATATTCTAATGGACCGAAACTTTCTGTTAATGGTCAAAGATGTGAT

TATAGGGAGGATGCAAACGGTGCTATCCATGGTATGTAGAATAGACAACCTGTTCTCAGAGCAAGACATC

TTCTCCCTTCTAAATATCTACAGAATTGGAGATAAAATTGTGGAGAGGCAGGGAAATTTTTCTTATGACT

TGATTAAAATGGTGGAACCGATATGCAACTTGAAGCTGATGAAATTAGCAAGAGAATCAAGGCCTTTAGT

CCCACAATTCCCTCATTTTGAAAATCATATCAAGACTTCTGTTGATGAAGGGGCAAAAATTGACCGAGGT

ATAAGATTCCTCCATGATCAGATAATGAGTGTGAAAACAGTGGATCTCACACTGGTGATTTATGGATCGT

TCAGACATTGGGGTCATCCTTTTATAGATTATTACACTGGACTAGAAAAATTACATTCCCAAGTAACCAT

GAAGAAAGATATTGATGTGTCATATGCAAAAGCACTTGCAAGTGATTTAGCTCGGATTGTTCTATTTCAA CAGTTCAATGATCATAAAAAGTGGTTCGTGAATGGAGACTTGCTCCCTCATGATCATCCCTTTAAAAGTC

ATGTTAAAGAAAATACATGGCCCACAGCTGCTCAAGTTCAAGATTTTGGAGATAAATGGCATGAACTTCC

GCTGATTAAATGTTTTGAAATACCCGACTTACTAGACCCATCGATAATATACTCTGACAAAAGTCATTCA

ATGAATAGGTCAGAGGTGTTGAAACATGTCCGAATGAATCCGAACACTCCTATCCCTAGTAAAAAGGTGT

TGCAGACTATGTTGGACACAAAGGCTACCAATTGGAAAGAATTTCTTAAAGAGATTGATGAGAAGGGCTT

AGATGATGATGATCTAATTATTGGTCTTAAAGGAAAGGAGAGGGAACTGAAGTTGGCAGGTAGATTTTTC

TCCCTAATGTCTTGGAAATTGCGAGAATACTTTGTAATTACCGAATATTTGATAAAGACTCATTTCGTCC

CTATGTTTAAAGGCCTGACAATGGCGGACGATCTAACTGCAGTCATTAAAAAGATGTTAGATTCCTCATC

CGGCCAAGGATTGAAGTCATATGAGGCAATTTGCATAGCCAATCACATTGATTACGAAAAATGGAATAAC

CACCAAAGGAAGTTATCAAACGGCCCAGTGTTCCGAGTTATGGGCCAGTTCTTAGGTTATCCATCCTTAA

TCGAGAGAACTCATGAATTTTTTGAGAAAAGTCTTATATACTACAATGGAAGACCAGACTTGATGCGTGT

TCACAACAACACACTGATCAATTCAACCTCCCAACGAGTTTGTTGGCAAGGACAAGAGGGTGGACTGGAA

GGTCTACGGCAAAAAGGATGGAGTATCCTCAATCTACTGGTTATTCAAAGAGAGGCTAAAATCAGAAACA

CTGCTGTCAAAGTCTTGGCACAAGGTGATAATCAAGTTATTTGCACACAGTATAAAACGAAGAAATCGAG

AAACGTTGTAGAATTACAGGGTGCTCTCAATCAAATGGTTTCTAATAATGAGAAAATTATGACTGCAATC

AAAATAGGGACAGGGAAGTTAGGACTTTTGATAAATGACGATGAGACTATGCAATCTGCAGATTACTTGA

ATTATGGAAAAATACCGATTTTCCGTGGAGTGATTAGAGGGTTAGAGACCAAGAGATGGTCACGAGTGAC

TTGTGTCACCAATGACCAAATACCCACTTGTGCTAATATAATGAGCTCAGTTTCCACAAATGCTCTCACC

GTAGCTCATTTTGCTGAGAACCCAATCAATGCCATGATACAGTACAATTATTTTGGGACATTTGCTAGAC

TCTTGTTGATGATGCATGATCCTGCTCTTCGTCAATCATTGTATGAAGTTCAAGATAAGATACCGGGCTT

GCACAGTTCTACTTTCAAATACGCCATGTTGTATTTGGACCCTTCCATTGGAGGAGTGTCGGGCATGTCT

TTGTCCAGGTTTTTGATTAGAGCCTTCCCAGATCCCGTAACAGAAAGTCTCTCATTCTGGAGATTCATCC

ATGTACATGCTCGAAGTGAGCATCTGAAGGAGATGAGTGCAGTATTTGGAAACCCCGAGATAGCCAAGTT

TCGAATAACTCACATAGACAAGCTAGTAGAAGATCCAACCTCTCTGAACATCGCTATGGGAATGAGTCCA

GCGAACTTGTTAAAGACTGAGGTTAAAAAATGCTTAATCGAATCAAGACAAACCATCAGGAACCAGGTGA

TTAAGGATGCAACCATATATTTGTATCATGAAGAGGATCGGCTCAGAAGTTTCTTATGGTCAATAAATCC

TCTGTTCCCTAGATTTTTAAGTGAATTCAAATCAGGCACTTTTTTGGGAGTCGCAGACGGGCTCATCAGT

CTATTTCAAAATTCTCGTACTATTCGGAACTCCTTTAAGAAAAAGTATCATAGGGAATTGGATGATTTGA

TTGTGAGGAGTGAGGTATCCTCTTTGACACATTTAGGGAAACTTCATTTGAGAAGGGGATCATGTAAAAT

GTGGACATGTTCAGCTACTCATGCTGACACATTAAGATACAAATCCTGGGGCCGTACAGTTATTGGGACA

ACTGTACCCCATCCATTAGAAATGTTGGGTCCACAACATCGAAAAGAGACTCCTTGTGCACCATGTAACA

CATCAGGGTTCAATTATGTTTCTGTGCATTGTCCAGACGGGATCCATGACGTCTTTAGTTCACGGGGACC

ATTGCCTGCTTATCTAGGGTCTAAAACATCTGAATCTACATCTATTTTGCAGCCTTGGGAAAGGGAAAGC

AAAGTCCCACTGATTAAAAGAGCTACACGTCTTAGAGATGCTATCTCTTGGTTTGTTGAACCCGACTCTA

AACTAGCAATGACTATACTTTCTAACATCCACTCTTTAACAGGCGAAGAATGGACCAAAAGGCAGCATGG GTTCAAAAGAACAGGGTCTGCCCTTCATAGGTTTTCGACATCTCGGATGAGCCATGGTGGGTTCGCATCT

CAGAGCACTGCAGCATTGACCAGGTTGATGGCAACTACAGACACCATGAGGGATCTGGGAGATCAGAATT

TCGACTTTTTATTCCAAGCAACGTTGCTCTATGCTCAAATTACCACCACTGTTGCAAGAGACGGATGGAT

CACCAGTTGTACAGATCATTATCATATTGCCTGTAAGTCCTGTTTGAGACCCATAGAAGAGATCACCCTG

GACTCAAGTATGGACTACACGCCCCCAGATGTATCCCATGTGCTGAAGACATGGAGGAATGGGGAAGGTT

CGTGGGGACAAGAGATAAAACAGATCTATCCTTTAGAAGGGAATTGGAAGAATTTAGCACCTGCTGAGCA

ATCCTATCAAGTCGGCAGATGTATAGGTTTTCTATATGGAGACTTGGCGTATAGAAAATCTACTCATGCC

GAGGACAGTTCTCTATTTCCTCTATCTATACAAGGTCGTATTAGAGGTCGAGGTTTCTTAAAAGGGTTGC

TAGACGGATTAATGAGAGCAAGTTGCTGCCAAGTAATACACCGGAGAAGTCTGGCTCATTTGAAGAGGCC

GGCCAACGCAGTGTACGGAGGTTTGATTTACTTGATTGATAAATTGAGTGTATCACCTCCATTCCTTTCT

CTTACTAGATCAGGACCTATTAGAGACGAATTAGAAACGATTCCCCACAAGATCCCAACCTCCTATCCGA

CAAGCAACCGTGATATGGGGGTGATTGTCAGAAATTACTTCAAATACCAATGCCGTCTAATTGAAAAGGG

AAAATACAGATCACATTATTCACAATTATGGTTATTCTCAGATGTCTTATCCATAGACTTCATTGGACCA

TTCTCTATTTCCACCACCCTCTTGCAAATCCTATACAAGCCATTTTTATCTGGGAAAGATAAGAATGAGT

TGAGAGAGCTGGCAAATCTTTCTTCATTGCTAAGATCAGGAGAGGGGTGGGAAGACATACATGTGAAATT

CTTCACCAAGGACATATTATTGTGTCCAGAGGAAATCAGACATGCTTGCAAGTTCGGGATTGCTAAGGAT

AATAATAAAGACATGAGCTATCCCCCTTGGGGAAGGGAATCCAGAGGGACAATTACAACAATCCCTGTTT

ATTATACGACCACCCCTTACCCAAAGATGCTAGAGATGCCTCCAAGAATCCAAAATCCCCTGCTGTCCGG

AATCAGGTTGGGCCAATTACCAACTGGCGCTCATTATAAAATTCGGAGTATATTACATGGAATGGGAATC

CATTACAGGGACTTCTTGAGTTGTGGAGACGGCTCCGGAGGGATGACTGCTGCATTACTACGAGAAAATG

TGCATAGCAGAGGAATATTCAATAGTCTGTTAGAATTATCAGGGTCAGTCATGCGAGGCGCCTCTCCTGA

GCCCCCCAGTGCCCTAGAAACTTTAGGAGGAGATAAATCGAGATGTGTAAATGGTGAAACATGTTGGGAA

TATCCATCTGACTTATGTGACCCAAGGACTTGGGACTATTTCCTCCGACTCAAAGCAGGCTTGGGGCTTC

AAATTGATTTAATTGTAATGGATATGGAAGTTCGGGATTCTTCTACTAGCCTGAAAATTGAGACGAATGT

TAGAAATTATGTGCACCGGATTTTGGATGAGCAAGGAGTTTTAATCTACAAGACTTATGGAACATATATT

TGTGAGAGCGAAAAGAATGCAGTAACAATCCTTGGTCCCATGTTCAAGACGGTCGACTTAGTTCAAACAG

AATTTAGTAGTTCTCAAACGTCTGAAGTATATATGGTATGTAAAGGTTTGAAGAAATTAATCGATGAACC

CAATCCCGATTGGTCTTCCATCAATGAATCCTGGAAAAACCTGTACGCATTCCAGTCATCAGAACAGGAA

TTTGCCAGAGCAAAGAAGGTTAGTACATACTTTACCTTGACAGGTATTCCCTCCCAATTCATTCCTGATC

CTTTTGTAAACATTGAGACTATGCTACAAATATTCGGAGTACCCACGGGTGTGTCTCATGCGGCTGCCTT

AAAATCATCTGATAGACCTGCAGATTTATTGACCATTAGCCTTTTTTATATGGCGATTATATCGTATTAT

AACATCAATCATATCAGAGTAGGACCGATACCTCCGAACCCCCCATCAGATGGAATTGCACAAAATGTGG

GGATCGCTATAACTGGTATAAGCTTTTGGCTGAGTTTGATGGAGAAAGACATTCCACTATATCAACAGTG

TTTAGCAGTTATCCAGCAATCATTCCCGATTAGGTGGGAGGCTGTTTCAGTAAAAGGAGGATACAAGCAG

AAGTGGAGTACTAGAGGTGATGGGCTCCCAAAAGATACCCGAATTTCAGACTCCTTGGCCCCAATCGGGA ACTGGATCAGATCTCTGGAATTGGTCCGAAACCAAGTTCGTCTAAATCCATTCAATGAGATCTTGTTCAA

TCAGCTATGTCGTACAGTGGATAATCATTTGAAATGGTCAAATTTGCGAAGAAACACAGGAATGATTGAA

TGGATCAATAGACGAATTTCAAAAGAAGACCGGTCTATACTGATGTTGAAGAGTGACCTACACGAGGAAA

ACTCTTGGAGAGATTAAAAAATCATGAGGAGACTCCAAACTTTAAGTATGAAAAAAACTTTGATCCTTAA

GACCCTCTTGTGGTTTTTATTTTTTATCTGGTTTTGTGGTCTTCGTGGGTCGGCATGGCATCTCCACCTC

CTCGCGGTCCGACCTGGGCATCCGAAGGAGGACGTCGTCCACTCGGATGGCTAAGGGAGGGGCCCCCGCG

GGGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAAC

CCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGGAGGAACTATATCCGGATCGAGAC

CTCGATACTAGAGCGGCCGCCACCGCGGTGGAGCTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG

GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGC

TGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG

AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG

ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC

GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCA

GCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA

CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT

GGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT

CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGC

AAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG

CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGAT

CCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAC

CAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC

CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGA

CCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT

CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAG

TTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGC

TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT

AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAG

CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA

GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCG

CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCT

TACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT

CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG

AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT

GCCACCTG

SEQ ID NO: 20 HIVenv/EboTM nucleic acid sequence based on pNL4.3 ( underlined sequence represents the transmembrane (nucleotides 2044-2109) and twice underlined sequence (nucleotides 2110-2124) represents the short cytoplasmic tail of the construct with stop codon (nucleotides 2125-2127).

ATGAGAGTGAAGGAGAAGTATCAGCACTTGTGGAGATGGGGGTGGAAATGGGGCACCATGCTCCTTGGGA

TATTGATGATCTGTAGTGCTACAGAAAAATTGTGGGTCACAGTCTATTATGGGGTACCTGTGTGGAAGGA

AGCAACCACCACTCTATTTTGTGCATCAGATGCTAAAGCATATGATACAGAGGTACATAATGTTTGGGCC

ACACATGCCTGTGTACCCACAGACCCCAACCCACAAGAAGTAGTATTGGTAAATGTGACAGAAAATTTTA

ACATGTGGAAAAATGACATGGTAGAACAGATGCATGAGGATATAATCAGTTTATGGGATCAAAGCCTAAA

GCCATGTGTAAAATTAACCCCACTCTGTGTTAGTTTAAAGTGCACTGATTTGAAGAATGATACTAATACC

AATAGTAGTAGCGGGAGAATGATAATGGAGAAAGGAGAGATAAAAAACTGCTCTTTCAATATCAGCACAA

GCATAAGAGATAAGGTGCAGAAAGAATATGCATTCTTTTATAAACTTGATATAGTACCAATAGATAATAC

CAGCTATAGGTTGATAAGTTGTAACACCTCAGTCATTACACAGGCCTGTCCAAAGGTATCCTTTGAGCCA

ATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTCTAAAATGTAATAATAAGACGTTCAATGGAA

CAGGACCATGTACAAATGTCAGCACAGTACAATGTACACATGGAATCAGGCCAGTAGTATCAACTCAACT

GCTGTTAAATGGCAGTCTAGCAGAAGAAGATGTAGTAATTAGATCTGCCAATTTCACAGACAATGCTAAA

ACCATAATAGTACAGCTGAACACATCTGTAGAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAA

GTATCCGTATCCAGAGGGGACCCGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATGAGACAAGC

ACATTGTAACATTAGTAGAGCAAAATGGAATGCCACTTTAAAACAGATAGCTAGCAAATTAAGAGAACAA

TTTGGAAATAATAAAACAATAATCTTTAAGCAATCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTT

TTAATTGTGGAGGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGTTTAATAGTAC

TTGGAGTACTGAAGGGTCAAATAACACTGAAGGAAGTGACACAATCACACTCCCATGCAGAATAAAACAA

TTTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGGACAAATTAGATGTT

CATCAAATATTACTGGGCTGCTATTAACAAGAGATGGTGGTAATAACAACAATGGGTCCGAGATCTTCAG

ACCTGGAGGAGGCGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAA

CCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAG

GAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGT

ACAGGCCAGACAATTATTGTCTGATATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAA

CAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAACAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGAT

ACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCC

TTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATAACATGACCTGGATGGAGTGGGAC

AGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGA ATGAACAAGAATTATTGG_AAT_TAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCT

GTGGTATATAAAGCAATGGATACCGGCAGGTATCGGAGTCACAGGCGTTATCATCGCAGTTATCGCTTTG

TTCTGTATCTGCAAGTTCGTCTTCTAG SEQ ID NO: 21 HIVenv/EboTM amino acid sequence based on pNL4.3

MRVKEKYQHLWRWGWKWGTMLLGILMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWA THACVPTDPNPQEWLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNT NSSSGRMIMEKGEIKNCSFNISTSIRDKVQKEYAFFYKLDIVPIDNTSYRLISCNTSVITQACPKVSFEP IPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEDWIRSANFTDNAK TIIVQLNTSVEINCTRPNNNTRKSIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNATLKQIASKLREQ FGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQ FINMWQEVGKAMYAPPISGQIRCSSNITGLLLTRDGGNNNNGSEIFRPGGGDMRDNWRSELYKYKWKIE PLGVAPTKAKRRWQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSDIVQQQNNLLRAIEAQ QHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWD REINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKQWIPAGIGVTGVIIAVIAL FCICKFVF*

Claims

CLAIMS:

1. Particles or viral particles comprising vesicular stomatitis virus (VSV) nucleocapsid, phosphoprotein, matrix protein, and polymerase L, an Ebola virus glycoprotein and an antigen or antigens.

2. The viral particles of claim 1, further comprising the VSV glycoprotein.

3. The viral particles of claim 1 or 2, wherein the Ebola virus glycoprotein is a natural Ebola virus glycoprotein, a mutated Ebola virus glycoprotein, a chimeric Ebola virus glycoprotein or a portion thereof.

4. The viral particles of any one of claims 1 to 3, wherein the Ebola virus glycoprotein does not contain a functional a mucin-like domain.

5. The viral particles of claim 4, wherein the mucin-like domain is deleted or mutated.

6. The viral particles of any one of claims 1 to 5, wherein the Ebola virus glycoprotein is at least 80% identical to the Zaire Ebola virus glycoprotein.

7. The viral particles of claim 6, wherein the Zaire Ebola virus glycoprotein is from the Makona strain, the Mayinga strain or the Kikwit strain.

8. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

9. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

10. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

11. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

12. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

13. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

14. The viral particles of any one of claims 1 to 7, wherein the Ebola virus glycoprotein comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

15. The viral particles of claims 1 to 14, wherein the antigen or antigens are from one or more pathogens.

16. The viral particles of claim 15, wherein the pathogen is selected from the group consisting of viruses, bacteria and parasites.

17. The viral particles of any one of claims 1 to 14, wherein the antigen or antigens are tumor-specific antigen or antigens.

18. The viral particles of any one of claims 1 to 17, wherein the antigen or antigens comprise a sequence allowing anchoring at the surface of vesicular stomatitis viruses.

19. The viral particles of claim 18, wherein the anchoring sequence is from the Ebola glycoprotein.

20. The viral particles of claim 18, wherein the anchoring sequence is from the VSV glycoprotein.

21. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from the Human Immunodeficiency Virus (HIV).

22. The viral particles of claim 21, wherein the antigen or antigens comprise the HIV envelope or a portion thereof.

23. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from the Lassa virus.

24. The viral particles of claim 23, wherein the antigen or antigens comprise Lassa virus glycoproteins or a portion thereof.

25. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from the Nipah virus.

26. The viral particles of claim 25, wherein the antigen or antigens comprise Nipah virus glycoproteins, Nipah fusion proteins or a portion thereof.

27. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from the Crimean Congo Hemorrhagic Fever CCHF) virus.

28. The viral particles of claim 27, wherein the antigen or antigens comprise CCHF glycoprotein or a portion thereof.

29. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from the Middle East Respiratory coronavirus (MERS-CoV).

30. The viral particles of claim 29, wherein the antigen or antigens comprise MERS-CoV S protein or a portion thereof.

31. The viral particles of any one of claims 1 to 20, wherein the antigen or antigens are from a tick.

32. The viral particles of any one of claims 1 to 30, wherein the pseudotyped vesicular stomatitis virus is replicative.

33. A viral preparation comprising the viral particles of any one of claims 1 to 31.

34. The viral preparation of claim 32, for increasing an immune response against an antigen or antigens in a host, wherein the viral preparation comprises substantially purified viral particles.

35. A pharmaceutical composition comprising the viral particles of any one of claims 1 to 24 or the viral preparation of claim 32 or 33.

36. The pharmaceutical composition of claim 34, further comprising an adjuvant.

37. A vial or vials comprising the pharmaceutical composition of claim 33 or 34.

38. A kit comprising the vial or vials of claim 36 and instructions for the use of the pharmaceutical composition.

39. A method of increasing an immune response towards an antigen or antigens, the method comprising administering the pharmaceutical composition of claim 33 or 34 to a host in need.

40. The method of claim 38, wherein the pharmaceutical composition is administered by injection.

41. The method of claim 39, wherein the injection is performed intramuscularly, subcutaneously intradermally.

42. The method of claim 38, wherein the pharmaceutical composition is delivered transdermally.

43. The method of claim 38, wherein the pharmaceutical composition is delivered to a mucosa.

44. The method of any one of claims 38 to 42, wherein the pharmaceutical composition is administered in a single-dose regimen.

45. A vector for recombinant expression of a VSV positive-sense genome (antigenome) comprising genes encoding vesicular stomatitis virus (VSV) proteins, an Ebola virus glycoprotein and an antigen or antigens, wherein the genes are operably linked.

46. The vector of claim 44, wherein the vector comprises sequences allowing for transcription of the genome and/or for expression of the genes.

47. The vector of claim 44 or 45, wherein the vector is a VSV-based vector expressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, and the VSV polymerase L.

48. The vector of claim 44 or 45, wherein the vector is a VSV-based vector expressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, the VSV surface glycoprotein and the VSV polymerase L.

49. The vector of claim 46 or 47, wherein the VSV nucleocapsid, the VSV phosphoprotein, the VSV matrix protein, the antigen or antigens, the Ebola virus glycoprotein, and the polymerase L are arranged sequentially in a 5’to 3’ fashion.

50. The vector of any one of claims 44 to 48, wherein the Ebola virus glycoprotein is the Zaire Ebola virus glycoprotein.

51. The vector of claim 49, wherein the Zaire Ebola virus glycoprotein is from the Makona strain, the Mayinga strain or the Kikwit strain.

52. The vector of any one of claims 44 to 50, wherein the antigen or antigens are from one or more pathogens.

53. The vector of claim 51, wherein the pathogen is selected from the group consisting of viruses, bacteria and parasites.

54. The vector of any one of claims 44 to 50, wherein the antigen or antigens are tumor-specific antigen or antigens.

55. The vector of any one of claims 44 to 53, wherein the antigen or antigens comprise a sequence allowing anchoring at the surface of VSV-based viral particles.

56. The vector of any one of claims 44 to 54, wherein the antigen or antigens are from Human Immunodeficiency Virus (HIV).

57. The vector of claim 55, wherein the antigen or antigens comprise the HIV envelope or a portion thereof.

58. The vector of any one of claims 44 to 54, wherein the antigen or antigens are from the Lassa virus.

59. The vector of claim 57, wherein the antigen or antigens comprise Lassa virus glycoproteins or a portion thereof.

60. The vector of any one of claims 44 to 54, wherein the antigen or antigens are from the Nipah virus.

61. The vector of claim 59, wherein the antigen or antigens comprise Nipah virus glycoproteins or a portion thereof.

62. The vector of any one of claims 44 to 54, wherein the antigen or antigens are from the Crimean Congo Hemorrhagic Fever CCHF) virus.

63. The vector of claim 61, wherein the antigen or antigens comprise CCHF glycoprotein or a portion thereof.

64. The vector of any one of claims 44 to 54, wherein the antigen or antigens are from the Middle East Respiratory coronavirus (MERS-CoV).

65. The vector of claim 63, wherein the antigen or antigens comprise MERS-CoV S protein or a portion thereof.

66. An isolated cell comprising the vector of any one of claims 44 to 64.

67. The isolated cell of claim 65, further comprising helper plasmids for overexpressing the VSV nucleocapsid, the VSV phosphoprotein, the VSV polymerase L or combination thereof.

68. The isolated cell of claim 65 or 66, wherein the cell comprises a plasmid for expressing a bacteriophage RNA polymerase under the control of a bacteriophage RNA polymerase promoter.

69. A nucleic acid or a set of nucleic acids suitable for expressing a VSV genome, an Ebola glycoprotein and an antigen or antigens.

70. The nucleic acid or set of nucleic acids of claim 69, wherein the VSV genome does not encode a functional VSV glycoprotein.

71. The nucleic acid or set of nucleic acids of claim 70, wherein the VSV genome is a VSVAG genome.

72. The nucleic acid or set of nucleic acids of any one of claims 69 to 71, wherein the nucleic acids expressing the VSV genome and the Ebola glycoprotein are on a single vector and wherein the nucleic acid expressing the antigen or antigens is on a separate vector or vectors.

73. The nucleic acid or set of nucleic acids of any one of claims 69 to 71, wherein the nucleic acids expressing the VSV genome and the antigen or antigens are on a single vector and wherein the nucleic acid expressing the Ebola glycoprotein is on a separate vector.

74. The nucleic acid or set of nucleic acids of any one of claims 69 to 71, wherein the nucleic acids expressing the antigen or antigens and the Ebola glycoprotein are on a single vector and wherein the nucleic acid expressing the VSV genome is on a separate vector.

75. The nucleic acid or set of nucleic acids of any one of claims 69 to 71, wherein the nucleic acids expressing the VSV genome, the Ebola glycoprotein and the antigen or antigens are on a single vector and are operably linked.

76. The nucleic acid or set of nucleic acids of any one of claims 69 to 75, wherein the Ebola glycoprotein is expressed from the VSV genome.

77. The nucleic acid or set of nucleic acids of any one of claims 69 to 75, wherein the antigen or antigens are expressed from the VSV genome.

78. A vector or set of vectors comprising the nucleic acid or set of nucleic acids of any one of claims 69 to 76.

79. A kit comprising the nucleic acid or set of nucleic acid of any one of claims 69 to 77 or the vector or set of vectors of any one of claims 45 to 65 or 78.

80. A kit comprising a vector or a set of vectors for recombinant expression of vesicular stomatitis viruses pseudotyped with an Ebola virus glycoprotein and the antigen or antigens.

81. The kit of claim 80, wherein kit comprises a vial containing a vector comprising gene encoding a vesicular stomatitis virus (VSV) positive-sense genome (antigenome) including genes encoding VSV proteins, an Ebola virus glycoprotein and an antigen or antigens.

82. The kit of claim 81, further comprising one or more vial comprising a vector or vectors encoding the VSV nucleocapsid, the VSV phosphoprotein, and/or the VSV polymerase L.

83. A method of making pseudotyped vesicular stomatitis viral particles, the method comprising allowing expression in a cell of a) a VSV positive-sense genome comprising genes encoding VSV proteins, b) an Ebola glycoprotein and c) an antigen or antigens.

84. The method of claim 83, wherein the Ebola glycoprotein is expressed from the VSV positive-sense genome.

85. The method of claim 83 or 84, wherein the antigen or antigens are expressed from the V SV positive- sense genome.

86. The method of any one of claims 83 to 85, further comprising overexpressing the VSV nucleocapsid, the VSV phosphoprotein, and/or the VSV polymerase L.

87. The method of any one of claims 83 to 86, further comprising overexpressing the Ebola glycoprotein.

88. The method of any one of claims 83 to 87, further comprising overexpressing the antigen or antigens.

89. The method of any one of claims 83 to 88, further comprising the purification of viral particles.