Vesicular stomatitis virus marburg virus vaccine

Abstract

The invention relates to a vesicular stomatitis virus vaccine vector (rVSV [delta] G-MARV-GP) for coding MARV glycoprotein. Vaccination using as few as 200 plaque-forming units may be 100% effective in preventing MARV lethal and preventing viremia occurrence. The rVSV [delta] G-MARV-GP vaccination induces serum IgG specific to MARV GP, and the virus neutralizing activity in the serum can be detected in animals vaccinated with the highest dose.

Description

[0001] Related Applications and Incorporation by Reference

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 374,408, filed on September 2, 2022.

[0003] The aforementioned applications and all documents cited therein or cited during prosecution ("application cited documents") and all documents cited or referenced herein ("herein cited documents") and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any documents incorporated by reference herein, are incorporated herein by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Federal Funding Description

[0005] This invention was made with government support under Grant No. MCDC-18-06-17-001 provided by the Defense Threat Reduction Agency (DTRA) through the Medical CBRN Defense Consortium and Grant No. UC7AI094660 provided by the Department of Health and Human Services, National Institutes of Health. The government has certain rights in this invention. Field of the Invention

[0006] The present invention relates to a vesicular stomatitis virus vaccine vector (rVSVΔG-MARV-GP) encoding MARV glycoprotein. Background of the Invention

[0008] Filoviruses are a major threat to global health and continue to affect health security and geopolitical stability in Central and West Africa. The Filoviridae family consists of the genera Ebolavirus and Marburgvirus. The Ebolavirus genus includes Zaire ebolavirus and six other species, while the Marburgvirus genus contains a single species, Marburg marburgvirus. https: / / ictv.global / taxonomy]. In 2014-2016, a large-scale outbreak of Ebola virus (EBOV) occurred in West Africa, followed by worrying outbreaks in the Democratic Republic of the Congo and Guinea [Sun, J. et al., Ann Med Surg (Lond), 2022.79: p. 103958. EBOV recurrence-related events in non-endemic areas of West Africa and recent zoonotic transmission in endemic areas have led to outbreaks [Keita, AK et al., Nature, 2021.597(7877): p. 539-543 and WHO. Ebolavirus disease–Democratic Republic of the Cong o. 2022 [cited on July 8, 2022]; available from: https: / / www.who.int / emergencies / disease-outbreak-news / item / 2022-DON377 ]. Other filoviruses remain endemic in animal hosts throughout Africa, including Marburg virus (MARV) and other members of the Ebolavirus genus, particularly Sudan virus (SUDV), which causes fatal hemorrhagic fever in humans and has epidemic potential similar to EBOV [Munster, VJ et al., N Engl J Med, 2018.379(13): p. 1198-1201]. Modeling suggests that the geographic area that could support MARV transmission is quite extensive, highlighting the risk of zoonotic transmission [Pigott, DM et al., Trans R Soc Trop Med Hyg, 2015.109(6): p. 366-78]. Additionally, transmission was first detected in a Guinean patient with no travel history in West Africa [Koundouno et al., NEJM, 2022. 386(26): pp. 2528-2530] and was recently detected in Ghana [WHO. Ghana reports first-ever suspected cases of Marburg virus disease. 2022 [cited July 12, 2022]; available from: https: / / www.afro.who.int / countries / ghana / news / ghana-reports-first-ever- suspected-cases-marburg-virus-disease ]. Outbreaks of filoviruses, including MARV, will continue to occur at an accelerated rate in the future, with factors such as climate change, increased intercontinental travel, population growth, and expansion of zoonotic host ranges increasing the likelihood of future disease transmission events [Carlson, CJ et al., Climate change increases cross-species viral transmission risk. Nature, 2022].

[0009] Vaccination against filoviruses, both in response to outbreaks and as a routine public health measure, has the potential to help further control health security threats in Africa. The vaccine against EBOV (rVSVΔG-ZEBOV-GP, produced by Merck Vaccines) is available as a The success of the first phase of the vesicular stomatitis virus (VSV) vaccine vector technology has provided a strong theoretical basis for efforts to develop other recombinant live attenuated vaccines based on vesicular stomatitis virus (VSV) vaccine vector technology [Tell, JG et al., Vaccines (Basel), 2020.8(4) and Wolfe, DN et al., Hum Vaccin Immunother, 2020.16(11): pp. 2855-2860]. The performance of ERVEBO in an outbreak setting clearly demonstrates that VSV-based technology has multiple features required for the development of other effective filovirus vaccines, including 1) acceptable safety and tolerability; 2) efficacy after a single dose; and 3) the ability to elicit rapidly developing protective immunity [Tell, JG et al., Vaccines (Basel), 2020.8(4), Wolfe, DN et al., Hum Vaccin Immunother, 2020.16(11): pp. 2855-2860, Wolf, J. et al., Development of Pandemic Vaccines: ERVEBO Case Study. Vaccines (Basel), 2021.9(3), Santoro, F. et al., Vaccines (Basel), 2021.9(2) and Pinski, AN and I. Messaoudi, To B or Not to B: Mechanisms of Protection Conferred by rVSV-EBOV-GP and the Roles of Innate and Adaptive Immunity. Microorganisms, 2020.8(10)].

[0010] In addition to the key rVSVΔG-ZEBOV-GP performance characteristics mentioned above, it is also important to consider factors that affect the access of filovirus vaccines to populations endemic to the virus in West and Central Africa. Since outbreaks of EBOV and other filoviruses (such as MARV or SUDV) cannot be accurately predicted, it is important to obtain vaccine materials that are safe for human use and can be quickly deployed [Wolf, J. et al., Development of Pandemic Vaccines:ERVEBO Case Study. Vaccines (Basel), 2021]. Filovirus vaccines must also be cost-effective, and therefore it is important to evaluate dose-saving conditions and efficacy after a single dose. Finally, due to the sporadic nature of filovirus outbreaks, traditional human efficacy trials are not feasible. Therefore, obtaining novel filovirus vaccines may require the use of existing alternative regulatory pathways, such as the US Food and Drug Administration (FDA) animal rule (https: / / www.fda.gov / emergency-preparedness-and-response / mcm-regulatory-science / animal-rule-information) and accelerated approval (https: / / www.fda.gov / drugs / information-health-care-professionals-drugs / accelerated-approval-program) and the generation of innovative data packages to demonstrate adequate safety, immunogenicity, and efficacy through preclinical animal studies and human clinical trials [Finch, CL et al., Vaccines (Basel), 2022.10(3)].In the case of VSV-based filovirus vaccines, this can be seen through the preclinical and clinical records of rVSVΔG-ZEBOV-GP [Tell, JG et al., Vaccines (Basel), 2020.8(4), Wolfe, DN et al., Hum Vaccin Immunother, 2020.16(11): pp. 2855-2860, and Wolf, J. et al., Development of Pandemic Vaccines: ERVEBO Case Study. Vaccines (Basel), 2021.9(3)] as well as the large number of previous preclinical studies on MARV and other filovirus vaccines based on rVSVΔG-ZEBOV-GP design [Dulin, N. et al., Vaccine, 2021.39(2): pp. 202-208, Geisbert, TW and H. Feldmann, J Infect Dis, 2011.204 Suppl 3: pages S1075-81 and Fathi, A. et al., Hum Vaccin Immunother, 2019.15(10): pages 2269-2285].

[0011] In response to the first identified human MARV case in West Africa, the World Health Organization (WHO) convened a group of filovirus experts, including infectious disease scientists, epidemiologists, public health experts, and vaccine developers, with the task of developing a research and development blueprint to strengthen the WHO Strategic Agenda for Filoviruses Research and Monitoring (AFIRM) [WHO. A WHO Strategic Agenda for Filovirus Research and Monitoring (AFIRM)-Roadmap Meeting. 2022 [cited 8 July 2022]; available from: https: / / www.who.int / news-room / events / detail / 2022 / 03 / 30 / default-calendar / save-the- date-a-who-strategic-agenda-for-filovirus-research-and-monitoring-(afirm)--- roadmap-meeting]. Central to the blueprint is an understanding of what experimental MARV vaccines are available and their development status, and what preclinical data are available to support their use in an outbreak setting. In addition, the blueprint will cover the development of clinical trial approaches that can be used during public health emergencies due to MARV outbreaks. In response to the emergence of MARV or other filovirus threats, it may be possible to implement ring vaccination early, a strategy in which those most likely to be infected receive immediate vaccination, and the availability of clinical trial materials (as was the case with Zaire Ebola) will help respond to future filovirus outbreaks [Dean, NE and I. M. Longini, Clin Trials, 2022: p. 17407745211073594]. Without the availability of vaccines prepared in accordance with Good Manufacturing Practices (GMP) and available for immediate use, the response to the 2014-2016 Zaire Ebola outbreak in West Africa would have been much slower and the consequences in terms of casualties and economic losses would have been more far-reaching.

[0012] Citation or identification of any document in this application does not constitute an admission that such document is available as prior art to the present invention. Summary of the invention

[0013] The present invention relates to a recombinant vaccine, which may include a nucleic acid sequence encoding a glycoprotein (GP) from the Musoke strain of Marburg virus (MARV) encoded in a VSV (VSVΔG) that does not include a glycoprotein G gene. In one embodiment, the nucleic acid may include a nucleic acid encoding an open reading frame of MARV GP, such as SEQ ID NO: 1. In another embodiment, the nucleic acid may include a nucleic acid encoding an open reading frame of MARV GP in VSVΔG, such as SEQ ID NO: 2.

[0014] The present invention also relates to a method of vaccinating a mammal in need thereof with a vaccine, which may comprise administering to the mammal about 10 2 -10 7 The administration may be about 10 4 PFU, about 10 5 PFU or about 10 6 PFU of vaccine.

[0015] In one embodiment of the present invention, the mammal may be a bat or a primate. The primate may be a bonobo, a chimpanzee, a gibbon, a gorilla, a monkey, an orangutan or a human. Advantageously, the primate is a human.

[0016] In another embodiment of the invention, administration may be intramuscular (IM), intranasal (IN) or by oral bait drops.

[0017] In another embodiment, the vaccines of the invention may be administered as part of a combination vaccine, such as with an Ebola virus vaccine or a Sudan virus vaccine.

[0018] Therefore, it is not the intention of the present invention to include any previously known product, process of making a product, or method of using a product within the scope of the present invention, and the applicant reserves the right and hereby discloses a disclaimer of any previously known product, process, or method. It should also be noted that the present invention is not intended to include any product, process of making a product, or method of using a product that does not meet the written description and feasibility requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Art. 83 of the EPC) within the scope of the present invention, and the applicant reserves the right and hereby discloses a disclaimer of any previously described product, process of making a product, or method of using a product. In the practice of the present invention, it may be advantageous to comply with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to expressly disclaim any embodiment of the subject matter of any one or more of the applicant's granted patents in this series of applications or any other series or any previously filed application of any third party are expressly reserved. Nothing herein shall be construed as a commitment.

[0019] It is worth noting that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises, comprised, comprising”, etc. may have the meanings assigned to them in U.S. patent law; for example, they may mean “includes, included, including”, etc.; and terms such as “consisting essentially of, consists essentially of”, etc. have the meanings assigned to them in U.S. patent law, for example, they allow elements not expressly recited but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention.

[0020] These and other embodiments are disclosed or apparent from the following detailed description and are encompassed therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following detailed description is given by way of example and is not intended to limit the invention to the particular embodiments described, which are best understood in conjunction with the accompanying drawings.

[0022] Figure 1. Schematic diagram showing the design of the rVSVΔG-MARV-GP investigational vaccine.

[0023] Figure 1A. Included at the top is a linear representation of the MARV RNA genome, showing that it encodes seven polypeptides (Abir, MH et al. (2022). "Pathogenicity and virulence of Marburg virus." Virulence 13(1):609-633). A schematic of the MARV virion is shown below the genome, with the transmembrane GP incorporated into the membrane envelope and exposed on the particle surface.

[0024] Figure 1B. Schematic diagram showing the VSV RNA genome map, which encodes five structural proteins, with the virion shown below (Lyles, DS et al. (2013). Rhabdoviridae. Fields Virology .DM Knipe and P.M. Howley. Philadelphia, Lippincott Williams and Wilkins. 1:885-922). The rVSVΔG-MARV-GP vaccine was developed by replacing the gene encoding VSV glycoprotein (G) with the gene encoding MARV GP from the Mosok isolate (Garbutt, M. et al. (2004)). "Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses." JVirol 78(10):5458-5465 and Jones, SM et al. (2005). "Liveattenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses." NatMed 11(7):786-790).

[0025] FIG. 1C . The VSVΔG-MARV-GP vaccine is a replication-competent chimeric virus in which MARV GP is incorporated on the surface of the virion.

[0026] Figure 2. Overview of the steps taken to regenerate the rVSVΔG-MARV-GP vaccine. RNA was extracted from samples of a research vaccine expressing GP from the MARV Mosok variant that had previously been shown to be effective (Garbutt, M. et al. (2004). "Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses." JVirol 78(10):5458-5465 and Jones, SM et al. (2005). "Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses." Nat Med 11(7):786-790) and was used to determine the genomic nucleotide sequence. The GP gene nucleotide sequence was then used to synthesize a novel GP gene and inserted into a VSV (Indiana serotype) genomic clone (Garbutt, M. et al. (2004). "Properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses." J Virol 78(10):5458-5465; Schnell, MJ et al. (1996). "The minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus." J Virol 70(4):2318-2323 and Lawson, ND et al. (1995). "Rec ombinant vesicular stomatitis viruses from DNA." Proc Natl Acad Sci USA 92(10):4477-4481) to generate genomic plasmid DNA, which can be used to regenerate recombinant virus under conditions that support the development of human vaccines.

[0027] Figure 3. Generation and characterization of the rVSVΔG-MARV-GP vaccine for use in humans.

[0028] Figure 3A Schematic diagram outlining the steps during rederivation of rVSVΔG-MARV-GP chimeric viruses suitable for human vaccine development. Figure 2 ), followed by 3 rounds of viral plaque isolation to develop clonal virus isolates. The pre-master virus seed (preMVS) was then amplified and characterized to support good manufacturing practice (GMP) manufacturing. The virus derived from preMVS was further evaluated by producing purified vaccine material for preclinical efficacy studies.

[0029] Figure 3B .Purified vaccine viruses used in preclinical efficacy studies were analyzed by nanoflow cytometry or flow virometry to assess the homogeneity of particles in the purified vaccine material [Ricci, G. et al., SciRep, 2021.11(1): p. 7432].

[0030] Figure 3C Nanoflow cytometry or flow virometry has also been used as an analytical tool during multiple stages of vaccine virus production, as shown in the analysis of virus produced in independent vaccine production runs. Nanoflow characterization of multiple rVSVΔG-MARV-GP production stages are shown, including: harvesting of medium from infected Vero cell culture (HM), clarification of the harvest (CH), purification and concentration using tangential flow filtration (TFF), followed by nuclease treatment (post-universal nuclease treatment; PBT), post-buffer exchange (BEP) by TFF, and final purified product (FP).

[0031] Figure 3D .Vaccine product characterization included analysis of MARV GP gene integrity by RT-PCR. The complete MARV-GP insert is expected to amplify a 2.9Kb band by primers that bind to the VSV M and L genes, as shown below the agarose gel image. Lanes: 1, 1Kb marker; 2, positive control VSVΔG-MARV-GP genomic plasmid; 3, VSV genomic plasmid DNA, in which the G gene is moved to the 5' end of the genome so that there is no transcription unit between M and L [Rabinovich, S. et al., PLoS One, 2014. 9(9): p. e106597]; 4, no template negative control; 5, HM; 6, no sample; 7, FP.

[0032] Figure 3E. Western blotting was used to analyze rVSVΔG-MARV-GP peptides from different vaccine production stages. Rabbit polyclonal antiserum was used to detect VSV N (Rabinovich, S. et al. (2014). "A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice." PLoS ONE 9(9):e106597) and MARV GP (IBT Bioservices). The anti-GP antiserum is specific for the GP2 subunit of MARV GP.

[0033] Figure 3F .Expression of MARV GP and VSV N during infection of Vero cells. Vero cells infected with three different purified batches of rVSVΔG-MARV-GP were analyzed by flow cytometry to detect the expression of MARV GP (monoclonal antibody 5C1; IBT Bioservices, Inc) on the cell surface and VSV N (monoclonal 10G4; Kerafast, Inc) within the cell. Red is characteristic of uninfected Vero cells, and blue is infected cells.

[0034] Figure 4A . Design of a preclinical dose-ranging efficacy study of VSVΔG-MARV-GP in cynomolgus monkeys. The upper portion of A illustrates the study schedule, and the table shows the study groups and VSVΔG-MARV-GP doses. A control VSVΔG-based Lassa virus vaccine (rVSVΔG-LASV-GPC; [Garbutt, M. et al., J Virol, 2004. 78(10): p. 5458-65 and Geisbert, TW et al., PLoS Med, 2005. 2(6): p. e183.]) was produced from amplified virus (data not shown).

[0035] Figure 4B .The graph shows the survival rate after challenge with the MARV Angola isolate.

[0036] Figure 4C Plaque assays were performed to assess viremia after MARV challenge. Infectious MARV was detected only in blood collected from control animals vaccinated with rVSVΔG-LASV-GPC.

[0037] Figure 5 MARV RNA detected in blood after MARV Angola challenge. RNA was extracted from whole blood and quantified by RT-qPCR. The MARV genome copy number in the sample was calculated using a genome equivalent standard. The detection limit was 1,000 copies / mL.

[0038] Figure 6. Characterization of antibody responses induced by rVSVΔG-MARV-GP vaccination.

[0039] Fig. 6A .Enzyme-linked immunosorbent assay (ELISA) was performed using plates coated with a soluble form of MARV GP from an Angolan isolate. Endpoint serum antibody titers are shown in the graph. The vaccine dose in PFU is included on the right side of the graph. The lower limit of detection was 100.

[0040] Figure 6B . Plaque reduction assay based on serum neutralization of rVSVΔG-MARV-GP (Mosok). Although similar, the VSV-based chimeric virus used for the neutralization assay was developed using a different VSV (Indiana) genomic clone and the GP (MARV Mosok) gene (which was optimized using VSV codon preference and previously described procedures) (Rabinovich, S. et al. (2014). "A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice." PLoS ONE 9(9): e106597 and Espeseth, AS et al. (2022). "Preclinical immunogenicity and efficacy of a candidate COVID-19 vaccine based on a vesicular stomatitis virus-SARS-CoV-2 chimera." EBioMedicine 82: 104203). The serum dilution that reduced the number of rVSVΔG-MARV-GP plaques by 50% (neutralization titer 50 or NT50) is plotted. The lower limit of detection was 20. DETAILED DESCRIPTION

[0041] A new ERVEBO-based TMThe MARV vaccine candidate (rVSVΔG-MARV-GP) based on recombinant VSV technology used is for human use. The rVSVΔG-MARV-GP investigational vaccine has been shown to be safe and effective in multiple preclinical studies. In order to advance rVSVΔG-MARV-GP as a globally available vaccine candidate for human use, the applicant regenerated the recombinant vaccine strain using conditions that can support future human vaccine development and tested the immunogenicity and efficacy of the vaccine against MARV challenge at a range of doses in the cynomolgus macaque animal model of MARV disease. The rVSVΔG-MARV-GP vaccine is 100% effective against Marburg disease after a single IM injection and protects against the development of MARV viremia, even with doses as low as 200 PFU. Vaccination with rVSVΔG-MARV-GP induces MARV GP-specific humoral responses that can be further studied to better understand the correlates of protection, and this data will provide an important bridge for future human safety and immunogenicity studies.

[0042] The present invention relates to a recombinant MARV vaccine encoding a MARV protein or a non-natural mutant thereof. Advantageously, the MARV protein is a MARV glycoprotein or a non-naturally occurring mutant thereof.

[0043] Marburg virus is one of two members of the Marburg virus species, belonging to the order Monoretrovirales, family Filoviridae, genus Marburgvirus. The Marburg virus body consists of seven structural proteins. The center is a helical ribonucleocapsid, which consists of genomic RNA wrapped around a nucleoprotein (NP) polymer. Associated with the ribonucleoprotein is an RNA-dependent RNA polymerase (L) as well as a polymerase cofactor (VP35) and a transcription activator (VP30). The ribonucleoprotein is embedded in a matrix formed by a major matrix protein (VP40) and a minor matrix protein (VP24). These particles are surrounded by a lipid membrane from the host cell membrane. A glycoprotein (GP1,2) is anchored to the membrane, which extends from the membrane surface with a spike of 7 to 10 nm. Any structural protein can be considered for use in vaccines. Advantageously, the structural protein considered for use in vaccines is a glycoprotein (GP).

[0044] Marburg virus (MARV) isolates include Angola, Musoke and Ozolin. Marburg virus Musoke (MARV-Mus) and Angola (MARV-Ang) have highly similar genome sequences. Advantageously, the strain is a MARV Musoke isolate.

[0045] The present invention encompasses eliciting an immune response, which may include systemically administering to an animal in need thereof an effective amount of any non-naturally occurring protein of the present invention or any nucleic acid encoding the non-naturally occurring protein, including nucleic acids that may have at least 80% or 85% or 90% or 95% homology or identity with the nucleotide sequence of the non-naturally occurring protein of the present invention. The animal may be a mammal, advantageously a primate, advantageously a human.

[0046] The present invention relates to the identification, design, synthesis and isolation of the MARV protein disclosed herein and the nucleic acid encoding the MARV protein. The present invention also relates to homologs, derivatives and variants of the MARV protein sequence and the nucleic acid sequence encoding the MARV protein, wherein preferably, the homolog, derivative or variant has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the MARV protein sequence or the nucleic acid sequence encoding the MARV protein. It is worth noting that in the present specification, the homology with the sequence of the mutant protein and the nucleic acid encoding it refers to the homology of the binding site of the homolog, derivative or variant with the mutant protein and the nucleic acid encoding it.

[0047] The present invention further relates to nucleic acid sequences expressing the MARV proteins disclosed herein, or homologues, variants or derivatives thereof. Those skilled in the art will know, appreciate and understand the techniques for creating such materials. In addition, those skilled in the art will be able to incorporate such nucleic acid sequences into appropriate vectors, thereby allowing the production of the amino acid sequence of the mutant protein and the nucleic acid encoding the mutant protein, or homologues, variants or derivatives thereof.

[0048] As used herein and unless expressly stated otherwise, the following terms are intended to have the following meanings, in addition to any broader (or narrower) meanings they may have in the art:

[0049] The terms "isolated" or "non-naturally occurring" are used herein to indicate that the isolated portion (e.g., a peptide or compound) is present in a physical environment that is different from the physical environment in which it occurs in nature. For example, an isolated peptide can be substantially isolated relative to the complex cellular environment in which it occurs in nature. The absolute level of purity is not important, and one skilled in the art can readily determine the appropriate level of purity based on the intended use of the peptide. The term "isolated" when used in reference to a step in a process should be interpreted accordingly.

[0050] In many cases, the isolated fraction will form part of a composition (e.g. a more or less crude extract containing numerous other molecules and substances), a buffer system, a matrix or an excipient, which may, for example, contain other components (including proteins such as albumin).

[0051] In other cases, the isolated fractions may be purified to substantial homogeneity, e.g., as determined by PAGE or column chromatography (e.g., HPLC or mass spectrometry). In preferred embodiments, the isolated peptides or nucleic acids of the invention are substantially the only peptides or nucleic acids in a given composition.

[0052] In an advantageous embodiment, the tag can be used for purification or biotinylation. The tag used for purification can be a his tag. In another embodiment, the tag used for biotinylation can be an avi-tag. Other tags are also contemplated for purification, but purification can also be accomplished without using a tag. In another embodiment, an antibody (such as, but not limited to, a broadly neutralizing antibody) affinity column is contemplated. In another embodiment, a lectin column is contemplated.

[0053] The term "pharmaceutical composition" is used herein to define a solid or liquid composition having a form, concentration and purity level suitable for administration to a patient (e.g., a human patient), which can cause the desired physiological changes after administration. The terms "immunogenic composition" and "immunological composition" and "immunogenic or immunological composition" encompass any composition that induces an immune response to the targeted pathogen Marburg virus. Terms such as "vaccinal composition" and "vaccine" and "vaccine composition" encompass any composition that induces a protective immune response to a targeted pathogen or effectively prevents a pathogen; for example, after administration or injection, a protective immune response to a targeted pathogen is induced or effective protection against the pathogen is provided. Therefore, an immunogenic or immunological composition induces an immune response that can be, but is not necessarily, a protective immune response. Immunogenic or immunological compositions can be used to treat individuals infected by pathogens, for example, to stimulate an immune response to the pathogen, such as by stimulating antibodies to the pathogen. Therefore, an immunogenic or immunological composition can be a pharmaceutical composition. In addition, when the text refers to "immunogen, antigen or epitope", the immunogen can be an antigen or an epitope of an antigen. A diagnostic composition is a composition containing a compound or an antibody (such as a labeled compound or antibody) for detecting the presence of an antibody that binds to the compound or an immunogen, antigen or epitope that binds to the antibody in a sample (such as a biological sample, such as blood, semen, vaginal fluid, etc.); for example, an anti-MARV antibody or a MARV immunogen, antigen or epitope.

[0054] A "conservative amino acid change" is an amino acid change in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged amino acids or polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0055] The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or a biologically active component.

[0056] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, usually a protein, that is capable of inducing an immune response in a subject. The term also refers to an immunologically active protein insofar as it is capable of eliciting a humoral and / or cellular type immune response against the protein upon administration to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to the subject).

[0057] As used herein, the terms "nucleotide sequence" and "nucleic acid sequence" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including but not limited to messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. Nucleic acids can be single-stranded or partially or completely double-stranded (duplex). Duplex nucleic acids can be homoduplexes or heteroduplexes.

[0058] As used herein, the term "transgenic" may be used to refer to a "recombinant" nucleotide sequence that may be derived from any nucleotide sequence encoding a protein of the present invention. The term "recombinant" means a nucleotide sequence that has been "artificially" manipulated and does not exist in nature, or is linked to another nucleotide sequence or exists in a different arrangement in nature. It should be understood that "artificial" manipulation means manipulation by some artificial means, including the use of machines, codon optimization, restriction enzymes, etc.

[0059] For example, in one embodiment, the nucleotide sequence can be mutated so that the activity of the encoded protein in vivo is eliminated. In another embodiment, the nucleotide sequence can be codon optimized, for example, codons can be optimized for human use. In a preferred embodiment, the nucleotide sequence of the present invention is both mutated to eliminate the normal in vivo function of the encoded protein and codon optimized for human use. For example, each sequence of the present invention (such as the MARV protein) can be altered in these ways.

[0060] As for codon optimization, the nucleic acid molecule of the present invention has a nucleotide sequence encoding the antigen of the present invention and can be designed to adopt the codon used in the gene of the subject to be produced antigen. Many viruses use a large number of rare codons, and by changing these codons to correspond to the codons commonly used in the expected subject, enhanced antigen expression can be achieved. In a preferred embodiment, the codons used are "humanized" codons, that is, the codons are those codons that frequently occur in highly expressed human genes (Andre et al., J.Virol.72:1497-1503,1998), rather than those codons frequently used by MARV. The use of such codons provides effective expression of transgenic MARV proteins in human cells. Any suitable codon optimization method can be used. Such methods and the selection of such methods are well known to those skilled in the art. In addition, there are several companies that optimize the codons of the sequence, such as Geneart (geneart.com). Therefore, the nucleotide sequence of the present invention can be easily codon optimized.

[0061] The present invention also encompasses nucleotide sequences encoding functional equivalent variants of the antigen of the present invention and / or antigen equivalent variants and derivatives and functional equivalent fragments thereof. These functionally equivalent variants, derivatives and fragments show the ability to retain antigenic activity. For example, changes in the DNA sequence that do not change the encoded amino acid sequence, and those changes that result in conservative substitutions of amino acid residues, deletions or additions of one or more amino acids, and substitutions of amino acid residues by amino acid analogs, are those changes that do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan. In one embodiment, the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest, or the nucleotide sequence encoding the same.

[0062] For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. Specifically, sequence identity can be determined using any of a variety of mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, as modified in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

[0063] Another example of a mathematical algorithm used for the comparison of sequences is the algorithm of Myers and Miller, CAB IOS 1988; 4: 11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm, as described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

[0064] The WU-BLAST (Washington University BLAST) version 2.0 software is advantageously used in accordance with the present invention.WU-BLAST version 2.0 executable programs are available for download for multiple UNIX platforms from ftp: / / blast.wustl.edu / blast / executables. This program is based on WU-BLAST version 1.4, which is in turn based on the public domain NCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266:460-480; Altschul et al., Journal of Molecular Biology 1990; 215:403-410; Gish and States, 1993; Nature Genetics 3:266-272; Karlin and Altschul, 1993; Proc. Natl. Acad. Sci. USA 90:5873-5877; all of which are incorporated herein by reference).

[0065] The various recombinant nucleotide sequences and immunogens of the present invention are made using standard recombinant DNA and cloning techniques. Such techniques are well known to those skilled in the art. See, for example, "Molecular Cloning: A Laboratory Manual," Second Edition (Sambrook et al., 1989).

[0066] The nucleotide sequence of the present invention can be inserted into a "vector". The term "vector" is widely used and understood by those skilled in the art, and as used herein, the term "vector" is used in accordance with the meaning understood by those skilled in the art. For example, the term "vector" is often used by those skilled in the art to refer to a medium that allows or facilitates the transfer of nucleic acid molecules from one environment to another or allows or facilitates the manipulation of nucleic acid molecules.

[0067] Any vector that allows expression of the immunogen of the invention can be used in accordance with the present invention. In certain embodiments, the immunogen of the invention can be used in vitro (such as using a cell-free expression system) and / or in cultured cells grown in vitro to produce an encoded immunogen, which can then be used in a variety of applications, such as the production of proteinaceous vaccines. For such applications, any vector that allows expression of the immunogen in vitro and / or in cultured cells can be used.

[0068] For applications where it is desired that immunogens be expressed in vivo, such as when the transgenics of the invention are used in DNA or DNA-containing vaccines, any vector that allows expression of the antibodies of the invention and is safe for use in vivo can be used. In a preferred embodiment, the vector used is safe for use in humans, mammals, and / or laboratory animals.

[0069] For the immunogen of the present invention to be expressed, the protein coding sequence should be "operably linked" to a regulatory sequence or nucleic acid control sequence that directs the transcription and translation of the protein. As used herein, a coding sequence is referred to as "operably linked" to a nucleic acid control sequence or promoter when the coding sequence is covalently linked in a manner such that the expression or transcription and / or translation of the coding sequence is affected or controlled by the nucleic acid control sequence. A "nucleic acid control sequence" can be any nucleic acid element, such as, but not limited to, a promoter, an enhancer, an internal ribosome entry site (IRES), an intron, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. For VSV, the gene may also be operably linked to an intergenic region that controls gene expression.

[0070] Vectors used in accordance with the invention should generally be selected such that they contain appropriate gene regulatory regions, such as promoters or intergenic regions, allowing expression of the immunogens of the invention.

[0071] Any suitable vector may be used depending on the application. For example, plasmids, viral vectors, bacterial vectors, protozoan vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, etc. may be used. A skilled person may select a suitable vector by considering the characteristics of the vector and the requirements for expressing the immunogen under identified circumstances.

[0072] In a preferred embodiment of the present invention, a viral vector is used. Viral expression vectors are well known to those skilled in the art and include viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpes viruses, retroviruses and poxviruses, including fowl poxviruses, attenuated poxviruses, vaccinia viruses and in particular modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when used as expression vectors, are essentially non-pathogenic to a selected subject (such as a human), or have been modified to render them non-pathogenic to a selected subject. For example, replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.

[0073] Advantageously, the vector is a vesicular stomatitis virus (VSV) vector.

[0074] VSV is a very practical, safe, and immunogenic vector for animal studies and is an attractive candidate for the development of vaccines for human use, such as the commercially available Ebola virus vaccine. VSV is an enveloped virus member of the Rhabdoviridae family that contains a non-segmented negative sense RNA genome. The genome consists of five genes arranged in a 3′-NPMGL-5′ sequence, each encoding a polypeptide present in the mature virion. Notably, the surface glycoprotein G is a transmembrane polypeptide that is present as a homotrimer in the viral envelope and, like MARV GP, mediates cell attachment and infection.

[0075] Advantageously, the VSV vector is replication-defective due to the deletion of the glycoprotein G gene (VSVΔG). In one embodiment, the VSV G gene is replaced by a gene encoding a MARV protein or a fragment thereof. In a second embodiment, VSV G is a carrier or scaffold for the MARV epitope. The disclosures of U.S. Pat. Nos. 9,610,346, 9,802,986, and 10,844,095 are incorporated by reference. In another embodiment, the MARV GP replacing G is functional.

[0076] The VSV vector may be replication-defective due to the missing glycoprotein G gene (VSVΔG). In one embodiment, VSV G is replaced by a gene encoding a MARV protein or a fragment thereof. The disclosures of U.S. Pat. Nos. 9,610,346, 9,802,986, and 10,844,095 are incorporated by reference. Also contemplated is the VSVΔG vector of Espeseth et al. (eBioMedicine 2022; 00: 104203, published online at https: / / doi.org / 10.1016 / j.ebiom.2022.104203 ).

[0077]

[0078]

[0079] For example, if the purpose is to express MARV antigens in cells so as to produce and separate expressed proteins, such as from cells grown in culture, the nucleotide sequences and vectors of the present invention can be delivered to cells. For expressing antigens in cells, any suitable transfection, transformation or gene delivery method can be used. Such methods are well known to those skilled in the art, and those skilled in the art can easily select suitable methods according to the properties of the nucleotide sequences, vectors and cell types used. For example, transfection, transformation, microinjection, infection, electroporation, lipofection or liposome-mediated delivery can be used. The expression of antigens can be carried out in any type of host cell, such as bacterial cells, yeast, insect cells and mammalian cells. The antibodies of the present invention can also be expressed using in vitro transcription / translation systems. All such methods are well known to those skilled in the art, and those skilled in the art can easily select suitable methods according to the properties of the nucleotide sequences, vectors and cell types used.

[0080] Alternatively, methods well known to those skilled in the art can be used to construct expression vectors containing nucleic acid molecules encoding polypeptides or their homologues or derivatives under appropriate transcription / translation control signals for expression. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., 1989.

[0081] The compound or composition can be administered orally, subcutaneously or parenterally (including intravenous, intraarterial, intramuscular (IM), intraperitoneal and intranasal administration), as well as intrathecal and infusion techniques. IM is preferred, but other routes, such as subcutaneous or applied to the mucosal surface of the nasal or oral cavity, may also be used.

[0082] In an advantageous embodiment, administration is IM. Dosage is measured in PFU. The present invention shows that low doses of the vaccine are as effective as high doses. Applicants have demonstrated that a single vaccination dose of 2x10 7 The rVSVΔG-MARV-GP vaccine with as low as 200 PFU can effectively protect cynomolgus monkeys from Marburg virus infection. The administration dose can be about 10 2 -10 7 Advantageously, the dosage may be about 10 4 , 10 5 , 10 6 PFUs.

[0083] Combination vaccines are also contemplated. The Marburg virus vaccine of the present invention can be combined with another vaccine, such as a vaccine against a Filoviridae virus. Ebola (EBOV), Sudan (SUDV) and Marburg (MARV) viruses are the three filamentous viruses that cause the most human deaths. It has been demonstrated that a combination of MARV or SUDV with an EBOV vaccine can be formulated to produce a bivalent vaccine that retains full efficacy (Lehrer et al., Front Immunol. 2021 Aug 18; 12:703986.doi:10.3389 / fimmu.2021.703986.eCollection 2021).

[0084] It is worth noting that humans may require higher vaccine doses than mice or other experimental animals to induce an effective immune response. The dose can be a single dose or multiple doses over a period of time, but a single dose is preferred. Therefore, the technology in this disclosure and the documents cited herein and the knowledge in the art can be extended from animal experiments (e.g., rats, mice, etc.) to humans without excessive experimentation.

[0085] In another embodiment, primate immunization is also contemplated. For non-human primate immunization, oral administration (such as via bait drops) is contemplated. Non-human primates that can be immunized by the vaccine of the present invention include, but are not limited to, chimpanzees and bonobos, gorillas, orangutans, gibbons, and monkeys. It is also contemplated that other animals (such as bats) carrying Marburg virus are immunized. In this case, bait drops are contemplated. In one embodiment, bait drops can include hollow plastic bags. In another embodiment, the composition can be inserted into hollow polymer cubes. For example, bait drops can include hollow fish meal polymer cubes (1.25 inches * 0.75 inches). A pouch or plastic bag containing the vaccine can be inserted into the hollow area of ​​the bait and sealed with wax.

[0086] When the therapeutic agent of the present invention is administered parenterally, it is usually formulated into a unit dose injectable form (solution, suspension, emulsion). Pharmaceutical preparations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injection solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and a vegetable oil.

[0087] In addition, various additives for enhancing the stability, sterility and isotonicity of the composition can also be added, including antibacterial preservatives, antioxidants, chelating agents and buffers. Various antibacterial agents and antifungal agents (such as parabens, chlorobutanol, sorbic acid, etc.) can be used to ensure that the effect of microorganisms is prevented. In many cases, it is also desirable to include isotonic agents, such as sugar, sodium chloride, etc. The absorption of the injectable drug form can be extended by using an agent that delays absorption, such as aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent or additive used must be compatible with the vaccine preparation. The preparation of stable viruses is also considered. Additives such as but not limited to carbohydrates (such as sucrose or trehalose), gelatin, hydrolyzed gelatin, amino acids, etc. are considered.

[0088] Sterile injectable solutions can be prepared by incorporating the compounds used to practice the invention in the required amount of an appropriately buffered solution along with varying amounts of the other ingredients as required.

[0089] For example, the pharmacological preparations of the present invention, which may contain the therapeutic compounds or polypeptides of the present invention, can be administered to patients in the form of injection preparations containing any compatible carriers (such as various vehicles, adjuvants, additives and diluents); alternatively, the compounds utilized in the present invention can be administered to patients parenterally in the form of polymer matrices, liposomes and microspheres.

[0090] The pharmacological preparations of the compounds and compositions that may contain the polypeptides used in the present invention can be administered orally to the patient. The compounds can be administered in the form of tablets, suspensions, solutions, emulsions, capsules, powders, syrups, etc. by conventional methods. Known techniques for delivering compounds orally or intravenously and retaining biological activity are preferred.

[0091] In one embodiment, the preparation of the present invention can be first administered and then maintained by further administration. For example, the preparation of the present invention can be administered in one type of composition and then further administered in a different or same type of composition. For example, the preparation of the present invention can be administered by intravenous injection to bring the blood level to a suitable level. The patient's level is then maintained by an oral dosage form, although other forms of administration may also be used depending on the patient's condition. In the case of a vaccine composition, the vaccine can be administered as a single dose, or the vaccine can be incorporated into a set booster dose.

[0092] The amount to be administered will vary depending on the patient being treated and whether the administration is for treatment or prevention, and will be about 10 2 to 10 7 The vaccine may be administered in an amount of about 10 plaque forming units (PFU). 4 PFU, about 10 5 PFU or about 10 6PFUs.

[0093] Of course, for any composition (including components thereof) to be administered to animals or humans and for any particular method of administration, it is therefore preferred to determine: toxicity, such as by determining the lethal dose (LD ) and LD in a suitable animal model (e.g., rodents such as mice). 50 ;And the dosage of one or more compositions that induce a suitable immune response, the concentration of the components therein and the time of applying the one or more compositions, such as by serum titration and analysis of its antibodies or antigens, such as by ELISA and / or Rapid Fluorescent Foci Inhibition Test (RFFIT) analysis. According to the knowledge of the technician, the disclosure and the documents cited herein, such determinations do not require excessive experiments. And, the time of sequential administration can be determined without excessive experiments. For example, those skilled in the art can easily determine the dosage according to the disclosure and the knowledge in the art. Therefore, those skilled in the art can easily determine the amount of the compound and optional additives, vehicles and / or carriers in the composition and to be applied in the method of the present invention. Typically, adjuvants or additives are typically used in 0.001 to 50% by weight solutions in phosphate buffered saline, and the active ingredient is present in micrograms to milligrams, such as about 0.0001 to about 5% by weight, preferably about 0.0001 to about 1% by weight, most preferably about 0.0001 to about 0.05% by weight or about 0.001 to about 20% by weight, preferably about 0.01 to about 10% by weight, and most preferably about 0.05 to about 5% by weight. According to the knowledge of the technician, the present disclosure, and the documents cited herein, such determination does not require excessive experimentation. And, the time of sequential administration can be determined without excessive experimentation.

[0094] Examples of compositions that may contain therapeutic agents of the invention include liquid formulations for administration to an orifice (e.g., oral, nasal, anal, vaginal, perioral, gastric, mucosal (e.g., via the tongue, alveolar, gum, olfactory or respiratory mucosa), etc.), such as suspensions, syrups or elixirs; and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., administration by injection), such as sterile suspensions or emulsions. Such compositions may be admixed with suitable carriers, diluents or excipients (e.g., sterile water, saline, glucose, etc.). The composition may also be lyophilized. Depending on the route of administration and the desired preparation, the composition may contain auxiliary substances, such as wetting agents or emulsifiers, pH buffers, gelling agents or viscosity enhancers, preservatives, flavoring agents, pigments, etc. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable formulations without undue experimentation.

[0095] The composition of the present invention is conveniently provided with liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions or viscous compositions, which can be buffered to a selected pH. If digestive tract absorption is preferred, the composition of the present invention can be in the "solid" form of pills, tablets, capsules, caplets, etc., including timed release or "solid" preparations with liquid fillers, such as liquids covered with gelatin, whereby gelatin is dissolved in the stomach to be delivered to the intestinal tract. If it is necessary to administer nasally or through the respiratory tract (mucosal), the composition can be in the form of an extruded spray dispenser, a pump dispenser or an aerosol dispenser and distributed therethrough. Aerosols are usually under pressure by means of hydrocarbons. A pump dispenser can preferably distribute a quantitative dose or a dose with a specific particle size.

[0096] The composition of the present invention may contain pharmaceutically acceptable flavors and / or pigments to make it more attractive, especially if it is administered orally. Viscous compositions may be in the form of gels, lotions, ointments, creams, etc. (e.g., for transdermal administration), and generally contain a sufficient amount of thickener to give a viscosity of about 2,500 to 6,500 cps, although more viscous compositions, even up to 10,000 cps, may also be used. The viscosity of the viscous composition is preferably 2,500 to 5,000 cps, because beyond this range, they become more difficult to administer. However, beyond this range, the composition may be close to a solid or gelatin form, which can then be easily administered orally as a swallowing pill.

[0097] Liquid preparations are generally easier to prepare than gels, other viscous substances, and solid compositions. In addition, liquid compositions are more convenient to administer to some extent, especially by injection or oral administration. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with mucous membranes (such as the lining of the stomach or the nasal mucosa).

[0098] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel or other liquid form), or a solid dosage form (e.g., whether the composition is formulated as a pill, tablet, capsule, caplet, sustained release form or liquid-filled form).

[0099] Solutions, suspensions and gels usually contain, in addition to the active compound, a large amount of water (preferably purified water). Small amounts of other ingredients may also be present, such as pH adjusters (e.g., bases such as NaOH), emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), pigments and / or flavors. The composition may be isotonic, i.e., it may have the same osmotic pressure as blood and tears.

[0100] The isotonicity required for the composition of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. For buffers containing sodium ions, sodium chloride is particularly preferred.

[0101] Pharmaceutically acceptable thickeners can be used to maintain the viscosity of the composition at a selected level. Methylcellulose is preferred because it is easily available and economically feasible, and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The concentration of the preferred thickener depends on the selected agent. Emphasis is on using an amount that will reach the selected viscosity. Viscous compositions are usually prepared from solution by adding such thickeners.

[0102] Pharmaceutically acceptable preservatives may be used to increase the shelf life of the composition. Benzyl alcohol may be suitable, although a variety of preservatives may be used, including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride. Suitable preservative concentrations are 0.02% to 2% based on total weight, although this may vary significantly depending on the agent selected.

[0103] Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert with respect to the active compound. For those skilled in the art of chemical and pharmaceutical principles, this does not present any problem, or can be easily avoided by reference to standard texts or simple experiments (not involving undue experimentation), according to the present disclosure and the documents cited herein.

[0104] It is generally envisioned that the compounds and compositions of the present invention will be administered by injection, as such compounds will elicit anti-MARV antibodies, and those skilled in the art can, based on this disclosure and the knowledge in the art, formulate the compounds and compositions identified by the methods herein for administration by injection, and administer such compounds and compositions by injection.

[0105] The present composition of the present invention is prepared by mixing each component according to a generally accepted procedure. For example, the selected components can be simply mixed in a blender or other standard equipment to produce a concentrated mixture, which can then be adjusted to final concentration and viscosity by adding water or thickening agents and possible buffers to control pH or additional solutes to control tension. Generally speaking, the pH can be about 3 to 8.5. Compositions can be administered according to dosages and techniques well known to medical technicians, taking into account factors such as the age, sex, body weight and condition of a particular patient and the form of the composition used (such as solid vs. liquid). Technicians can determine the dosage for humans or other mammals without excessive experimentation based on the disclosure, the documents cited herein, and the knowledge in the art.

[0106] Suitable regimens for initial administration and further doses or sequential administration are also variable and may include an initial administration followed by sequential administration; but nonetheless can be determined by one skilled in the art based on this disclosure, documents cited herein, and the knowledge in the art.

[0107] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

[0108] The present invention will be further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the present invention in any way.

[0109] Example: Vaccination against Marburg virus disease using recombinant vesicular stomatitis virus and dosing potential

[0110] Vaccines are needed to interrupt or prevent ongoing filovirus outbreaks, including MARV outbreaks, in humans throughout West and Central Africa. As part of a filovirus vaccine product development program, it is important to study dose responses early in preclinical development to determine the optimal dose range for safety, immunogenicity, and efficacy, and to potentially demonstrate that the use of lower doses is feasible, which would improve access to the product. To determine the effective dose range of a producible live VSV vaccine vector (rVSVΔG-MARV-GP) encoding the MARV glycoprotein, a dose-ranging study was conducted in cynomolgus macaques, and the results showed that a single vaccination of as few as 200 PFU was 100% effective in preventing lethality and the development of viremia when animals were challenged with the MARV Angola variant. Vaccination with rVSVΔG-MARV-GP induced MARV GP-specific serum IgG, and virus neutralizing activity was detectable in the sera of animals vaccinated with the highest dose. The data suggest that the use of lower doses of MARV vaccines should be further investigated.

[0111] Filoviruses are a significant threat to global health and continue to impact health security and geopolitical stability in Central and West Africa. In 2014–2016, a large outbreak of EBOV occurred in West Africa, followed by alarmingly frequent outbreaks in the Democratic Republic of the Congo and Guinea [1]. EBOV resurgence-related events have led to outbreaks in non-endemic areas of West Africa and, more recently, zoonotic transmission in endemic areas [2,3]. Other filoviruses remain endemic in animal reservoirs throughout Africa, including MARV, SUDV, and others that cause fatal hemorrhagic fevers in humans and have similar epidemic potential to EBOV [4]. Modeling suggests that the geographic area that could support MARV transmission is quite extensive, highlighting the risk of zoonotic transmission [5]. In addition, the first transmission event was detected in a Guinean patient in West Africa with no travel history [6] and has recently been detected in Ghana [7]. Outbreaks of filoviruses, including MARV, will continue to occur at an accelerating rate in the future, as factors such as climate change, increased intercontinental travel, population growth, and expansion of zoonotic host ranges increase the likelihood of future disease transmission events [8].

[0112] Vaccination against filoviruses, both in response to outbreaks and as a routine public health measure, has the potential to help further control health security threats in Africa. The ZEBOV vaccine (rVSVΔG-ZEBOV-GP, produced by Merck Vaccines) is currently available for clinical trials in Africa. The success of ERVEBO (available for sale) provides a strong rationale for efforts to develop additional recombinant live attenuated vaccines based on VSV vaccine vector technology[9,10]. The performance of ERVEBO in an outbreak setting clearly demonstrated that VSV-based technology possesses multiple properties required for the development of other effective filovirus vaccines, including 1) acceptable safety and tolerability; 2) efficacy after a single dose; and 3) rapid development of protective immunity[9-13].

[0113] In addition to the key rVSVΔG-ZEBOV-GP performance characteristics mentioned above, it is also important to consider factors that influence access to filovirus vaccines in populations where the virus is endemic in West and Central Africa. Because outbreaks of EBOV and other filoviruses (such as MARV or SUDV) cannot be accurately predicted, it is important to have vaccine materials that are safe for human use and can be rapidly deployed

[10] . Filovirus vaccines must also be cost-effective, and therefore it is important to evaluate dose-sparing conditions and efficacy after a single dose. Finally, due to the sporadic nature of filovirus outbreaks, traditional human efficacy trials are not feasible. Therefore, access to novel filovirus vaccines may require the use of existing alternative regulatory pathways, such as animal regulations and accelerated approval, as well as the generation of innovative data packages to demonstrate adequate safety, immunogenicity, and efficacy through preclinical animal studies and human clinical trials

[14] . In the case of VSV-based filovirus vaccines, this is driven by the preclinical and clinical record of rVSVΔG-ZEBOV-GP [9–11] and the extensive preclinical studies previously conducted on MARV and other filovirus vaccines based on the rVSVΔG-ZEBOV-GP design [15–17].

[0114] In response to the identification of the first human MARV case in West Africa, WHO convened a Filovirus Expert Group of infectious disease scientists, epidemiologists, public health experts, and vaccine developers to develop a research and development blueprint to strengthen WHO’s Strategic Agenda for Filoviruses Research and Monitoring (AFIRM)

[18] . Central to the blueprint is an understanding of what experimental MARV vaccines are available and their development status, as well as what preclinical data are available to support their use in an outbreak setting. In addition, the blueprint will cover the development of clinical trial approaches that can be used during public health emergencies due to MARV outbreaks. Early implementation of a ring vaccination strategy in response to the emergence of MARV or other filovirus threats, and the availability of clinical trial materials (as was the case with Zaire Ebola) will help to respond to future filovirus outbreaks

[19] . Without a vaccine that was prepared according to GMP and available for immediate use, the response to the 2014–2016 Zaire Ebola outbreak in West Africa would have been much slower and the consequences in terms of human loss and economic losses would have been much more far-reaching.

[0115] A MARV vaccine candidate based on the VSV technology used in ERVEBO is currently in development (rVSVΔG-MARV-GP; Figure 1 ), and the investigational vaccine has been shown to be safe and effective in multiple preclinical studies. To advance rVSVΔG-MARV-GP toward becoming a globally available vaccine candidate for human use, the applicant regenerated the recombinant vaccine strain using conditions that would support future human vaccine development and tested the vaccine’s immunogenicity and efficacy against MARV challenge over a range of doses in a cynomolgus macaque animal model of MARV disease

[20] . The rVSVΔG-MARV-GP vaccine was 100% effective against Marburg disease caused by an Angolan isolate after a single IM injection and protected against MARV viremia even with doses as low as 200 PFU. Vaccination with rVSVΔG-MARV-GP induced MARV GP-specific humoral responses that can be further studied to better understand the correlates of protection, and these data help provide an important bridge to future human safety and immunogenicity studies. The value of the VSVΔG-based MARV vaccine approach and the next steps in filovirus vaccine development are discussed.

[0116] Cell culture and recombinant VSV. Figure 2 As outlined, the rVSVΔG-MARV-GP research library encoding the GP from the MARV Mosok strain was obtained from the Public Health Agency of Canada (PHAC)

[21] , which was used in multiple previous preclinical studies [22-24]. The nucleotide sequence of the viral RNA genome was determined by Sanger sequencing as previously described

[25] , and then a DNA fragment encoding the MARV-GP gene was synthesized at GenScript (Piscataway). The GP gene was then transferred to the VSV Indiana genomic plasmid as previously described

[21] . The nucleotide sequence of the new genomic clone was confirmed by Sanger sequencing. Animal product-free culture media and reagents were used to propagate and purify genomic plasmid DNA.

[0117] Recovery of rVSVΔG-MARV-GP from plasmid DNA was initiated by electroporation of Vero cells

[25] . A cell bank previously qualified for human vaccine production was used at all stages of the recombinant virus work

[26] (Figure 3). The cell bank was derived from the WHO working cell bank (WHO 10-87) deposited at the European Collection of Authenticated Cell Cultures, Vero [WHO], ECACC 88020401). Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM; Sigma) supplemented with 4 mM L-glutamine and 10% gamma-irradiated fetal bovine serum (FBS; Sigma Aldrich). Electroporation was performed using a method modified from those described previously [25, 27, 28]. Briefly, approximately 2.5 × 10 7 The cells were electroporated (low voltage mode, 3 pulses, 70 ms, 140 V, 900 ms interval), and then cultured at 37 ° C, 5% CO2 and 85% humidity. The cell supernatant was harvested 3 days after electroporation and used to infect Vero cell monolayers cultured in supplemented DMEM to amplify the new recombinant virus. Two days later, the culture medium containing the virus was collected and stored at <60 ° C. After confirming that the rescued virus population had the expected genomic consensus sequence, three rounds of plaque isolation were performed.

[0118] Isolated viral plaques were picked from infected Vero cell monolayers covered with DMEM (containing 2% FBS and 0.5% agarose (Lonza)) containing the above supplements. Viruses from multiple individual plaques were amplified in Vero cells and then subjected to genomic sequence analysis to identify leading candidates with the expected genomic sequence. The leading candidates were then subjected to two additional rounds of plaque isolation, after which the selected candidates were used to infect Vero cells cultured in 5-layer Cell Stacks (Corning). The culture medium was harvested approximately 40 hours after infection and clarified by low-speed centrifugation, then aliquoted and stored at <-60°C. The viral stock was characterized using a variety of assays to confirm the expected genomic sequence, MARV GP expression, and the absence of contaminants. The selected candidate was then designated as preMVS.

[0119] Vaccine materials for preclinical studies derived from preMVS are produced in Vero cell culture and purified by a TFF-based process. In short, Vero cells were inoculated in a 5-layer CellStack containing DMEM supplemented as described above and incubated for 72 hours to obtain a near-confluent monolayer. Before infection, the cell monolayer was washed twice with DMEM, and then 375 ml of virus-containing virus production serum-free medium (Virus-Production Serum-Free Medium, VP-SFM; Thermo Fisher Scientific) was added to reach an infection multiplicity (MOI) of 0.001. At 40 hours after infection, the virus-containing culture medium was harvested and then filtered with a 1.2 μm filter (Sartorius) and a 0.8 / 0.45 μm depth filter (Pall Corporation) in sequence to clarify. The virus was concentrated using TFF and further purified using a 750 kDa hollow fiber membrane (Repligen Corporation). This was followed by the addition of MgCl2 (InVitrogen, Thermo Fisher Scientific) to a final concentration of 1.5 mM and universal nuclease (200 U / ml; Sigma-Aldrich) while continuing TFF for 30 minutes at room temperature. Buffer exchange was performed using 50 mM Tris-HCl, 150 mM NaCl and 10% sucrose buffer (pH 8.0). The purified viral vaccine candidates were aliquoted and stored at <-80°C. Figure 3 illustrates some characterizations of the vaccine candidates, such as flow virology, genome integrity and MARV GP expression.

[0120] Flow cytometry (Apogee). The purified virus from the VSVΔG-MARV-GP vaccine was run on an A60-MicroPLUSApogee flow cytometer using highly purified Milli-Q water as the sheath fluid. The samples were diluted 1:300 in sterile HBSS buffer and run at 1.5 μL / min with an autocycler set to 200,000 total events. The 405 nm violet laser was set to 150 mW, and the VSV virus peak features were successfully resolved using a Large-Angle Light Scatter detector.

[0121] Genome integrity analysis and sequencing. The genome integrity of MARV GP was assessed by RT-PCR using the Superscript IV One-Step RT-PCR System (Invitrogen), in which the forward and reverse primers were located in the VSV M and L genes, respectively. RT-PCR was performed at 60°C for 10 minutes and at 98°C for 2 minutes. Then 40 cycles were performed, each cycle was 98°C for 10 seconds, 70°C for 10 seconds and 72°C for 1.5 minutes, and finally extended at 72°C for 5 minutes. A 2.9Kb band was detected in a 0.8% agarose gel and excised for DNA extraction (Qiagen). The BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and BigDye XTerminator were used. TM Purification kit (ThermoFisher Scientific) and Sanger sequencing were performed using ABI3500XL Genetic Analyzer (ThermoFisher Scientific).

[0122] Analysis of GP expression. GP incorporation into virions was monitored at various stages of production using Western blotting and methods similar to those described previously

[25] . Samples containing rVSVΔG-MARV-GP were denatured and separated using 4-12% Bis-Tris denaturing polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen) using the iBLOT2 system. For detection of virion proteins, rabbit polyclonal anti-MARV GP ( Catalog No. 0303-007, IBT Bioservices) and anti-VSV N (produced in-house

[29] ) were used as primary antibodies, and goat anti-rabbit HRP (Santa Cruz) was used as secondary antibody. Signals were detected using SuperSignal West Femto Maximum Sensitivity (ThermoFisher Scientific) and the ChemiDoc Imaging System (BioRad).

[0123] Flow cytometry was used to evaluate the cell surface expression of MARV GP and the intracellular expression of VSV N. At 48 hpi, adherent infected cells were separated by scraping them from the plate into a washing solution containing PBS and supplemented with 0.5% BSA (PBS / BSA). The cell suspension was distributed into a 96-deep well tissue culture plate and then collected by low speed centrifugation at 860 x g for 5 minutes. To stain MARV GP on the cell surface, cells were first suspended in PBS / BSA containing mouse monoclonal anti-MARV GP (Catalog No. 0203-023, 5C1, IBT Bioservices), rabbit polyclonal anti-MARV GP (Catalog No. 0303-007, IBT Bioservices) or pan-filamentous virus chimeric anti-GP mAb (Catalog No. 0200-003, IBT Bioservices) at a final concentration of 1 μg / ml and incubated at room temperature for 25 minutes. The cells were collected by centrifugation, resuspended in PBS / BSA, and centrifuged repeatedly to remove free anti-GP antibodies. The precipitated cells were resuspended in a cell fixation / cell permeabilization solution (Cytofix / CytopermSolution, BD Biosciences) and incubated for 20 minutes in the dark at 4°C. The permeabilized cells were collected by centrifugation and resuspended in a permeabilization / wash buffer (Perm / Wash Buffer, BD Biosciences), and then centrifuged repeatedly. In order to stain intracellular VSV N, the cells were resuspended in a permeabilization / wash buffer containing an anti-VSV N mouse monoclonal antibody (Catalog No. EB0009, 10G4, Kerafast) at a final concentration of 1 μg / ml and incubated in the dark at room temperature for 25 minutes. After incubation, cells were collected and washed with permeabilization / washing solution as described above, and cells were resuspended in goat anti-mouse IgG1 or goat anti-rabbit Alexa 555 and goat anti-mouse IgG2a Alexa 647 secondary antibody solution (respectively ThermoFisher catalog number A-21127, A-32732, A21241, ThermoFisher), and incubated in the dark at room temperature for 25 minutes. Permeabilization / washing buffer (BD Biosciences) was used to perform a washing step and resuspend cells. Flow cytometry was performed using a BD SORP LSRII flow cytometer (BD Biosciences).

[0124] VSVΔG-MARV-GP vaccination. Vaccination was performed in the ABSL2 suite at the University of Texas Medical Branch (UTMB). The study design was approved by the UTMB Institutional Biosafety Committee (IACUC), and all animal studies were conducted in compliance with the UTMB IACUC, the Animal Welfare Act, and other federal statutes and regulations regarding animal care. The UTMB Animal Research Facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

[0125] This study included 6 groups (n = 4 / group). Five groups were vaccinated with different doses of rVSV-MARV-GP, ranging from 2x10 7 PFU to 200 PFU. A control group was vaccinated with a vaccine prepared from preMVS developed from a similar VSV-based vaccine expressing the Lassa virus glycoprotein (IAVI unpublished) (rVSVΔG-LASV-GPC [21, 30]). Animals received a single IM injection into the quadriceps muscle. Diluted, unused vaccine material was back-titrated to confirm that the targeted vaccine dose had been delivered.

[0126] Analysis of anti-MARV GP serum IgG. Before vaccination and on the 10th and 27th day after vaccination, blood was drawn via peripheral venous puncture using serum separation tubes (Greiner Bio-One, Monroe, NC). Serum was stored frozen (-20°C) until analyzed by indirect ELISA or plaque reduction assay to assess neutralizing antibody titers. Anti-MARV GP IgG endpoint titers were quantified using ELISA plates (96 half-well plates; Corning), which were coated with recombinant MARV Angola GP (obtained by the U.S. Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological al and Nuclear Defense, CBRN-JPEO) diluted to 1 μg / ml in ELISA coating buffer (Biolegend, San Diego, CA) at 4°C overnight. After coating, the plates were blocked with blocking buffer (3% BSA in PBS containing 0.05% Tween-20) for 1.5 hours at 37°C and then washed with 150 μl of washing buffer (PBS containing 0.05% Tween-20). Serum samples were then added in three-fold dilutions starting from a 1:100 dilution and incubated for 1 hour at 37°C. After incubation, the plates were washed and incubated with anti-human IgG (H+L)-HRP (Jackson Immunoresearch) at a 1:6,000 dilution at 37°C for 1 hour, washed again, developed using 1-step Ultra TMB substrate (Thermo Fisher Scientific, Waltham MA), and stopped after 10 minutes with 5N sulfuric acid (Thermo Fisher Scientific, Walt ham MA). The plate was read at 450 nm using a Molecular Devices (San Jose, CA) VersaMax microplate reader using SoftMax Pro GxP data acquisition software (SoftMax Pro GxP) within 30 minutes. Serum from unvaccinated animals or serum collected before vaccination was included to determine the assay background. Titers were defined as serum dilutions that resulted in an absorbance greater than 2 times the standard deviation of the background wells. Commercially available anti-MARV GP antibodies (IBTBioservices, Rockville MD) were used as positive controls in the assay.

[0127] rVSVΔG-MARV-GP (Mosok) was used as a target virus to quantify virus neutralization and anti-MARV GP serum antibodies (nAb). The serum collected 27 days after vaccination was heat inactivated at 56°C for 30 minutes, clarified by centrifugation at 9,300xg for 10 minutes, and then serially diluted from 1:20 to 1:327,680 and incubated with an appropriate amount of rVSVΔG-MARV-GP on a shaker at 300RPM in a 37°C incubator for 1 hour to produce approximately 100 plaques per well. After incubation, the Vero cell monolayer in a 96-well tissue culture plate was infected with the serum-virus mixture. After the cells were incubated on a shaker at 300RPM in a 37°C incubator for 2 hours, the cells were covered with DMEM (ThermoFisher Scientific) containing 1% FBS and 0.5% methylcellulose (ThermoFisher Scientific). Plaques were allowed to develop at 37°C for 44 hours, then the methylcellulose overlay was removed and the cells were fixed with 7% v / v formaldehyde prepared in water (200 μl per well) and incubated for 1 hour at room temperature. Plaques were stained by adding 200 μl of crystal violet solution (0.33% in water) to each well and incubated for 1 hour at room temperature. The staining solution was removed, the plaques were rinsed and dried, and then the plaques were counted using Cytation 5Imager Gen53.08 software (Agilent). The lowest serum dilution that reduced plaques by 50% or more was reported.

[0128] MARV challenge virus and vaccine efficacy. The challenge virus was prepared at UTMB using MARV Angola (200501379) isolated from an 8-month-old female patient in Uige, Angola. The challenge virus stock was developed from a virus obtained from the CDC (CDC 810820) and passaged twice in Vero E6 cells at UTMB

[31] . On day 28 post-vaccination, 10 3PFU infected macaques. Animals were monitored daily, and MARV disease progression was scored using the humane endpoint filovirus disease score table approved by UTMB IACUC. Score changes from baseline measurements included posture and activity level, attitude and behavior, food intake, respiration and disease manifestations, such as visible rash, hemorrhage, ecchymosis or skin flushing. Animals were also monitored for central nervous system abnormalities. Score ≥ 10 indicated that the animal met the criteria for euthanasia. Blood was collected on days 4, 7, 10, 11, 13, 15, 21 and 28 after MARV attack to evaluate blood chemistry and quantitative infectivity MARV. UTMB facilities were certified by the Association for Assessment and Accreditation of Laboratory Animal Care International and adhered to the principles set forth in the eighth edition of the National Research Council's Guide for the Care and Use of Laboratory Animals.

[0129] Infectious MARV (viremia) in blood was quantified using plasma collected from macaques and a plaque assay

[30] . Briefly, ten-fold increasing dilutions of plasma samples were used to infect Vero E6 monolayers (ATCC, Manassas, VA) in duplicate wells (200 μl per well). The detection limit for serum was 25 PFU / ml.

[0130] To monitor MARV genomes (RNAemia) in blood by RT-qPCR, RNA was isolated from whole blood using a viral RNA mini kit (Qiagen) using 100 μl of blood mixed with 600 μl of viral lysis buffer AVL

[32] . Primers targeting the NP gene of MARV were used for real-time quantitative PCR (RT-qPCR) using a 6-carboxyfluorescein (6FAM)–5′-CCCATAAGGTCACCCTCTT-3′–6-carboxy-tetramethylrhodamine (TAMRA) probe. The thermocycler run was set to 50°C for 10 min; 95°C for 10 s; and 40 cycles of 95°C for 10 s plus 59°C for 30 s. Primers were synthesized by Integrated DNA Technologies, and labeled probes were prepared by Life Technologies. MARV genomes in the sample were enumerated using a genome equivalent standard. The detection limit of the assay was 1,000 copies / ml.

[0131] Generation of rVSVΔG-MARV-GP to support human vaccine development. The rVSVΔG-MARV-GP[21,23] vaccine is based on a replication-competent chimeric virus design (Figure 1) in which the gene encoding the native VSV glycoprotein (G) is deleted (VSVΔG) and replaced with the coding sequence of a functional glycoprotein from a heterologous virus [21,33]. The rVSVΔG-MARV-GP genomic clone was generated using a genomic plasmid of laboratory-adapted VSV serotype Indiana[34,35] and the GP coding sequence from the MARV Mosok strain ([21,23]). To generate an rVSVΔG-MARV-GP strain suitable for human vaccine development, a novel recombinant virus ( Figure 2 ).

[0132] The applicant chose to advance VSVΔG-MARV-GP, which expresses MARV Mosok GP, as a candidate vaccine for several important reasons, including: 1) a single vaccination of the VSVΔG-MARV-GP Mosok vaccine has been shown to be highly effective and to rapidly induce protective immunity in macaques [23,36,37]; 2) protective immunity is long-lasting

[38] ; 3) preclinical studies have shown that vaccination-induced immunity can protect macaques from MARV Mosok, MARV Angola, or Ravn viruses [22,23]; 4) preclinical studies have shown that the vaccine can induce effective protective immunity against aerosolized MARV

[39] ; and 5) in neurovirulence studies in macaques, the rVSVΔG-MARV-GP Mosok vaccine was evaluated together with the investigational rVSVΔG-ZEBOV-GP vaccine and showed that it had no neurovirulence potential, which provides valuable safety data for advancing the vaccine for human use

[24] .

[0133] After the novel rVSVΔG-MARV-GP strain was recovered from plasmid DNA, it was isolated by three rounds of plaque isolation (Methods and Figure 3A ) to generate multiple clonal virus isolates while maintaining the laboratory and documentation practices necessary to support human vaccine development. Subsequently, several lead candidates were selected based on comparison of the genomic nucleotide sequence of the research virus

[21] determined by the applicant with the clonal virus isolates and confirmation of GP expression by Western blot and flow cytometry (data not shown). After amplification and storage of multiple seed stocks designated as candidate preMVS, the preMVS candidates were analyzed using the various assays described below to select the lead candidates.

[0134] FIG3 summarizes the assays and methods used to evaluate preMVS candidates. Viruses from preMVS candidates were serially propagated in Vero cells to simulate manufacturing expansion, during which titers were monitored by plaque assay (data not shown) and genetic stability was assessed using genomic nucleotide sequencing and RT-PCR assays (e.g., Figure 3D The assay detected the intact GP gene but also confirmed the absence of a minority of unexpected GP deletion variants. Figure 3E ) and by evaluating the expression on the cell surface using flow cytometry ( Figure 3F ) confirmed GP expression.

[0135] Viruses derived from several preMVS candidates were also amplified and purified to produce preclinical vaccine material and to evaluate how each preMVS performs during the manufacturing process. Vaccine material was prepared by infecting Vero cells, and the virus was then purified and concentrated using a scalable TFF-based method (see Methods), which is consistent with the intended manufacturing process. Figure 3A In addition to the assays described in , DF, Applicants also used nanoflow cytometry to quantify virion particles during all stages of the preclinical vaccine production process ( Figure 3B and C) to ensure that a single major virion peak was detected and that there was no accumulation of smaller particles or larger aggregates.

[0136] Based on the performance of the preMVS candidates in the laboratory evaluations briefly outlined above, a lead preMVS was selected and transferred to the applicant's GMP manufacturing partner. Thereafter, qualified master virus seeds (MVS) have been produced to support manufacturing. Preclinical vaccine material produced from the lead preMVS is also used in the preclinical vaccine efficacy studies described below.

[0137] A single vaccination with multiple doses of rVSVΔG-MARV-GP (Mosok) protected against challenge with MARV Angola virus. A preclinical study was conducted in cynomolgus monkeys ( Figure 4A ) to investigate the range of doses of rVSVΔG-MARV-GP for immunogenicity and efficacy against a lethal dose of low-passage MARV Angola challenge. Briefly, six groups of cynomolgus monkeys (n=4 / group) were injected IM with doses ranging from 2×10 2 Up to 2x10 7 The control group received IM injection of 2x10 7Another rVSVΔG-based vaccine encoding the Lassa virus glycoprotein (rVSVΔG-LASV-GPC) was also developed by the applicant from a promising investigational vaccine (data not shown)

[30] . After vaccination, samples were collected ( Figure 4A ) for immunogenicity measurements, then challenged macaques with a lethal dose of MARV angola 28 days after vaccination and monitored for clinical signs of MARV disease ( Figure 4B ).

[0138] like Figure 4B As shown in the figure, all animals vaccinated with the control rVSVΔG-LASV-GPC vaccine developed MARV disease symptoms

[20] and were euthanized on day 8 or 9 based on the humane endpoint clinical score table. The development of MARV disease in the control animals suggests that anti-VSV vector immune responses do not interfere with MARV challenge. However, all animals vaccinated with the rVSVΔG-MARV-GP vaccine ( Figure 4A ) of the animals survived ( Figure 4B ) and did not develop clinical features of MARV disease.

[0139] To evaluate how vaccination affects the presence of infectious MARV in peripheral blood after challenge, Applicants quantified the titer of MARV in serum by plaque assay ( Figure 4C Determining the titer of infectious virus is important because quantification of RNA copy number by RT-qPCR does not measure viable viral progeny circulating in the blood, and importantly, an informative early preclinical study showed that protection from Ebola virus disease progression in macaques was associated with maintenance of infectious titers in the blood that were less than approximately 1x10 5 The threshold titer for tissue culture infectious dose 50 (TCID50)

[40] was quantified in the presence of viremia in all control animals in samples collected on day 4 post-challenge, with titers increasing to 10 by day 7. 6 PFU / ml or higher. When euthanasia is performed according to the protocol, the titer is as high as about 10 8 PFU / ml( Figure 4C In contrast, infectious MARV could not be detected in the serum at any time in animals vaccinated with either the high-dose or low-dose VSVΔG-MARV-GP vaccine.

[0140] RT-qPCR evaluation of MARV RNA copies was also performed using RNA extracted from whole blood ( Figure 5 As expected based on viral titers in serum, RNA copy numbers were higher in control macaques and increased in parallel with the titers of infectious virus ( Figure 4CConsistent with detectable viremia prevented by vaccination, MARV RNA was detectable in only 5 of 20 vaccinated animals at any time point analyzed post-challenge, and if RNA was detected, it was transient ( Figure 5 For example, the lowest dose of 2x10 2 Two animals in the group vaccinated with 10 PFU of VSVΔG-MARV-GP showed transient signals in the RT-qPCR assay. One of these animals was positive on days 4 and 7 but resolved by day 10, while the other animal was positive only at a single time point on day 7. These RNA signals did not increase as expected if extensive MARV replication was occurring in these vaccinated macaques, further suggesting that defective virions may have caused the transient RNA signals, as has been previously detected during filovirus infection [41-43]. Transient RNA copies detected on day 10 post-challenge were present in the viable samples of the macaques vaccinated with 2x10 4 PFU vaccination group, and 2x10 5 In one animal in the PFU vaccination group ( Figure 5 The presence of transient low RT-qPCR signals on days 4 or 7 post-challenge may indicate that MARV infection is rapidly controlled or aborted, resulting in little infectious virus being released into the circulation, which is consistent with Figure 4C The explanation for the transient RNA signal in 3 animals on day 10 is more speculative, especially because RNA copies were detected late and did not correlate with the presence of infectious virus ( Figure 4C ), but this may be related to clearance of the initial viral inoculum rather than to progeny virions produced by active MARV replication. Overall, RNAemia analysis is consistent with viremia, indicating that vaccination prevents MARV replication and the release of infectious viral progeny into the blood.

[0141] Humoral immune responses against MARV GP induced by VSVΔG-MARV-GP vaccination. Serum was collected from all animals before and after vaccination ( Figure 4A ) to assess the presence of anti-GP serum IgG. Binding antibody titers ( Fig. 6A ) were quantified. Binding antibodies to Angola GP were detected in all animals, indicating that even at a low dose, a single vaccination resulted in seroconversion. 7The median titers were highest in the group vaccinated with 100 PFU of VSVΔG-LASV-GPC and generally decreased proportionally with the tested dose. When compared to the control group vaccinated with VSVΔG-LASV-GPC, the median titers of all groups were statistically significant except for the group vaccinated with only 200 PFU of VSVΔG-MARV-GP.

[0142] Applicants also analyzed the virus neutralization activity in serum using a plaque reduction assay based on VSVΔG-MARV-GP (Mosok) neutralization ( Figure 6B The same type of VSV-based assay has been used previously to assess neutralizing antibodies against EBOV GP [44-46]. Although the neutralizing titers were lower (GMT approximately 100 in the high-dose group), the assay did detect neutralizing serum antibodies in 6 of 7 animals vaccinated with the higher vaccine dose ( Figure 3B ; 2x10 7 and 2x10 5 Neutralizing serum antibodies were also detected in some animals vaccinated with the lower dose, but low pre-vaccination background neutralizing activity was also observed in some macaques from these groups.

[0143] Early preclinical studies have demonstrated that the replication-competent VSVΔG-MARV-GP vaccine is safe and highly effective [16,22-24,36-39]. Applicants have developed a novel recombinant virus and pre-MVS under conditions that will support the production of vaccine material for human trials. Applicants have also replicated previous preclinical efficacy results for their vaccine candidates and generated important new information. The dose-saving potential of a particular vaccine technology is an important consideration for better access to vaccines, as large quantities of vaccine material can be produced at a lower cost. Applicants have included a study in the preclinical evaluation of preMVS in which less than 2x10 7 The applicants found that a single dose of as little as 200 PFU of rVSVΔG-MARV-GP (Mosok) could prevent clinical signs of MARV disease after challenge with the Angolan MARV strain ( Figure 4B ), which raises the possibility that lower doses may be effective in people.

[0144] Applicants’ data suggest that low doses of rVSVΔG-MARV-GP are effective, which is consistent with earlier studies showing that very low doses of the rVSVΔG-EBOV-GP (Kikwit variant GP) vaccine can prevent Ebola virus disease in cynomolgus macaques

[40] . In this study, Marzi et al. demonstrated that as few as 1–10 PFU of rVSVΔG-EBOV-GP protected against lethal disease. Although preclinical studies of VSVΔG-MARV-GP and VSVΔG-EBOV-GP clearly demonstrated that lower doses of rVSVΔG-based filovirus vaccines are highly protective in macaques, the Human data from clinical studies have shown that dose-dependent humoral responses are 5

[49] However, in the absence of human efficacy studies with lower doses, it is not possible to determine whether decreased circulating IgG titers are associated with reduced efficacy against EBOV or EVD disease.

[0145] In addition to the fact that vaccination prevented the emergence of MARV disease, the applicants also found that in vaccinated animals, infectious MARV could not be detected in the blood by plaque assay. This suggests that immunity induced by VSVΔG-MARV-GP provides a very effective barrier to the emergence of viremia. This is an important finding because early preclinical studies of dosing and efficacy using rVSVΔG-EBOV-GP by Marzi et al. showed that immune control of EBOV replication (controlling viremia to approximately 1x10 5 A threshold level of TCID50 / ml is critical to prevent disease progression

[40] .

[0146] The immune responses associated with protection against MARV disease are not fully understood. Studies of humans vaccinated with rVSVΔG-ZEBOV-GP have shown that total serum IgG titers as well as neutralizing antibody titers (toward the vaccine virus) provide some of the strongest correlates of protection in humans

[46] . The potential importance of vaccination-induced functional antibodies is perhaps highlighted by studies showing that antibodies with direct virus neutralizing activity were isolated from humans vaccinated with VSVΔG-ZEBOV-GP [50,51], but antibodies capable of mediating Fc-directed innate immune effector functions may also play a role in protection against EBOV disease

[52] . It is therefore encouraging that the applicants were able to detect some direct virus neutralizing activity specific for MARV GP in the sera of macaques vaccinated with higher doses of VSVΔG-MARV-GP and that serum IgG titers were detectable even at the lowest vaccine dose. Therefore, a more detailed assessment of the functional properties of anti-MARV antisera would be informative, particularly because innate immune effector functions mediated by the antibody Fc domain are thought to play an important role in protection against MARV disease

[53] , and neutralizing monoclonal antibodies specific for MARV GP are known to be protective [54,55].

[0147] The inability to conduct traditional efficacy studies on pathogens that sporadically infect humans, such as highly virulent filoviruses, is a challenge for vaccine developers. Therefore, in order to advance vaccines such as VSVΔG-MARV-GP as products for use in humans, novel regulatory strategies are needed that rely more on animal efficacy models, detailed knowledge of the characteristics of protective immune responses induced in animals, and the availability of licensed vaccines using the same technology (such as )

[14] . Although there is increasing evidence that IgG titers and neutralizing antibodies are associated with Although rVSVΔG-MARV-GP induces protective immunity,

[46] the humoral responses induced by rVSVΔG-MARV-GP are modest, and vaccination is highly protective in macaques even at low vaccine doses. Therefore, to support the advancement of future VSVΔG-based filovirus vaccines (such as rVSVΔG-MARV-GP) toward regulatory approval, it will be important to conduct additional studies to better understand the functional antiviral glycoprotein adaptive immune responses that contribute to protection and to develop immunological signatures associated with VSVΔG-based vaccination take and efficacy. This can be accomplished by combining the immunogenicity of glycoproteins derived from effective vaccines (such as rVSVΔG-MARV-GP) with the immunogenicity of glycoproteins derived from ... This will be achieved by bridging human clinical data from VSV with preclinical and clinical data from different VSV filovirus vaccine candidates.

[0148] To enable future access to filovirus vaccines, the rVSVΔG-MARV-GP candidate was produced and evaluated in a manufacturing-ready format and is currently being used to generate clinical trial material. Without the availability of rVSVΔG-EBOV-GP clinical trial material in 2014, the international community would not have had an effective EBOV vaccine to combat the continued emergence and spread of EBOV in Central and West Africa [56,57]. Even with this material, unprecedented coordination, collaboration, communication, and clinical capacity would have been required among international and national public health agencies (such as WHO, NIH, and CDC), vaccine developers, and local public health authorities and experts in West Africa to manage and evaluate investigational vaccines during public health emergencies

[58] . The 2014–2016 West African EBOV outbreak generated sufficient data to evaluate the human efficacy of the rVSVΔG-EBOV-GP vaccine. However, the vaccine was used in West and Central Africa for several years in an expanded access capacity before full licensure in 2019. Important lessons from the rVSVΔG-EBOV-GP experience should be applied to prevent future outbreaks of MARV, SUDV, or other filoviruses. Establishing a stockpile of investigational vaccines is urgently needed to prevent unnecessary future human morbidity and mortality in areas where human disease transmission is limited but the potential impact of an outbreak is high if it occurs.

[0149] Recent MARV cases in Guinea and Ghana highlight the need for continued preparedness so that applicants can better respond to these emerging infectious agents. rVSV technology has been evaluated against a variety of pathogens

[59] , and It has been licensed in the United States, Europe, and several African countries

[60] . Here, the applicants demonstrate an effective vaccine against another filovirus, MARV, with dose-sparing properties observed in a well-characterized non-human primate model. The vaccine was effective in cynomolgus macaques at doses as low as 200 PFU, as was the protection mediated by rVSVΔG-EBOV-GP at lower doses (Figures 3 and 4)

[40] . If lower doses of the vaccine are shown to be effective, the commodity costs used in vaccine production will be reduced, allowing larger doses to be produced at a lower cost, which would allow more people to receive the vaccine as needed.

[0150] Applicants' results highlight the potential value of VSV vaccine technology for another important filovirus disease. Similarities to this rVSVΔG-MARV-GP vaccine in terms of preliminary characterization and preclinical data provide a strong basis for stockpiling GMP materials for inclusion in emergency response to MARV outbreaks. To effectively advance rVSVΔG-MARV-GP as a public health tool for future MARV outbreaks, clinical trial material stockpiles need to be generated, similarities between immune responses to EBOV vaccination and MARV need to be explored, and clinical trial designs need to be adjusted and prioritized in advance for emergency outbreak scenarios. The relatedness and similar human disease presentations of EBOV, SUDV, MARV, and other filoviruses suggest that immune responses common to the entire family of filoviruses can be identified as efficacious, which may provide new avenues for licensable vaccines. If these goals for MARV can be achieved, better protection against filoviruses will be possible. The only way to better prepare for future outbreaks is to increase and plan and execute appropriate evaluation and data generation for filovirus vaccine candidates when there are no active outbreaks; it is critical that applicants learn from their experiences and remain vigilant to avoid being caught off guard again.

[0151] References:

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[0154] 3.WHO.Ebola virus disease-Democratic Republic of the Congo.2022[cited2022July 8]; Available from: https: / / www.who.int / emergencies / disease-outbreak- news / item / 2022-DON377 .

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[0212] ***

[0213] Having thus described in detail the preferred embodiments of the present invention, it should be understood that the invention defined in the preceding paragraphs is not limited to the specific details set forth in the foregoing description, since obvious variations are possible without departing from the spirit or scope of the present invention.

Claims

1. A recombinant vaccine comprising a nucleic acid encoding a glycoprotein (GP) of the Mosok isolate Marburg virus (MARV) encoded in a vesicular stomatitis vector (VSV) that does not contain the glycoprotein G gene (VSVΔG).

2. The vaccine of claim 1, wherein the nucleic acid comprises SEQ ID NO:

1.

3. The vaccine of claim 1 or claim 2, wherein the nucleic acid comprises SEQ ID NO:

2.

4. A method of vaccinating a mammal in need thereof with a vaccine as claimed in any one of the preceding claims, comprising administering to said mammal about 10 2 -10 7 PFU of the vaccine.

5. The method of claim 4, comprising administering about 10 4 PFU of the vaccine.

6. The method of claim 4, comprising administering about 10 5 PFU of the vaccine.

7. The method of claim 4, comprising administering about 10 6 PFU of the vaccine.

8. The method of any one of claims 4-7, wherein the mammal is a bat or a primate.

9. The method of claim 8, wherein the primate is a bonobo, chimpanzee, gibbon, gorilla, human, monkey or orangutan.

10. The method of claim 9, wherein the primate is a human.

11. The method of any one of claims 4-10, wherein the administration is intramuscular.

12. The method of any one of claims 4-10, wherein the administration is intranasal.

13. The method of any one of claims 4-10, wherein the administration is intradermal.

14. The method of any one of claims 4-10, wherein the administering is by oral bait drops.

15. The method of any one of claims 4-14, wherein the vaccine of the present invention is administered together with an Ebola virus vaccine or a Sudan virus vaccine.

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