WO2024091853A1

WO2024091853A1 - Vesicular stomatitis virus (vsv)-based vaccine against sudan virus

Info

Publication number: WO2024091853A1
Application number: PCT/US2023/077444
Authority: WO
Inventors: Andrea M. MARZI; Heinrich U. FELDMANN
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2022-10-26
Filing date: 2023-10-20
Publication date: 2024-05-02

Abstract

A recombinant vesicular stomatitis virus (VSV) in which the VSV G gene is replaced with the glycoprotein (GP) gene of Sudan virus (SUDV) is described. The recombinant VSV, referred to as VSV-SUDV, can be used as a live attenuated vaccine for the treatment or prophylaxis of Sudan virus disease (SVD). VSV-SUDV replicates in inoculated subjects, which induces strong innate and adaptive immune responses. Efficacy studies in non-human primates demonstrated that a single intramuscular vaccination protected animals from a lethal challenge dose of SUDV even when vaccination occurred only seven days prior to challenge. In addition, pre-exposure to the VSV vector did not inhibit a robust response to the SUDV GP component of the vaccine.

Description

VESICULAR STOMATITIS VIRUS (VSV)-BASED VACCINE AGAINST SUDAN VIRUS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/517,246, filed August 2, 2023, and U.S. Provisional Application No. 63/419,637, filed October 26, 2022, which are herein incorporated by reference in their entireties.

FIELD

This disclosure concerns a live attenuated Sudan virus (SUDV) vaccine comprised of a vesicular stomatitis virus (VSV) vector expressing SUDV glycoprotein (GP) and methods of its use.

INCORPORATION OF ELECTRONIC SEQUENCE LISTING

The electronic sequence listing, submitted herewith as an XML file named 4239- 109164-03. xml (22,819 bytes), created on September 25, 2023, is herein incorporated by reference in its entirety.

BACKGROUND

Six distinct species of ebolavirus have been described (Kuhn et al., J GenVirol 100(6): 911-912, 2019), of which Zaire, Sudan, Bundibugyo, and Tai Forest ebolaviruses are known causes of human hemorrhagic disease (Jacob et al., Nat Rev Dis Primers 6(1): 13, 2020). Sudan virus (SUDV), the single virus member in the Sudan ebolavirus species, was co-discovered with Ebola virus (EBOV) in 1976 during an outbreak of viral hemorrhagic disease (now designated Sudan virus diseases (SVD)) in what is now The Republic of South Sudan. SUDV remerged in The Republic of South Sudan in 1979 and 2004 causing smaller SVD outbreaks. In 2000-2001, SUDV emerged in Gulu, Uganda causing the largest SVD outbreak on record with 425 cases and a case fatality rate of 53% (Okware et al., Trop Med Int Health 2002; 7(12): 1068-1075, 2002). This was followed by smaller outbreaks in Uganda in 2011, 2012, and 2012-2013. The overall case fatality rate of SUDV infections is -50%. Apart from outbreaks caused by SUDV, Uganda has previously reported outbreaks caused by Bundibugyo virus in 2007 and EBOV in 2019 which had average case fatality rates of 25% (Kadanali and Karagoz, North Clin Istanb 2(1): 81-86, 2015) and 50%, respectively.

The 2022 SUDV outbreak started with a 24-year-old male who was diagnosed on September 19, 2022 after visiting several health clinics. Cases have been reported from the Buyangabu, Kampala, Wasiko, Kagadi, Kyegegwa, Mubende, and Kassanda districts in Central and West Uganda approximately 160 kilometers west of Kampala. Although on-site laboratory testing and medical support to identify and manage case patients are available, specific treatments and vaccines do not currently exist to assist with outbreak management. Thus, a need exists for effective therapeutic and prophylactic treatments for SUDV. SUMMARY

Described herein is a live attenuated vesicular stomatitis virus (VSV) vector encoding the glycoprotein (GP) from Sudan virus (VSV-SUDV). The disclosed VSV-SUDV vector replicates in inoculated subjects, which induces strong innate and adaptive immune responses. In vitro studies demonstrated efficient replication of the VSV-SUDV vector and proper antigen (GP) presentation. Efficacy studies in non-human primates demonstrated that a single intramuscular vaccination protected animals from a lethal challenge dose of SUDV even when vaccination occurred as little as seven days prior to challenge. In addition, pre-exposure to the VSV vector did not inhibit a robust response to the SUDV GP component of the vaccine.

Provided herein is a recombinant VSV-SUDV that has a deletion of the VSV G gene (such as a complete deletion or a partial deletion that results in the absence of G protein expression) and an insertion of a SUDV glycoprotein gene. The VSV-SUDV includes VSV nucleocapsid protein (N), phosphoprotein (P), matrix protein (M) and polymerase protein (L) genes, and a SUDV GP gene. In some aspects, the SUDV GP gene is inserted between the VSV M and L genes. In some aspects, the GP gene is from the Gulu strain of SUDV. Immunogenic compositions that include a pharmaceutically acceptable carrier (such as water or saline) and a recombinant VSV-SUDV disclosed herein are also provided. In some aspects, the immunogenic composition further includes an adjuvant.

Also provided are methods of eliciting an immune response against SUDV in a subject and/or immunizing a subject against SUDV. In some aspects, the methods include administering to the subject a therapeutically effective or prophylactically effective amount of a recombinant VSV-SUDV or immunogenic composition described herein. In some examples, the subject is human. In some examples, the subject is administered a single dose of the recombinant VSV-SUDV or immunogenic composition.

Further provided are nucleic acid molecules that encode a recombinant VSV-SUDV described herein. Immunogenic compositions that include a recombinant VSV-SUDV encoding nucleic acid are also provided.

Also provided are kits for immunizing a subject against SUDV. In some aspects, the kit includes a recombinant VSV-SUDV, immunogenic composition, or nucleic acid molecule disclosed herein. In some examples, the kit further includes equipment (e.g., a sterile needle, sterile syringe, or both) for administering the recombinant VSV-SUDV, immunogenic composition or nucleic acid molecule, an adjuvant and/or instructions.

The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I: Survival and clinical changes in NHPs after SUDV challenge. Groups of NHPs were vaccinated with VSV-SUDV (n=6) or control vaccine (VSV-MARV; n=5) and challenged 4 weeks later with SUDV. (FIG. 1 A) Survival, (FIG. IB) clinical scores, (FIG. 1C) lymphocytes, (FIG. ID) viremia by RT-qPCR, and (FIG. IE) viremia by titration. Serum levels of (FIG. IF) aspartate aminotransferase (AST), (FIG. 1G) alanine phosphatase (ALP), (FIG. 1H) blood urea nitrogen (BUN), and (FIG. II) albumin were determined. Significant differences in the survival curves were determined by performing Log-Rank analysis. Statistical significance is indicated as ** p<0.01.

FIGS. 2A-2B: Histopathology after SUDV challenge. (FIG. 2A) At the time of euthanasia, liver and spleen were collected (6-8 days post challenge (DPC) control; 40 DPC vaccinated), inactivated, processed, and stained with hematoxylin and eosin (H&E). SUDV antigen (VP40) was detected in control NHPs only. Immunoreactive cells are brown. Sections from a representative animal in each group are shown. Magnification 200x; scale bar = 50 pm. (FIG. 2B) SUDV titers in control NHPs at the time of euthanasia.

FIGS. 3A-3D: Humoral immune responses after vaccination and SUDV challenge. Serum IgG levels specific for (FIG. 3A) EBOV GP, (FIG. 3B) SUDV GP, and (FIG. 3C) VSV were determined over time. Geometric mean and geometric SD are depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons and is indicated as follows: p<0.0001 (****), p<0.001 (***), and /? <0.01 (**). (FIG. 3D) Serum neutralization presented as 50% fluorescence reduction (FRNTso) of GFP-positive cells at the time of vaccination (day -28), challenge (day 0) and euthanasia (day 40; study end). Dotted line presents the limit of detection.

FIGS. 4A-4B: Characterization of VSV-SUDV. (FIG. 4A) Schematic of the placement of the viral antigen in the VSV genome. Top shows the VSV-SUDV used for vaccination. The VSV-SUDV-GFP (bottom) was used for neutralization testing. N, nucleoprotein; P, phosphoprotein; M, matrix protein; G, glycoprotein; L, polymerase; SUDV GP, Sudan virus glycoprotein; GFP, green fluorescent protein. (FIG. 4B) Antigen expression by Western blot. SUDV GP (-150 kDa), SUDV soluble GP (sGP; ~45 kDa), and VSV-M (~30 kDa) were detected by monoclonal antibody staining.

FIGS. 5A-5B: Study outline. (FIG. 5A) Timeline of the EBOV and SUDV aspects of this study. (FIG. 5B) Details on NHP vaccination with VSV-EBOV and EBOV GP-specific IgG titers at the end of the EBOV challenge study (day 42). On day 290, a serum sample was collected before the NHPs were vaccinated with VSV-SUDV or VSV-MARV (control). Reciprocal endpoint titers of IgG specific for SUDV GP, EBOV GP, or VSV are listed.

FIGS. 6A-6B: Gating strategy for flow cytometry. (FIG. 6A) Cell-only control was used to gate for GFP+ cells. (FIG. 6B) Gate was kept for all sample analysis (example depicted).

FIGS. 7A-7I: Survival and clinical changes in NHPs after SUDV challenge. NHPs (n=6 per group) were vaccinated 28 days prior to challenge (d-28) with either VSV-SUDV, VSV-EBOV or a control vaccine (VSV-LASV). Another group was vaccinated with VSV-SUDV seven days prior to challenge (d-7). On day 0, all 24 NHPs were challenge with a lethal dose of SUDV. (FIG. 7 A) VSV RNA in the blood after vaccination. (FIG. 7B) Clinical scores, (FIG. 7C) survival, (FIG. 7D) viremia by RT-qPCR, and (FIG. 7E) SLDV sGP levels in serum after challenge are shown. (FIG. 7F) Platelet count in whole blood samples after challenge. Serum levels of (FIG. 7G) aspartate aminotransferase (AST), (FIG. 7H) blood urea nitrogen (BUN) and (FIG. 71) calcium were determined. Significant differences in the survival curves were determined by performing Log-Rank analysis. All other data were evaluated for statistical significance using two-way Anova with Tukey’s multiple comparisons.

FIGS. 8A-8B: Humoral immune responses after vaccination and SUDV challenge. (FIG. 8 A) SUDV GP-specific serum IgG levels were determined over time. (FIG. 8B) Serum neutralization presented as 50% fluorescence reduction (FRNT50) of GFP-positive cells at the time of vaccination (day -28 or day -7), challenge (day 0) and study end (day 42). Geometric mean and geometric SD are depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 9A-9B: Cytokine responses after challenge. Expression levels of selected cytokines (FIG. 9A: GM-CSF, MCP-1, IL-10, IL-12p70, IFN-a2a and IFN-y; FIG. 9B: IL-4, IL-6, IL-15, IP-10, IL-1 , TNF-a) were determined after SUDV-challenge. Mean and standard error of the mean are depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 10A-10I: Changes in blood and serum parameters in NHPs after SUDV challenge. NHPs (n=6 per group) were vaccinated 28 days prior to challenge (d-28) with either VSV-SUDV, VSV-EBOV or control vaccine (VSV-LASV). Another group was vaccinated with VSV-SUDV seven days prior to challenge (d-7). On day 0, all 24 NHPs were challenge with a lethal dose of SUDV. Shown are levels of (FIG. 10A) white blood cells, (FIG. 10B) neutrophils, (FIG. 10C) lymphocytes, (FIG. 10D) K+, (FIG. 10E) ALP, (FIG. 10F) alanine transaminase (ALT), (FIG. 10G) albumin, (FIG. 10H) total protein, and (FIG. 101) creatinine. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 11A-11B: Tissue virus loads and EBOV GP-specific IgG levels. (FIG. 11 A) At the time of necropsy, selected tissue samples were collected and SUDV RNA levels were quantified. (FIG. 11B) EBOV GP-specific IgG responses in the serum of the three groups vaccinated 28 days prior to SUDV challenge. Geometric mean and geometric SD are depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 12A-12F: CD4 T cell responses after vaccination and challenge. PBMCs were stimulated with a SUDV GP-specific peptide pool and analyzed for CD4+ EM-RE T cells. Levels of CD69, IFN-y and IL-4 expression were analyzed at the time of vaccination (FIG. 12A), 14 days before challenge (FIG. 12B), at the time of challenge (FIG. 12C), 14 days post-challenge (FIG. 12D), 28 days post-challenge (FIG. 12E), and 42 days post-challenge (FIG. 12F). Geometric mean and geometric SD are depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 13A-13B: Levels of cytokines and chemokines in the serum after vaccination. Expression levels of selected cytokines (FIG. 13A: GM-CSF, MCP-1, IL-10, IL-12p70, IFN-a2a and IFN-y; FIG. 13B: IL-4, IL-6, IL-15, IP-10, IL-10, TNF-a) were determined after vaccination. Mean and standard error of the mean arc depicted. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons.

FIGS. 14A-14C: Levels of cytokines, chemokines and SUDV sGP in the lung of NHPs. (FIGS. 14A-14B) Expression levels of selected cytokines (FIG. 14A: GM-CSF, IFN-y, IL-6, IL-4, IP-10, and MCP- 1; FIG. 14B: TNF-a, IL-1 p, IL-10, IL-12p70, IL-15, and IFNcc2a) were determined in lung samples from NHPs collected at the time of necropsy. Geometric mean and geometric SD are depicted. Statistical significance was determined by Kruskal-Wallis test with Dunn’s multiple comparisons. (FIG. 14C) SUDV sGP levels in NHP lungs. Geometric mean and geometric SD are depicted. Statistical significance was determined by Mann-Whitney test.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of the VSV-SUDV vector.

DETAILED DESCRIPTION

I. Introduction

The Sudan virus (SUDV) outbreak in Uganda highlights the need for rapid response capabilities against emerging viruses with high public health impact. While such countermeasures have been established for Ebola virus (EBOV), they do not exist for SUDV or any other human-pathogenic filovirus.

There are currently two vaccines licensed for Ebola virus disease (EVD) caused by EBOV : the single shot VSV-EBOV vaccine (Ervebo®, Merck) and the Ad26.ZEBOV/MVA-BN-Filo prime-boost approach (Zabdeno® and Mvabea®, Johnson & Johnson). While VSV-EBOV would be immediately available, it is unlikely to cross-protect against SUDV infections due to antigenic differences of the viral glycoprotein (GP) (Geisbert er al., J Virol 83(14): 7296-304, 2009). Multiple vaccine candidates targeting SUDV specifically or targeting multiple different filoviruses including SUDV, are in preclinical development. The vaccines are mainly based on platforms that have previously been investigated for EBOV- specific vaccines. The platforms include viral vectors (e.g.. human and chimpanzee adenoviruses, VSV, human parainfluenza virus type 3, rabies virus, modified vaccinia Ankara (MVA), Venezuelan equine encephalitis virus), virus-like particles, protein subunit and DNA as outlined in WHO’ s Landscape of SUDV vaccine candidates. Those approaches tested in preclinical nonhuman primate (NHP) studies are listed in Table 2. Only three candidate vaccines have been or are currently in phase 1 a/b clinical trials addressing toxicity and immunogenicity: chimpanzee adenovirus serotype 3 (cAd3) expressing the SUDV GP (clinicaltrials.gov; NCT04041570 and NCT04723602), the chimpanzee adenovirus ChAdOxl-BiEBOV expressing the EBOV and SUDV GPs (clinicaltrials.gov; NCT0509750 and NCT05301504), and a DNA- based vaccine expressing Marburg virus GP, EBOV GP and SUDV GP (Sarwar et al. , J Infect Dis 211 (4): 549-57, 2015), the latter of which is no longer being pursued. Thus, together with the Ad26.ZEBOV/MVA- BN-Filo, there are only three candidates to consider for potential use in the current SUDV outbreak.

Described herein is the development of a VSV-based SUDV-specific vaccine candidate (“VSV- SUDV”) that expresses the SUDV-Gulu strain GP instead of the VSV glycoprotein (G). SUDV-Gulu is the causative strain of the largest recorded SVD outbreak from 2000-2001 in Uganda (Table 1) and phylogenetically closer to the current outbreak strain than the original SUDV -Boniface strain. It is shown herein in the cynomolgus macaque model that a single vaccination with VSV-SUDV is uniformly protective against SUDV challenge, that pre-existing EBOV immunity does not affect the protective efficacy of VSV- SUDV against SUDV challenge, and that pre-existing VSV-EBOV immunity does not protect against SUDV challenge despite cross-reactive immune responses, emphasizing the need for species-specific filovirus vaccines. Moreover, the data disclosed herein demonstrate that the VSV-SUDV vaccine can provide protection from lethal doses of SUDV even when vaccination occurs only seven days prior to challenge. Taken together, the data described herein demonstrates that VSV-SUDV is a fast-acting singledose vaccine ideal for use in future outbreaks. II. Abbreviations

ADCD antibody-dependent complement deposition

ADCP antibody-dependent cellular phagocytosis

AST aspartate aminotransferase

ALP alanine phosphatase

ALT alanine transaminase

BUN blood urea nitrogen

CFR case fatality rate

DPC days post challenge

EBOV Ebola virus

FBS fetal bovine serum

FRNTso50% fluorescence reduction

GM-CSF granulocyte-macrophage colony-stimulating factor

GP glycoprotein

IFN interferon

IL interleukin

IM intramuscular

IP- 10 interferon-inducible protein 10

MARV Marburg virus

MCP monocyte chemoattractant protein

MVA modified vaccinia Ankara

NHP non-human primate

ORF open reading frame

PBMC peripheral blood mononuclear cells

PFU plaque forming unit rVSV recombinant vesicular stomatitis virus sGP soluble glycoprotein

SUDV Sudan virus

SVD Sudan virus disease

TCID50 tissue culture infectious dose 50

TNF tumor necrosis factor

VSV vesicular stomatitis virus

III. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs el al. feds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided.

Adjuvant: A component of an immunogenic composition used to enhance antigenicity, in some aspects, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Adjuvants for use in the disclosed immunogenic compositions can include, for example, the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. Other adjuvants are also known (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), infusion, sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Conservative amino acid substitution: Amino acid substitutions in a protein that do not substantially affect or decrease a function of a protein (such as SUDV GP), such as the ability of the protein to elicit an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of a protein (such as SUDV GP), such as the ability to elicit an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing a recombinant virus in direct physical association with a cell.

Degenerate variant: A polynucleotide encoding a polypeptide (such as SUDV GP) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

Deletion: A recombinant VSV-SUDV that has a “deletion” of the VSV G gene in the viral genome refers to a VSV-SUDV having a complete deletion of the open reading frame (ORF) encoding the G gene, or a partial deletion of the ORF that results in the absence of expression of the G protein. In some aspects herein, the G gene is completely deleted. In other aspects, the G gene is partially deleted, such as 50%, 60%, 70%, 80%, 90%, 95% or 99% deleted.

Ebolavirus: A genus of enveloped, non-segmented, negative-sense, single-stranded RNA viruses that causes Ebolavirus disease (EVD), formerly known as Ebola hemorrhagic fever (EHF), in humans. Ebolaviruses spread through human-to-human transmission, with infection resulting from direct contact with blood, secretions, organs or other bodily fluids of infected people, and indirect contact with environments contaminated by such fluids.

The symptoms of Ebolavirus infection and EVD are well-known. Briefly, in humans, Ebolaviruses have an initial incubation period of 2 to 21 days (7 days on average, depending on the Ebolavirus species) followed by rapid onset of non-specific symptoms such as fever, extreme fatigue, gastrointestinal complaints, abdominal pain, anorexia, headache, myalgias and/or arthralgias. These initial symptoms last for about 2 to 7 days after which more severe symptoms related to hemorrhagic fever occur, including hemorrhagic rash, epistaxis, mucosal bleeding, hematuria, hemoptysis, hematemesis, melena, conjunctival hemorrhage, tachypnea, confusion, somnolence, and hearing loss. In general, the symptoms last for about 7 tol4 days after which recovery may occur. Death can occur 6 to 16 days after the onset of symptoms. People are infectious as long as their blood and secretions contain the virus, which in some instances can be more than 60 days.

Immunoglobulin M (IgM) antibodies to the virus appear 2 to 9 days after infection whereas immunoglobulin G (IgG) antibodies appear approximately 17 to 25 days after infection, which coincides with the recovery phase. In survivors of EVD, both humoral and cellular immunity are detected, however, their relative contribution to protection is unknown.

Six distinct species of Ebolavirus are known, including Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), Reston ebolavirus (RESTV), Tai' Forest ebolavirus (TAFV), and Bombali ebolavirus (BOMV). Bundibugyo ebolavirus, Sudan ebolavirus, and Zaire ebolavirus have been associated with large outbreaks of EVD in Africa and have reported case fatality rates of up to 90%.

The genome of Ebolaviruses includes about 19 kb, which encode seven structural proteins including NP (a nucleoprotein), VP35 (a polymerase cofactor), VP30 (a transcriptional activator), VP24, L (a RNA polymerase), and GP (a glycoprotein).

Effective amount: A quantity of a specific substance (such as a vaccine) sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit, prevent or treat a SUDV infection, or to measurably alter outward symptoms of the infection.

In some aspects, a therapeutically effective amount of a disclosed VSV-SUDV or immunogenic composition thereof is an amount necessary to inhibit SUDV replication or treat SUDV infection in a subject (for example, as measured by infection of cells, or by number or percentage of subjects infected by SUDV, or by an increase in the survival time of infected subjects, or by reduction in symptoms associated with the infection), for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SLDV infection), as compared to a suitable control.

In some aspects, a prophylactically effective amount refers to administration of an agent or composition (such as VSV-SUDV or a composition thereof) in an amount that inhibits or prevents establishment of an infection by SUDV. It is understood that to obtain a protective immune response against an antigen of interest, multiple administrations of a disclosed immunogen/immunogenic composition can be required, and/or administration of a disclosed composition as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogenic composition. Accordingly, a prophylactically effective amount of a disclosed immunogen/immunogenic composition can be the amount of the immunogen or immunogenic composition sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response. In some examples, a response is eliciting an immune response that inhibits or prevents SUDV infection. The SUDV-infected cells do not need to be completely eliminated or prevented for the composition to be effective. For example, administration of an effective amount of an immunogen (such as VSV-SUDV) or immunogenic composition can elicit an immune response that decreases the number of SUDV-infected cells (or prevents the infection of cells), for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SUDV infected cells), as compared to the number of SUDV-infected cells in the absence of the immunization.

Glycoprotein (GP): The virion-associated transmembrane glycoprotein of Ebolaviruses (such as GP of SUDV) is initially synthesized as a precursor protein of about 676 amino acids in size, designated GPo. Individual GPo polypeptides form a homotrimer and undergo glycosylation and processing to remove the signal peptide, as well as cleavage by a cellular protease between approximately positions 501/502 (from the initiating methionine) to generate separate GPi and GP2 polypeptide chains, which remain associated via disulfide bonds as GP1/GP2 protomers within the homotrimer. The extracellular GPi trimer (approximately 153 kDa) is derived from the amino-terminal portion of the GPo precursor. The GP2 trimer (approximately 59 kDa), which includes extracellular, transmembrane, and cytosolic domains, is derived from the carboxyl- terminal portion of the GPo precursor. GPi is responsible for attachment to host cells while GP2 mediates fusion with host cells. GPi contains a mucin-like domain from position 309-501 that is dispensable for infection. Given this, the domain is often removed to more efficiently produce viruses and proteins for assays and is referred to as GPOMUC or GPAMuc.

Comparisons of the predicted amino acid sequences for the GPs of the different species of Ebolavirus show conservation of amino acids in the amino-terminal and carboxy-terminal regions with a highly variable region in the middle of the protein (Sanchez et al., Virus Res 29(3): 215-240, 1993; Sanchez et al. Proc. Natl. Acad. Sci. U.S.A. 93(8): 3602-3607, 1996). The GPs of the Ebolaviruses are highly glycosylated and contain both N-linked and O-linked carbohydrates that contribute up to 50% of the molecular weight of the protein. Most of the glycosylation sites are found in the central variable region of GP.

Heterologous: Originating from a separate genetic source or species. For example, a promoter can be heterologous to an operably linked nucleic acid sequence.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some aspects, the response is specific for a particular antigen (an “antigenspecific response”), such as a SUDV GP. In some aspects, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other aspects, the response is a B cell response, and results in the production of specific antibodies. “Priming an immune response” refers to treatment of a subject with a “prime” immunogen/immunogenic composition to induce an immune response that is subsequently “boosted” with a boost immunogen/immunogenic composition. Together, the prime and boost immunizations produce the desired immune response in the subject. Immunogenic composition: A composition that includes an immunogen or a nucleic acid molecule or vector encoding an immunogen (such as SUDV GP), that elicits a measurable immune response (such as a T cell response and/or B cell response) against the immunogen, when administered to a subject. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this immunogen). For in vivo use, the immunogenic composition can include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Immunize: To render a subject protected from infection by a particular infectious agent, such as SUDV. Immunization does not require 100% protection. In some examples, immunization provides at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% protection against infection (such as infection by SUDV) compared to infection in the absence of immunization.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22^nd ed., Uondon, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

In general, the nature of the carrier depends on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

Recombinant: A recombinant nucleic acid, vector or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques.

Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs and variants of a SUDV GP are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest.

Methods of alignment of sequences for comparison are well known. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3): 443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(l):237-244, 1988; Higgins and Sharp, Bioinformatics. 5(2): 151-3, 1989; Corpet, Nucleic Acids Res. 16(22): 10881-10890, 1988; Huang et al. Bioinformatics. 8(2): 155- 165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

Subject: Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In some aspects, the subject is a human. In other aspects, the subject is a non-human primate (NHP). In some aspects, the subject is a subject with an SUDV infection or at risk of an SUDV infection.

Unit dosage form: A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a patient (such as a human patient), each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic or prophylactic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof.

Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents.

Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replicationincompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.

Vesicular stomatitis virus (VSV): A member of the Rhabdoviridae family of viruses. The genome of VSV is single-stranded, negative-sense RNA that encodes five proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (GP) and a viral polymerase protein (L). VSV can infect a wide variety of mammalian hosts, most commonly horses and cows, but occasionally VSV infects pigs, sheep, goats, llamas and alpacas. Tn rare instances, VSV can infect humans. VSV can be transmitted by insect bites.

IV. Live-Attenuated VSV-SUDV Vaccine

The 2022 SVD outbreak in Uganda has shown that the public health response to emerging/reemerging infectious diseases is vulnerable, lacking accredited and stockpiled vaccine and treatment options. Despite great effort and success for EBOV, there is a general lack of available countermeasures against infections with other human-pathogenic filoviruses. More than 8 years after the EBOV epidemic in West Africa, the situation with vaccines and treatment options against ebolaviruses in general is similar to when EBOV struck West Africa. Multiple SUDV vaccine candidates (Table 2) and some treatment options have finished preclinical evaluation and are awaiting clinical trials, yet nothing was ready for deployment when the SVD outbreak was declared in September 2022.

Clinically, SVD is indistinguishable from EVD and Marburg virus disease, but the pathogens are genetically and antigenically distinct enough to render specific vaccines and monoclonal antibody treatments ineffective against a heterologous infection. Hence, EBOV-specific vaccines, such as VSV-EBOV (Ervebo, Merck), are not expected to be effective against SUDV infections due to lack of cross-protective immune responses as demonstrated in the studies disclosed herein. Therefore, the present disclosure provides a VSV-SUDV vector to be used as a single-dose, live-attenuated vaccine against SVD. It is demonstrated that VSV-SUDV completely protected cynomolgus macaques against lethal challenge with SUDV. Vaccinated animals did not develop viremia, organ tissue viral loads or damage, or clinical disease, which was in strong contrast to the VSV-MARV- or VSV-Lassa virus-vaccinated control NHPs. This unique study design allowed deciphering of potential cross-reactive or cross-protective immune responses between EBOV and SUDV. All macaques had detectable EBOV GP-specific IgG antibody titers approximately one year after VSV-EBOV vaccination and EBOV challenge (FIG. 5B). They also maintained similar levels of VSV-specific immune responses prior to start of the VSV-SUDV vaccine study. Neither EBOV nor VSV pre-existing immunity hampered the development of SUDV GP-specific humoral immune responses. In contrast, the VSV-SUDV vaccination boosted the EBOV GP-specific responses likely adding a durability benefit of these protective responses. The SUDV GP-specific immune responses were protective against SUDV challenge whereas the EBOV GP-specific immune responses were not as demonstrated by the control group. Therefore, cross-reactive antibodies are generated between EBOV and SUDV but those are unlikely to cross-protect against heterologous challenge. Thus, the VSV-SUDV vaccine disclosed herein meets an unmet need by providing protection against SVD and enhancing immunity against EBOV in animals previously vaccinated against EBOV. Additional data in naive NHPs revealed a limited cross-protective potential of the VSV-EBOV vaccine against SVD but highlighted that protection from SVD can be achieved with the VSV-SUDV within seven days of vaccination.

Provided herein is a recombinant vesicular stomatitis virus-Sudan virus (VSV-SUDV) that includes a deletion of the VSV G gene and an insertion of a Sudan virus (SUDV) glycoprotein (GP) gene. The recombinant VSV-SUDV genome includes VSV nucleocapsid protein (N), phosphoprotein (P), matrix protein (M) and polymerase protein (L) genes, and a SUDV GP gene. In some aspects, the recombinant VSV-SUDV has a complete deletion of the VSV G gene (a complete deletion of the ORF encoding the VSV G protein). In other aspects, the recombinant VSV-SUDV has a partial deletion of the G gene (a partial deletion of the ORF encoding the VSV G protein) such as the G protein is not expressed. In some aspects, the SUDV GP is from the Gulu strain of SUDV. In some examples, the SUDV GP gene is inserted between the SUDV M and L genes (for example, the SUDV G gene is inserted in place of the VSV G gene), such that the order of genes is N-P-M-GP-L (see FIG. 4A). In some examples, the nucleic acid sequence of the SUDV GP gene is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In specific non-limiting examples, the nucleic acid sequence of the SUDV glycoprotein gene includes or consists of the nucleic acid sequence of SEQ ID NO: 2.

In some aspects, the recombinant VSV-SUDV expresses a SUDV GP having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In some examples, the recombinant VSV-SUDV expresses a SUDV GP having an amino acid sequence including or consisting of SEQ ID NO: 3.

In some aspects, the recombinant VSV-SUDV is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some examples, the recombinant VSV-SUDV is encoded by a nucleic acid sequence that includes or consists of SEQ ID NO: 1. Also provided are immunogenic compositions that include a pharmaceutically acceptable carrier and a recombinant VSV-SUDV disclosed herein. In some aspects, the immunogenic composition further includes an adjuvant. In other aspects, the immunogenic composition does not include an adjuvant. In some aspects, the immunogenic composition is formulated for intramuscular administration. In some examples, the immunogenic composition is administered in a single dose. In other examples, the immunogenic composition is administered in multiple doses, such as 1, 2, 3, 4 or 5 doses. In some aspects, the immunogenic composition is administered at a dose of about 1 x 10⁶ to about 1 x 10⁸ PFU, such as about 1.5 x 10⁶ to about 1.5 x 10⁷ PFU, for example about 1 x 10⁷ PFU VSV-SUDV.

Further provided are methods of eliciting an immune response against SUDV in a subject. In some aspects, the method includes administering to the subject a therapeutically effective or a prophylactically effective amount of a recombinant VSV-SUDV or immunogenic composition disclosed herein. Also provided are methods of immunizing a subject against SUDV. In some aspects, the method includes administering to the subject a prophylactically effective amount of a recombinant VSV-SUDV or immunogenic composition disclosed herein.

In some aspects of the disclosed methods, the recombinant VSV-SUDV or immunogenic composition is administered intramuscularly. In some aspects, the recombinant VSV-SUDV or the immunogenic composition is administered (such as intramuscularly) in a single dose. In other aspects, the recombinant VSV-SUDV or immunogenic composition is administered (such as intramuscularly) in multiple doses, such as 2, 3, 4 or 5 doses.

In some aspects, the recombinant VSV-SUDV or immunogenic composition is administered at a dose of about 1 x 10⁶ to about 1 x 10⁸ PFU, such as about 1.5 x 10⁶ to about 1.5 x 10⁷ PFU, for example about 1 x 10⁷ PFU VSV-SUDV.

In some aspects, the recombinant VSV-SUDV or immunogenic composition is administered as part of a prime-boost immunization protocol. In some examples, the recombinant VSV-SUDV or immunogenic composition is the prime dose. In other examples, the recombinant VSV-SUDV or immunogenic composition is the boost dose.

In some aspects of the disclosed methods, the subject is human.

In some aspects of the disclosed methods, the subject has been previously vaccinated with an Ebola virus (EBOV) vaccine. In other aspects, the subject has not been previously vaccinated with an EBOV vaccine.

In some aspects of the disclosed methods, the subject has been previously infected with EBOV. In other aspects, the subject has not been previously infected with EBOV.

Also provided herein are nucleic acid molecules encoding a recombinant VSV-SUDV that expresses SUDV GP. In some aspects, the nucleic acid molecule has a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleic acid sequence encoding the recombinant VSV-SUDV includes or consists of the nucleic acid sequence of SEQ ID NO: 1. Further provided are immunogenic compositions that include a nucleic acid molecule disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the immunogenic composition further includes an adjuvant. In some examples, the immunogenic composition is formulated for intramuscular administration.

Also provided herein are kits for immunizing a subject against SUDV. In some aspects, the kit includes a recombinant VSV-SUDV, immunogenic composition, or nucleic acid molecule disclosed herein. In some aspects, the kit further includes equipment for administering the recombinant VSV-SUDV, immunogenic composition or nucleic acid molecule; an adjuvant; and/or instructions. In particular aspects, the equipment includes a sterile needle and/or sterile syringe.

V. Immunogenic Compositions

Immunogenic compositions that include a disclosed immunogen (e.g., a recombinant VSV expressing a SUDV GP protein, or a recombinant VSV-SUDV nucleic acid vector that includes a SUDV GP coding sequence), and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to subjects by a variety of administration modes, for example, intramuscular, intranasal, inhalation, oral, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. Methods for preparing administrable compositions are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013.

Thus, an immunogen described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Exemplary carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually s=l% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The pharmaceutical composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AIPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), may be used as an adjuvant (Newman etal., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants can help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product. In some aspects, an adjuvant is not required and is thus not administered with the VSV-SUDV vaccine.

In some aspects, the composition can be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount which elicits an immune response without significant, adverse side effects. In some examples, the dose is about 1 x 10⁶ to about 1 x 10⁸ PFU, such as about 1.5 x 10⁶ to about 1.5 x 10⁷ PFU, for example about 1 x 10⁷ PFU VSV-SUDV.

In some aspects, the composition can be provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent SUDV infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof. In some examples, the unit dosage is about 1 x 10⁶ to about 1 x 10⁸ PFU, such as about 1.5 x 10⁶ to about 1.5 x 10⁷ PFU, for example about 1 x 10⁷ PFU VSV-SUDV.

VI. Methods of Eliciting an Immune Response

The disclosed immunogens (e.g., a recombinant VSV expressing a SUDV GP), polynucleotides and vectors encoding the disclosed immunogens, and compositions including same, can be used in methods of inducing an immune response to SUDV to prevent, inhibit (including inhibiting transmission), and/or treat a SUDV infection.

Provided herein are methods of eliciting an immune response against SUDV in a subject. In some aspects, the method includes administering to the subject an effective amount of a recombinant VSV-SUDV expressing SUDV GP or immunogenic composition disclosed herein. In some aspects, the recombinant VSV-SUDV or immunogenic composition is administered intramuscularly. In other aspects, the route of administration is intranasal (such as by nebulizer of aerosol). In yet other examples, the route of administration is subcutaneous.

When inhibiting, treating, or preventing SUDV infection, the methods can be used either to avoid infection in a SUDV seronegative subject (e.g.. by inducing an immune response that protects against SUDV infection), or to treat existing infection in a SUDV seropositive subject. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and/or characterize SUDV infection. These and other routine methods allow the clinician to select patients in need of therapy using the methods and immunogenic compositions of the disclosure. In accordance with these methods and principles, a composition can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

The disclosed immunogens can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. In certain aspects, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, for example each directed toward eliciting an anti-SUDV immune response, such as an immune response to SUDV GP. Separate immunogenic compositions that elicit the anti-SUDV immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol. In some aspects, the recombinant VSV-SUDV or immunogenic compositions disclosed herein are administered with one or more vaccines against other filoviruses, such as a vaccine against EBOV.

The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of SUDV infection, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, a dose of a disclosed immunogen can be increased, or the route of administration can be changed.

In some aspects, a disclosed immunogen can be administered to the subject simultaneously with the administration of an adjuvant. In other aspects, the immunogen can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response. In other aspects, no adjuvant is administered.

SUDV infection does not need to be completely inhibited for the methods to be effective. For example, elicitation of an immune response to SUDV can reduce or inhibit SUDV infection by for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SUDV infected cells), as compared to SUDV infection in the absence of immunization. In additional examples, SUDV replication can be reduced or inhibited by the disclosed methods. SUDV replication does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed immunogens can reduce SUDV replication by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SUDV replication), as compared to SUDV replication in the absence of the immune response.

Following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity, include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, single-cycle infection assays, and pseudovirus neutralization assays.

In some aspects, immunization is achieved by administration of recombinant VSV-SUDV nucleic acid. Immunization by nucleic acid constructs is taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell- mediated or a humoral response), U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression), and broadly described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42- 45, 1996.

EXAMPLES

The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

The following Examples describe the generation and characterization of a vesicular stomatitis virus (VSV)-based SUDV vaccine (VSV-SUDV) and demonstrate protective efficacy following a single-dose vaccination against lethal SUDV infection in nonhuman primates (NHPs). Using NHPs repurposed from a successful VSV-EBOV vaccine efficacy study, it is further demonstrated that VSV-SUDV can be used effectively in individuals previously vaccinated against EBOV. While the NHPs developed cross-reactive humoral responses to SUDV after VSV-EBOV vaccination and EBOV challenge, cross-protection was limited emphasizing the need for the development of specific countermeasures for each human-pathogenic ebolavirus. Additionally, the data described in the Examples provides evidence that while previous VSV- EBOV immunity is boosted after VSV-SUDV vaccination, it has only limited impact on the immunogenicity and protective efficacy of VSV-SUDV vaccination important for frontline outbreak workers. Example 1: Materials & Methods

This example describes the materials and experimental procedures for the studies described in Examples 2-5.

Ethics statement

All work involving EBOV and SUDV was performed in the maximum containment laboratory (MCL) at the Rocky Mountain Laboratories (RML), Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. RML is an AAALACi-accredited institution. All procedures followed RML Institutional Biosafety Committee (IBC)-approved standard operating procedures (SOPs). Animal work was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, the Office of Animal Welfare and the Animal Welfare Act, United States Department of Agriculture. This study was approved by the RML Animal Care and Use Committee (ACUC), and all procedures were conducted on anesthetized animals by trained personnel under the supervision of board-certified clinical veterinarians. The NHPs were observed at least twice daily for clinical signs of disease according to a RML ACUC-approved scoring sheet and humanely euthanized when they reached endpoint criteria.

NHPs were housed in adjoining individual primate cages that enabled social interactions, under controlled conditions of humidity, temperature, and light (12 hour light - dark cycles). Food and water were available ad libitum. NHPs were monitored and fed commercial monkey chow, treats, and fruit at least twice a day by trained personnel. Environmental enrichment consisted of commercial toys, music, video, and social interaction. All efforts were made to ameliorate animal welfare and minimize animal suffering in accordance with the Weatherall report on the use of NHPs in research.

Vaccine vectors

Previously described VSV-based vaccine vectors expressing the EBOV-Kikwit GP (VSV-EBOV; Marzi et al., Science 349(6249): 739-742, 2015) and VSV-MARV (Marzi et al., Front Immunol 9: 3071, 2019; Marzi et al., Front Immunol 12: 774026, 2021) were used in this study. The VSV-SUDV vector was generated by cloning the SUDV-Gulu GP gene (GenBank NC_006432.1; 8A version) into the VSV backbone (FIG. 4A, top) as previously described (Marzi et al., J Infect Dis 204 Suppl 3: S1066-S1074, 2011). Similarly, VSV-SUDV-GFP was generated by adding the GFP gene as an additional ORF between the SUDV GP and VSV-L genes (FIG. 4A, bottom). Antigen expression was verified by Western blot analysis using anti-EBOV GP (ZGP 42/3.7, 1:10,000), and anti- VSV M (23H12, 1:1,000; Kerafast Inc.).

Cells and challenge virus

Vero E6 cells (Mycoplasma negative; CVCL_0059) were grown at 37°C and 5% COz in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Wisent, St. Bruno, QC, Canada). 2 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin (all supplements from Thermo Fisher Scientific, MA, USA). EBOV-Makona Guinea C07 was used as challenge virus in the first study (Marzi et al., Science 349(6249): 739-742, 2015). SUDV-Gulu (GenBank NC_006432.1) was obtained from United States Army Medical Research Institute of Infectious Diseases. The virus was propagated once on Vero E6 cells, titered with median tissue culture infectious dose (TCID₅₀) assay on Vero E6 cells and stored in liquid nitrogen. Deep sequencing revealed no contaminants; however, 4 base pair changes (3 of them coding) were noted from this viral passage compared to its reference sequence (Table 3). A target dose of 10,000 TCID₅₀ (backtitered as 5,623 TCID₅₀) was used for the IM SUDV challenge.

NHP study design

Eleven male or female cynomolgus macaques, 6-11 years of age and 3.8-9.0 kg at the time of VSV- SUDV vaccination, were used in this study. The NHPs used in this study had been previously used in a published EBOV challenge study (Matassov etal., J Virol 92(3): eOl 190-17, 2018). In that study, nine animals were completely protected from disease after the EBOV challenge and never showed EBOV viraemia, and two animals developed mild disease with low level of EBOV viraemia (Matassov et al., J Virol 92(3): eOl 190-17, 2018). After the EBOV challenge, NHPs were rested for - 9 months and IM- vaccinated with 1 x10⁷ PFU VSV-SUDV or VSV-MARV (control) (FIG. 5A). The NHPs were divided into 2 study groups as outlined in FIG. 5B. All animals received a 1 ml IM injection for vaccination into 2 sites in the caudal thighs containing either 1x10⁷ PFU VSV-SUDV (n=6) or 1x10⁷ PFU VSV-MARV (control; n=5). 28 days after vaccination, all NHPs were challenged IM (day 0 after challenge) with IxlO⁴ TCID₅₀ SUDV- Gulu into 2 sites in the caudal thighs. Physical examination, rectal temperature measurement, body weight determination and blood draws were performed as outlined in FIG. 5A on days 28, 21, 14, and 7 before challenge; days 0, 3, 6, 9, 14, 21, 28, and after challenge; and at euthanasia (day 40 for survivors; humane endpoint for non-survivors). Blood and serum samples were used to do complete blood cell counts, serum chemistry analysis, viraemia levels, and humoral immune response analysis by ELISA and neutralization. Following euthanasia, a necropsy was performed, and samples of selected tissues including lymph nodes, liver, spleen, and adrenal gland were collected for virological and histopathological analysis.

Western Blot analysis

Virus stocks produced in Vero E6 cells were used to generate protein samples for protein expression analysis. Supernatant samples were mixed 1 : 1 with sodium dodecyl sulfate -polyacrylamide (SDS) gel electrophoresis sample buffer containing 20% P-mercaptoethanol and heated to 100°C for 10 minutes. SDS- PAGE was performed on TGX criterion pre-cast gels (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, proteins were transferred to a Trans-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked for 3 hours at room temperature in PBS with 3% powdered milk and 0.05% Tween 20 (Thermo Fisher Scientific, Waltham, MA, USA), and subsequently washed with PBS/0.05% Tween three times. Protein detection was performed using the following mouse monoclonal antibodies for 1 hour at room temperature: anti-EBOV GP (ZGP 42/3.7, 1 pg/ml) or anti-VSV M (clone 23H12, 1: 1,000; Kerafast Inc., Boston, MA, USA). After three washes with PBS/Tween, horseradish peroxidase (HRP) labeled secondary antibody staining was performed with an antimouse IgG (1 :10,000; cat. #715- 035-151; Jackson ImmunoResearch, West Grove, PA, USA). Finally, the blot was imaged using the SuperSignal West Pico chemiluminescent substrate and the iBright™ CL1500 Imaging System (both Thermo Fisher Scientific, Waltham, MA, USA).

Hematology and serum chemistries

Blood cell counts were determined from EDTA blood with the IDEXX ProCyte DX analyzer (IDEXX Laboratories, Westbrook, ME). Serum biochemistry (including AST, ALP, albumin, and BUN) was analyzed using the Piccolo Xpress Chemistry Analyzer and Piccolo General Chemistry 13 Panel discs (Abaxis, Union City, CA).

SUDV RNA and titer

SUDV RNA copy numbers in EDTA blood samples after challenge were determined using a RT- qPCR assay specific to the SUDV GP; sequences were as follows: forward primer CAAAGGGAAGAATCTCCGACC (SEQ ID NO: 4); reverse primer CAGGGGAATTCTTTGGAACC (SEQ ID NO: 5); probe GGCCACCAGGAAGTATTCGGACC (SEQ ID NO: 6). Blood samples were extracted with QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer specifications. One-step RT-qPCR was performed with QuantiFast Probe RT-PCR+ROX Vial Kit (Qiagen) on the Rotor-Gene Q (Qiagen). RNA from the SUDV stock was extracted the same way and used alongside samples as standards with known TCIDso concentrations. SUDV titers in macaque EDTA blood and tissue samples were determined on VeroE6 cells (Mycoplasma negative) using a TCID₅₀ assay as previously described for EBOV (Marzi et al., Emerg Infect Dis 21(10): 1777-1783, 2015). Titers were calculated using the Reed-Muench method (Reed and Muench, Am J Hyg 27(3): 493-497, 1938).

Histology and immunohistochemistry

Necropsies and tissue sampling were performed according to IBC-approved SOPs. Collected tissues were fixed, processed, and stained as previously described (Furuyama et al., mBio 2022: e0337921, 2022). Specific anti-VP40 immunoreactivity was detected using a cross-reactive anti-EBOV VP40 at a 1: 1,000 dilution. All tissue slides were evaluated by a board-certified veterinary pathologist.

Assessment of humoral immune response

Previous studies using the VSV-vcctorcd filovirus vaccines have demonstrated the importance of antigen-specific IgG for protection in NHPs with rather limited contributions from T cell responses (Feldmann etal., Am J Trop Med Hyg 100(5): 1275-1277, 2019; Grant- Klein et al., Am J Trop Med Hyg 101(1): 207-213, 2019; Nakayama et al., Clin Vaccine Immunol 17(11): 1723-1728, 2010). Therefore, the analysis in the present studies was focused on the humoral immune responses. Post-challenge NHP sera were inactivated by y-irradiation (4 MRad), a well-established method with minimal impact on serum antibody binding (Feldmann et al.. Am J Trop Med Hyg 100(5): 1275-1277, 2019; Grant-Klein et al., Am J Trap Med Hyg 101(1): 207-213, 2019), and removed from the MCL according to SOPs approved by the RML IBC. Titers for IgG specific to EBOV GP or SUDV GP were determined in endpoint dilution ELISAs using recombinant EBOV GPATM (#0501-001; 1BT Bioservices, Rockville, MD) or recombinant SUDV GPATM (#0502-001; IBT Bioservices) as described previously (Nakayama et al., Clin Vaccine Immunol 17(11): 1723-1728, 2010). Levels of VSV-specific IgG were determined by endpoint dilution ELISA using concentrated VSV wildtype particles lysed with 0.01% triton-XlOO in PBS as antigen.

Neutralization of irradiated and heat-inactivated serum samples were assessed in Vero E6 cells. Briefly, cells were seeded in 96-well round-bottom plates for 24 hours. On the day of neutralization, serial dilutions of heat-inactivated serum samples were performed in DMEM supplemented with 2% FBS, penicillin/streptomycin, and L-glutamine. Each plate contained a negative serum control, cell-only control, and virus-only control. VSV-SUDV-GFP was added to each well of the serum dilution plate and the serumvirus mix was incubated at 37°C for 1-hour. The mix was added to the cells and incubated at 37°C for 24 hours. The cells were then fixed with 4% paraformaldehyde at room temperature for 15-minutes and centrifuged at 600 x g for 5-minutes at room temperature. The supernatant was discarded and FACS+EDTA buffer was added. Samples were run on the FACSymphony A5 Cell Analyzer (BD Biosciences, Mississauga, ON, Canada) and FITC MFI was measured. Data were analyzed using FlowJo V10. Gating strategy is shown in FIGS. 6A-6B.

Statistical analysis

Differences in the survival curves was assessed using log-rank analysis. Human immune responses were compared after vaccination and SUDV challenge at all timepoints between both groups by two-way ANOVA with Tukey’s multiple comparison to evaluate statistical significance. Results with /i<0.05 were considered statistically significant. Statistical analysis was done in Prism (version 9; GraphPad).

Example 2: Vector and study design

VSV-SUDV was generated according to previously published methods (Garbutt et al., J Virol 78(10): 5458-5465, 2004; Marzi et al., J Infect Dis 204 Suppl 3: S1066-S1074, 2011). The SUDV-Gulu GP open reading frame was inserted into the VSV backbone replacing the VSV G (FIG. 4A, top). SUDV GP expression in VSV-SUDV-infected cells was confirmed by immunoblot (FIG. 4B). For in vivo efficacy testing of VSV-SUDV, survivors of a previous VSV-EBOV vaccine study were used. The animals were originally vaccinated intramuscularly (IM) with a single dose of 1x10⁷ PFU of VSV-EBOV either 28, 21, 14, 7 or 3 days prior to EBOV-Makona strain (10,000 TCID₅₀) challenge. The study design is shown in FIG. 5 A, and the outcome was previously published (Marzi et al., Science 349(6249): 739-742, 2015). Eleven animals that were protected from EBOV challenge were repurposed for the current vaccine study. Of these, 9 animals were completely protected from disease and never showed EBOV viremia and 2 animals developed mild disease with low level of EBOV viremia (Marzi et al., Science 349(6249): 739-742, 2015). All 11 macaques were rested for ~9 months prior to the VSV-SUDV study start.

Example 3: VSV-SUDV protects macaques from SUDV associated clinical disease

Approximately one year after the initial VSV-EBOV vaccination, the macaques were divided into two groups. NHPs of one group (n=6) were IM vaccinated with 1x10⁷ PFU of VSV-SUDV and NHPs in the control group (n=5) were IM vaccinated with 1x10⁷ PFU of VSV-MARV. Vaccination with VSV-SUDV and VSV-MARV did not result in any obvious or noticeable adverse effects. After 28 days, all NHPs in both groups were challenged with 10,000 TCID₅₀ of SUDV-Gulu by the IM route (FIG. 5A). While none of the VSV-SUDV vaccinated macaques showed any signs of disease in response to challenge and were completely protected, 4 of 5 animals (80%) in the control group developed characteristic clinical signs of SVD and had to be euthanized according to the endpoint scoring criteria (FIGS. 1A-1B). Control NHPs succumbing to SUDV infection developed lymphocytopenia (FIG. 1C) and high-titer viremia. However, the one surviving control NHP while developing lymphocytopenia was only weakly positive 6 days postchallenge (DPC) for SUDV RNA but no virus isolation from whole blood was possible (FIGS. ID- IE). Survival of this control animal may he explained by the published observation that the cynomolgus macaque model for SVD is not uniformly lethal (Bennett et al., Curr Top Microbiol Immunol 411: 171-193, 2017). Compared to the protected NHPs, the 4 control NHPs that succumbed to infection presented with elevated aspartate transaminase (AST), alkaline phosphatase (AFP) and blood urea nitrogen (BUN) (FIGS. 1F-1H) levels. In addition, these NHPs had hypoalbuminemia (FIG. II). All of these clinical chemistry changes are consistent with clinical SVD (Woolsey et al., Emerg Microbes Infect 11(1): 1635-1646, 2022). The single surviving control NHP showed serum clinical chemistries similar to the protected VSV-SUDV-vaccinated NHPs.

Example 4: VSV-SUDV prevents macaques from developing SVD-associated pathology

At the time of euthanasia, the control macaques presented with liver and spleen pathology as previously described for SUDV infections (Woolsey et al., Emerg Microbes Infect 11(1): 1635-1646, 2022). Histologically, the control NHPs demonstrated liver lesions characteristic for SVD including multifocal to coalescing hepatocellular degeneration and necrosis with acute inflammation and abundant micro-fibrin thrombi (FIG. 2A). In the spleen, white pulp necrosis and loss with abundant fibrin effacing the red pulp were observed. Immunohistochemical evaluation demonstrated abundant viral antigen associated with these hepatic and splenic lesions (FIG. 2A). High SUDV titers were found in target tissues such as liver, spleen, adrenal glands, lymphoid tissues, urinary bladder and muscle at the injection site (FIG. 2B). Vaccinated NHPs and the single survivor in the control group were necropsied on 40 DPC. All collected tissue samples were essentially normal with no evidence of immunoreactivity in liver or spleen (FIG. 2A). Example 5: Pre-existing immunity to EBOV and VSV has limited impact on the development of SUDV GP- specific IgG

Previous studies using the VSV-vectored filo virus vaccines have demonstrated the importance of antigen-specific IgG for protection in NHPs with rather limited contributions from T cell responses (Marzi et al., Proc Natl Acad Set USA 110(5): 1893-1898, 2013; Menicucci et al., Sci Rep 7(1): 919, 2017; Marzi et al., Front Immunol 9: 3071, 2018). Therefore, studies focused on the humoral immune response; peripheral blood mononuclear cells (PBMC) for T cell responses were not collected in this study. Prior to VSV-SUDV or VSV-MARV vaccination, all animals still showed an EBOV GP-specific IgG titer of > 1: 10³ (FIG. 5B). The response was significantly boosted with the VSV-SUDV vaccination by more than a magnitude. In contrast, no boosting effect was noticed with the VSV-MARV vaccination (FIG. 3A). The SUDV challenge did not further boost the EBOV GP-specific IgG titers except for the single survivor in the control group which showed a steep booster effect peaking at 14 DPC indicative of an anamnestic response (FIG. 3A).

Investigation of the SUDV GP-specific IgG response revealed that all macaques showed limited cross-reactive antibodies from the previous VSV-EBOV vaccination and EBOV challenge prior to VSV- SUDV and VSV-MARV vaccination (FIG. 3B; FIG. 5B). Following VSV-SUDV vaccination, the SUDV GP-specific IgG peaked 14 days after vaccination (titer of > 1 : 10⁴) and was slightly boosted by the SUDV infection, reaching its highest level at 6 DPC (1 :25, 600-1 : 102,400) (FIG. 3B). The sole survivor among the control animals again showed a steep booster effect following SUDV challenge peaking at 14 DPC indicative of an anamnestic response (FIG. 3B). When the VSV-specific IgG was compared over the course of this experiment (68 days), peak titers were observed 1-2 weeks after vaccination without a significant difference between the VSV-SUDV study group and VSV-MARV control group (FIG. 3C). These titers stabilized by 0 DPC and remained constant throughout the study.

Analysis of neutralizing immune responses using a VS V-SUDV-GFP -based assay revealed limited neutralizing activity in all vaccinated NHPs at the time of challenge and at study end (FIG. 3D). The serum of the single surviving control NHP demonstrated only a small increase in neutralizing activity on 40 DPC despite the strong increase of SUDV GP-specific IgG responses after challenge.

TABLES

Table 1. Outbreaks of Ebola disease in Uganda

CFR = case fatality rate.

’Okware et al. (Trop Med hit Health 2002; 7(12): 1068-1075, 2002)

²MacNeil et al. (J Infect Dis 204 Suppl 3: S761-S767, 2011)

³Shoemaker et al. (Emerg Infect Dis 18(9): 1480-1483, 2012) ⁴Albarino et al. (Virology 442(2): 97-100, 2013)

*These 4 cases occurred during the 2018-2020 Ebola virus outbreak in The Democratic Republic of the Congo (DRC). They were attributed to cross-border movement from DRC and are accounted for in the DRC outbreak statistics.

VLPs, virus-like particles: GP, glycoprotein; VEEV, Venezuelan equine encephalitis virus; MV A, modified vaccinia Ankara; TAFV, Tai Forest virus; IM, intramuscular; IAVI, International AIDS Vaccine Initiative;

D, days; *Days after vaccinations were completed. Table 3. Base pair changes SUDV-Gulu RML stock versus GenBank reference

NP, nucleoprotein; VP40, virion protein 40; GP, glycoprotein; bp, base pair.

Example 6: VSV-based Sudan virus vaccine provides rapid protection against lethal challenge in macaques In this study, cynomolgus macaques were vaccinated with a single intramuscular (IM) dose of VSV-

SUDV either one month or one week prior to SUDV challenge. A third group was vaccinated with a single IM dose of VSV-EBOV one month prior to SUDV challenge to assess its cross-protective potential since SUDV and EBOV are related filoviruses. All vaccinated nonhuman primates (NHPs) developed antigenspecific IgG within 2 weeks of vaccination, including cross-reactive responses as demonstrated by ELISA. After IM challenge with a lethal dose of SUDV, all VS V-SUDV- vaccinated NHPs were uniformly protected from disease. In contrast, the VSV-EBOV-vaccinated and control NHPs succumbed to disease between day 5 and 7 after challenge presenting with classical signs of disease including fever, high titer viremia, thrombocytopenia, and elevated liver enzyme levels. These animals also presented with systemic viral spread and dysregulated cytokine profiles. While the SUDV challenge boosted the humoral response in the NHPs vaccinated with VS V-SUDV one month before challenge, this was not observed for the NHPs vaccinated one week before challenge. Taken together, these data demonstrated that VSV-EBOV provided no relevant protection against SUDV infection in NHPs, highlighting the need for species-specific vaccines against filoviruses. Furthermore, the data show that VSV-SUDV is a fast-acting single-dose vaccine, providing protection from lethal challenge in as little as one week, making this vaccine ideal for use in future outbreaks.

Materials and Methods

All work involving SUDV was performed in the maximum containment laboratory (MCL) at the Rocky Mountain Laboratories (RML), Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. RML is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited institution. All procedures followed RML Institutional Biosafety Committee (IBC)-approved standard operating procedures (SOPs). Animal work was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health, the Office of Animal Welfare and the Animal Welfare Act, United States Department of Agriculture. This study was approved by the RML Animal Care and Use Committee (ACUC), and all procedures were conducted on anesthetized animals by trained personnel under the supervision of board-certified clinical veterinarians. The animals were observed at least twice daily for clinical signs of disease according to a RML ACUC-approved scoring sheet and humanely euthanized when they reached endpoint criteria.

Animals were housed in adjoining individual primate cages that enabled social interactions, under controlled conditions of humidity, temperature, and light ( 12 hour light - dark cycles). Food and water were available ad libitum. Animals were monitored and fed commercial monkey chow, treats, and fruit at least twice a day by trained personnel. Environmental enrichment consisted of commercial toys, music, video and social interaction.

Cells and viruses

Vero E6 cells (Mycoplasma negative) were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma- Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS) (Wisent Inc., St. Bruno, Canada), 2 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin (all supplements from Thermo Fisher Scientific, Waltham, MA). THP-1 cells (Mycoplasma negative) were grown at 37°C and 5% CO in Roswell Park Memorial Institute (RPMI) medium (Sigma- Aldrich, St. Louis, MO) containing 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 50 mg/mL. Previously described VSV-based vaccine vectors expressing the EBOV-Kikwit GP (VSV-EBOV), SUDV-Gulu GP (VSV- SUDV), VSV-SUDV-GFP, or Lassa virus GPC (VSV-LASV) were used in this study. For the NHP challenge, SUDV-Gulu at 10,000 median tissue culture infectious dose (TDCID50) (backtitered as 11,247 TCID₅₀) was used IM as previously described (Marzi et al., Lancet Microbe 4:el71-178, 2023).

NHP study design

Twenty-four male cynomolgus macaques of Chinese or Cambodian origin, 2.5-4.5 years of age and 2.9-5.2 kg in weight at the time of vaccination, were used in this study. The NHPs were randomly divided into 4 study groups of 6 NHPs each. On -28 DPC, NHPs received a 1 ml intramuscular (IM) injection for vaccination into 2 sites in the caudal thighs containing either 1x10⁷ PFU VSV-SUDV (n=6), 1x10⁷ PFU VSV-EBOV (n=6) or 1x10⁷ PFU VSV-LASV (control; n=6). After vaccination, physical examinations and blood draws were performed on -27, -25, -21, and -14 DPC. On -7 DPC the last vaccine group received Ix lO⁷ PFU VSV-SUDV (n=6). For this group, physical examinations and blood draws were performed on - 6, and -4 DPC. All 24 NHPs were challenged IM on 0 DPC with IxlO⁴ TCID₅₀ SUDV-Gulu into 2 sites in the caudal thighs as previously described (Marzi et al., Lancet Microbe 4:el71-178, 2023). Physical examinations and blood draws were performed on 0, 3, 6, 9, 14, 21, 28, and 35 DPC and at euthanasia (42 DPC for survivors; humane endpoint for non-survivors). Following euthanasia, a necropsy was performed, and tissue samples were collected for analysis.

Hematology and serum chemistries

Complete blood cell counts were determined from EDTA blood with the IDEXX ProCyte DX analyzer (IDEXX Laboratories, Westbrook, ME). Serum biochemistry was analyzed with a Vetscan 2 using Preventive care profile disks (Abaxis, Union City, CA).

VSV and SUDV RNA

VSV RNA copy numbers in EDTA blood samples after vaccination were determined as previously described (Bushmaker et al. , J Infect Dis jiad280, 2023). SUDV RNA copy numbers in EDTA blood and tissue samples after challenge were determined as previously described (Marzi et al., Lancet Microbe 4:el71-178, 2023).

SUDV sGP ELISA

The SUDV sGP-specific sandwich ELISA was performed as previously described (Furuyama et al., Microorganisms 8(10): 1535, 2020) with minor differences. SUDV sGP in serum and homogenized, cleared, gamma-irradiated lung samples at 1:100 dilution was captured using 1 Llg/ml polyclonal rabbit anti-SUDV sGP antibody (cat. # 0302-030, IBT Bioservices). Dilutions of recombinant SUDV sGP (cat. # 0570-001, IBT Bioservices) at known concentrations served as standards.

Assessment of humoral immune response

Post-challenge NHP sera were inactivated by y-irradiation (4 MRad), a well-established method with minimal impact on serum antibody binding, and removed from the MCL according to SOPs approved by the RML IBC. Titers for IgG specific to EBOV GP or SUDV GP were determined in serum samples using ELISA kits following the manufacturer’s instructions (Alpha Diagnostics, San Antonio, TX). Serum samples collected -28 to 0 DPC were diluted 1:200; samples collected 3 DPC and later in the study were assessed at 1: 1,000 dilution.

Neutralization assays with VSV-SUDV-GFP were performed as previously described (Marzi etal., Lancet Microbe 4:el71-178, 2023). The VSV-SUDV-GFP assay was optimized, and incubation of serum dilution mix on the Vero E6 cells lasted for 16 hours at 37°C. Samples were run on the FACSYMPHONY™ A5 Cell Analyzer (BD Biosciences, Mississauga, ON, Canada) and the GFP-positive cell count was determined.

Cellular Phenotyping Assays

PBMCs were isolated from whole blood samples, stored, and revived as previously described (O’Donnell etal., eBioMed 89:104463, 2023). For T cell response analysis, PBMCs were stimulated with 1.5 Llg/ml of either a SUDV GP peptide pool, media alone, or a SARS-CoV-2 nucleocapsid peptide pool as an unspecific control for 16 hours. Initial peptide pool activation media was then removed and 1.5 Llg/ml of the SUDV GP peptide pool, media alone, or the SARS-CoV-2 nucleocapsid peptide pool together with 2.5|lg/ml Brefeldin A (Biolegend) cell stimulation cocktail (containing PMA-Ionomycin, Biolegend) were added to the cells for 5 hours. Cells were then surface stained and analyzed as previously described (O’Donnell et al., eBioMed 89:104463, 2023). NK cell immune responses were measured as previously described (O’Donnell et al., eBioMed 89:104463, 2023).

Quantification of Antibody Effector Functions

Assays for antibody effector functions were adapted from previously established protocols (Lewis et al., Front Immunol 10:1025, 2019; O’Donnell et al., EBioMedicine 89: 104463, 2023). Recombinant SUDV- GP (IBT Bioservices) was tethered to Fluospheres NutrAvidin-Microspheres yellow-green or red (Thermo Fisher Scientific, Waltham, MA) using the EZ-link Micro Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher Scientific) and used in ADCD and ADCP assays as previously described (O’Donnell et al., eBioMed 89:104463, 2023). Cytokine and chemokine analysis

Cleared lung homogenates and serum samples were inactivated by y-irradiation. Levels of GM-CSF, IFN-y, IFN-a2a, IL-ip, IL-4, IL-6, IL-10, IL-12p70, IL-15, IP- 10, MCP-1, and TNF-a were assessed using a customized Meso Scale Discovery (MSD; Rockville, MD, USA) U-PLEX NHP multiplex assay according to the manufacturer’s instructions. Samples were read on the MSD MESO QuickPlex SQ 120MM with Methodical Mind software (v. 1.0.38) and analyzed with MSD Discovery Workbench software (v. 4.0).

Statistical analysis

Statistical analysis was performed in Prism 9 (GraphPad). Data from the VSV-EBOV group on 5 DPC were combined for statistical analysis only with the single 6 DPC data point and compared to all other groups on 6 DPC. Most data were analyzed by two-way ANOVA with Tukey’s multiple comparison to evaluate statistical significance at all timepoints between all groups. Data depicted in FIGS. 14A-14B was analyzed by Kruskal- Wallis test with Dunn’s multiple comparisons. Data in FIG. 14C was analyzed by Mann- Whitney test. Significant differences in the survival curves shown in FIG. 7C were determined performing Log-Rank analysis. Statistical significance is indicated as p<0.0001 (****), p<0.001 (***), p<0.01 (**), and * p<0.05.

Results

Survival and clinical changes in NHPs after SUDV challenge were evaluated and the results are shown in FIGS. 7A-7I. NHPs (n=6 per group) were vaccinated 28 days prior to challenge (d-28) with either VSV-SUDV, VSV-EBOV or a control vaccine (VSV-LASV). Another group was vaccinated with VSV- SUDV seven days prior to challenge (d-7). On day 0, all 24 NHPs were challenged with a lethal dose of SUDV. At various timepoints following vaccination, the following were measured: VSV RNA in the blood (FIG. 7A), clinical scores (FIG. 7B), survival (FIG. 7C), and viremia by RT-qPCR (FIG. 7D). In addition, SUDV sGP levels in serum after challenge are shown in FIG. 7E and platelet count in whole blood samples after challenge are shown in FIG. 7F. At various timepoints after infection, serum levels of aspartate aminotransferase (AST; FIG. 7G), blood urea nitrogen (BUN; FIG. 7H) and calcium (FIG. 71) were also determined. The results show that NHPs vaccinated with VSV-SUDV at either d-28 or d-7 maintained low clinical scores and survived challenge with a lethal dose of SUDV in contrast to control and VSV-EBOV- vaccinated NHPs. VSV-SUDV vaccination also resulted in significantly lower levels of SUDV RNA and soluble GP (sGP) compared to VSV-EBOV and control vaccinated animals. Additionally, VSV-SUDV vaccinated NHPs maintained normal levels of platelets, AST, BUN and calcium.

Next, humoral immune responses after vaccination and SUDV challenge were evaluated. SUDV GP-specific serum IgG levels were measured from day -28 to day 42 post-infection. The results show that vaccination with VSV-SUDV at either d-28 or d-7 resulted in greater levels of SUDV GP-specific IgG in NHPs compared to NHPs vaccinated with VSV-EBOV or a control vaccine (FIG. 8A). However, vaccination with VSV-SUDV at d-28 resulted in significantly greater SUDV GP-specific IgG than vaccination at d-7. Serum neutralization titers were also evaluated, and the results are shown in FIG. 8B. Serum neutralization is presented as 50% fluorescence reduction (FRNT50) of GFP-positive cells at the time of vaccination (day -28 or day -7), challenge (day 0) and study end (day 42). The results show that vaccination with VSV-SUDV at d-28 resulted in the highest serum neutralization titers.

In addition to the total antigen-specific humoral response and the neutralization capacity of that response, alternative Fc functions are induced. Antibody-dependent complement deposition (ADCD) and antibody -dependent cellular phagocytosis (ADCP) were assessed. NHPs vaccinated with VSV-SUDV at either time point demonstrated significantly more ADCP at the time of challenge which continued to the end point of the control NHPs. The magnitude of ADCP increased on days 14 and 28 indicating the maturation of the humoral response upon antigenic boost of the challenge virus. In contrast, ADCD was significantly different at the time of euthanasia of control NHPs compared to the vaccinated NHPs indicating a potential pathogenic role of complement activation.

Cytokine responses in NHPs after challenge were also assessed. Expression levels of GM-CSF, MCP-1, IL-10, IL-12p70, IFN-a2a, IFN-Υ, IL-4, IL-6, IL-15, IP-10, IL-1 β, and TNF-a were measured from 0 to 14 days after SUDV-challenge (FIGS. 9A-9B). Only the VSV-EBOV and control NHPs developed elevated cytokine levels indicative of a cytokine storm, a hallmark of SVD. The VSV-SUDV vaccinated NHPs maintained normal cytokine levels throughout the observation period

Additional tests were performed to evaluate changes in blood and serum parameters in NHPs after SUDV challenge. Shown in FIGS. 10A-10I are levels of white blood cells, neutrophils, lymphocytes, potassium (K+), ALP, ALT, albumin, total protein, and creatinine, respectively, measured from 0 to 14 days post-challenge. The VSV-EBOV group developed increases in WBCs and neutrophils on day 3 together with a decrease of lymphocytes, possibly contributing to the early euthanasia time points on day 5 and 6. The control NHPs did not develop this immune cell signature but were also euthanized on day 6 and 7. In addition, serum chemistry analysis for both groups revealed elevated enzyme levels indicative of liver and kidney damage. VSV-SUDV vaccination resulted in limited changes in all of these parameters throughout the observation period indicating protection from disease.

Viral load of SUDV after challenge in select NHP tissues (muscle, inguinal lymph nodes, axillary lymph nodes, lung, mediastinal lymph nodes, liver, spleen, adrenal gland and mesenteric lymph nodes) was quantified by measuring SUDV RNA collected at the time of necropsy. NHPs vaccination with VSV- SUDV showed significantly lower viral loads compared to VSV-EBOV and control vaccination animals (FIG. 11 A). EBOV GP-specific IgG levels were also determined in the serum of animals vaccinated with VSV-SUDV, VSV-EBOV and control vaccine 28 days prior to SUDV challenge. As shown in FIG. 11B, VSV-EBOV vaccinated animals had the highest levels of EBOV GP-specific IgG, followed by animals vaccinated with VSV-SUDV.

CD4 T cell responses after vaccination and challenge were also evaluated. PBMCs were stimulated with a SUDV GP-specific peptide pool and analyzed for CD4+ EM-RE T cells. Levels of CD69, IFN-y and IL-4 expression were analyzed at the time of vaccination (FIG. 12A), 14 days before challenge (FIG. 12B), at the time of challenge (FIG. 12C), 14 days post-challenge (FIG. 12D), 28 days post-challenge (FIG. 12E), and 42 days post-challenge (FIG. 12F). No significant differences were observed between the groups. However, upon further investigation, a shift was observed in the T cell polarity of the antigen-specific CD4⁺ EM-RE cells. Initially, this memory cell cohort was primarily Thl-driven in nature expressing IFNy. As the cellular immunity memory matured, the CD4⁺ EM-RE cells shifted to a balanced Thl/Th2 expressing either IFNy or IL-4. This is important in maintaining the humoral response as IL-4 activates mature B cells for antibody secretion and promotes the survival of B cells (Granato et al. , J Immunol 192( 12):5761 -5775, 2014; Illera etal., J Immunol 151 (61:2965-2973, 1993). This balance during maturation contributes to a robust vaccination response, in both direct antiviral effector functions of EM-RE Thl CD4⁺ T cells as well as the support of the humoral response via the Th2 EM-RE CD4⁺ T cell.

Levels of cytokines and chemokines in the serum of NHPs after vaccination were also tested. Expression levels of selected cytokines (GM-CSF, MCP-1, IL-10, IL-12p70, IFN-a2a, IFN-y, IL-4, IL-6, IL-15, IP-10, IL-ip, TNF-a) were determined 0 to 7 days after vaccination (FIGS. 13A-13B). All NHPs responded to the VSV vaccinations with an increase of cytokine expression one day after vaccination, however, the levels returned to normal values within one week.

As significant lung disease in NHPs succumbing to SVD was observed, expression levels of GM- CSF, IFN-y, IL-6, IL-4, IP-10, MCP-1, TNF-a, IL-ip, IL-10, IL-12p70, IL-15, and IFNa2a, as well as the amount of SUDV sGP, were determined in lung samples of NHPs collected at the time of necropsy (FIGS. 14A-14C). Only the NHPs in the VSV-EBOV and control groups had significantly higher levels of cytokine expression in the lung as they succumbed to acute disease. Similarly, SUDV replication in the lungs resulted in SUDV sGP levels only in NHPs euthanized during the acute disease phase (VSV-EBOV and control groups). All VSV-SUDV-vaccinated NHPs presented with normal cytokine levels.

Taken together, the data described herein demonstrates that a single vaccination with VSV-SUDV is 100% protective against SUDV challenge in the cynomolgus macaque model. The data further show that pre-existing EBOV immunity does not affect the protective efficacy of VSV-SUDV against SUDV challenge, and pre-existing EBOV immunity does not protect against SUDV challenge despite cross-reactive immune responses. Moreover, the studies disclosed herein demonstrate that the VSV-SUDV vaccine provides 100% protection from lethal doses of SUDV even when vaccination occurs only seven days prior to challenge. A fast-acting single-dose SUDV vaccine is highly advantageous for quelling an ongoing SUDV outbreak. All other SUDV vaccines in development are administered at least 28 days prior to challenge (see Table 2). NHPs vaccinated with VSV-SUDV either 7 days or 28 days prior to challenge exhibited normal cytokine levels and low viral loads compared to control and VSV-EBOV vaccinated animals. Taken together, the data described herein demonstrates that VSV-SUDV is an extremely effective and fast-acting singlc-dosc vaccine ideal for use in future outbreaks. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A recombinant vesicular stomatitis virus-Sudan virus (VSV-SUDV), wherein the genome of the recombinant VSV-SUDV comprises: a VSV nucleocapsid protein (N) gene, a VSV phosphoprotein (P) gene, a VSV matrix protein (M) gene and a VSV polymerase protein (L) gene; and a SUDV glycoprotein (GP) gene.

2. The recombinant VSV-SUDV of claim 1, wherein the SUDV GP gene is inserted between the VSV M and L genes.

3. The recombinant VSV-SUDV of claim 1 or claim 2, wherein the SUDV GP gene comprises a nucleic acid sequence at least 95% identical to SEQ ID NO: 2.

4. The recombinant VSV-SUDV of any one of claims 1-3, wherein the SUDV GP gene comprises the nucleic acid sequence of SEQ ID NO: 2.

5. The recombinant VSV-SUDV of any one of claims 1 -4, wherein the recombinant VSV- SUDV expresses a SUDV GP having an amino acid sequence at least 95% identical to SEQ ID NO: 3.

6. The recombinant VSV-SUDV of any one of claims 1-5, wherein the recombinant VSV- SUDV expresses a SUDV GP having an amino acid sequence comprising SEQ ID NO: 3.

7. The recombinant VSV-SUDV of any one of claims 1-6 encoded by a nucleic acid sequence at least 95% identical to SEQ ID NO: 1.

8. The recombinant VSV-SUDV of any one of claims 1-7 encoded by a nucleic acid sequence comprising SEQ ID NO: 1.

9. An immunogenic composition comprising a pharmaceutically acceptable carrier and the recombinant VSV-SUDV of any one of claims 1-8.

10. The immunogenic composition of claim 9, further comprising an adjuvant.

11. The immunogenic composition of any one of claims 9-10, wherein the composition is formulated for intramuscular administration or intranasal administration.

12. A method of eliciting an immune response against SUDV in a subject, comprising administering to the subject a therapeutically effective or a prophylactically effective amount of the recombinant VSV-SUDV of any one of claims 1-8 or the immunogenic composition of any one of claims 9- 11.

13. A method of immunizing a subject against SUDV, comprising administering to the subject a prophylactically effective amount of the recombinant VSV-SUDV of any one of claims 1-8 or the immunogenic composition of any one of claims 9-11.

14. The method of claim 12 or claim 13, wherein the subject is human.

15. The method of any one of claims 12-14, wherein the recombinant VSV-SUDV or immunogenic composition is administered intramuscularly or intranasally.

16. The method of anyone of claims 12-15, wherein the subject has been previously vaccinated with an Ebola virus (EBOV) vaccine.

17. The method of any one of claims 12-16, wherein the subject has been previously infected with EBOV.

18. The method of any one of claims 12-17, wherein the recombinant VSV-SUDV or the immunogenic composition is administered in a single dose.

19. A nucleic acid molecule encoding the recombinant VSV-SUDV of any one of claims 1-8.

20. The nucleic acid molecule of claim 19, comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1.

21. The nucleic acid molecule of claim 19 or claim 20, comprising the nucleic acid sequence of SEQ ID NO: 1.

22. An immunogenic composition comprising the nucleic acid molecule of any one of claims 19-21 and a pharmaceutically acceptable carrier.

23. The immunogenic composition of claim 22, further comprising an adjuvant.

24. A kit for immunizing a subject against Sudan virus (SUDV), comprising: the recombinant VSV-SUDV of any one of claims 1-8, the immunogenic composition of any one of claims 9-11, 22 and 23, or the nucleic acid molecule of any one of claims 19-21.

25. The kit of claim 23, further comprising: equipment for administering the recombinant VSV-SUDV, immunogenic composition or nucleic acid molecule; an adjuvant; and/or instructions.

26. The kit of claim 25, wherein the equipment comprises a sterile needle, a sterile syringe, or both.