Hemorrhagic fever vaccines

Abstract

Designed polypeptide sequences are described, and their use as vaccines against pathogens which cause hemorrhagic fever, such as viruses of the Ebolavirus, Lassa Fever virus and Marburg virus families. The designed sequences include those which comprise the designed Tri-LEMvac vaccine, and the bivalent Lassa virus vaccine. Nucleic acid molecules encoding the polypeptides, as well as vectors, fusion proteins, pseudotyped virus particles, pharmaceutical compositions, combined preparations, cells, and their use as vaccines against viruses of the Ebolavirus, Lassa Fever virus and Marburg virus families are also described.

Description

This invention relates to nucleic acid molecules, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, and combined preparation, and their use as vaccines against emerging or re-emerging pathogens such as viruses which cause hemorrhagic fever (particularly RNA viruses). The invention also relates to pseudotyped virus particles. The fundamental principal of a vaccine is to prepare the immune system for an encounter with a pathogen. A vaccine triggers the immune system to produce antibodies and T-cell responses, which help to combat infection. Historically, once a pathogen was isolated and grown, it was either mass produced and killed or attenuated, and used as a vaccine. Later recombinant genes from isolated pathogens were used to generate recombinant proteins that were mixed with adjuvants to stimulate immune responses. More recently the pathogen genes were cloned into vector systems (attenuated bacteria or viruses) to express and deliver the antigen in vivo. All of these strategies are dependent on pathogens isolated from past outbreaks to prevent future ones. For pathogens which do not change significantly, or slowly, this conventional technology is effective. However, some pathogens, are prone to mutating and antibodies do not always recognise different strains of the pathogen. New emerging and re-emerging pathogens often hide or disguise their vulnerable antigens from the immune system. Of the emerging and re-emerging diseases, a disproportionate number (37%) are caused by ribonucleic acid (RNA) viruses (Heeney, Journal of Internal Medicine 2006; 260: 399-408). An RNA virus is a virus that has RNA as its genetic material. This nucleic acid is usually single-stranded RNA (ssRNA) but may be double-stranded RNA (dsRNA). RNA viruses generally have very high mutation rates compared to DNA viruses, because viral RNA polymerases lack the proofreading ability of DNA polymerases. This is one reason why it is difficult to make effective vaccines to prevent diseases caused by RNA viruses. In most cases, current vaccine candidates against RNA viruses are limited by the viral strain used as the vaccine insert, which is often chosen based on availability of a wild-type strain rather than by informed design. Technical challenges for developing vaccines for enveloped RNA viruses include: i) viral variation of wild-type field isolate glycoproteins (GPs) provide limited breadth of protection as vaccine antigens; ii) selection of vaccine antigens expressed by the vaccine inserts is highly empirical; immunogen selection is a slow, trial and error process; ill) in an evolving or unanticipated viral epidemic, developing new vaccine candidates is timeconsuming and can delay vaccine deployment. Notable human diseases caused by RNA viruses include viral hemorrhagic fevers (VHFs), a group of illnesses that are caused by several distinct families of viruses. In general, the term “viral hemorrhagic fever” is used to describe a severe multisystem syndrome (i.e. multiple organ systems in the body are affected). Characteristically, the overall vascular system is damaged, and the body’s ability to regulate itself is impaired. These symptoms are often accompanied by hemorrhage (bleeding), although the bleeding is itself rarely life-threatening. While some types of hemorrhagic fever viruses can cause relatively mild illnesses, many of the viruses cause severe, life-threatening disease. VHFs are caused by viruses of at least five distinct families: Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, and Paramyxoviridae. The viruses of these families are all RNA viruses, and are all covered, or enveloped, in a fatty (lipid) coating. The survival of VHFs is dependent on an animal or insect host (the natural reservoir). The viruses are geographically restricted to the areas where their host species live, and humans are infected when they come into contact with infected hosts. With some of the viruses, after transmission from the host, humans can transmit the virus to one another. Human cases or outbreaks of hemorrhagic fevers caused by these viruses occur sporadically and irregularly. The occurrence of outbreaks cannot be easily predicted. With a few exceptions, there is no cure or established drug treatment for VHFs. VHFs caused by Arenaviruses and Filoviruses together cover a wide geographic region ranging from Western through to Central Africa and threaten adjacent regions where infected animal reservoirs may migrate but where human disease has not yet been reported. Filoviruses encode their genome in the form of single-stranded negative-sense RNA. Two members of the family that are commonly known are Ebola virus and Marburg virus. Ebola is an emerging and re-emerging RNA viral disease. Outbreaks are not always caused by the exact same virus, but by different relatives (types) of the same virus family of which there are close siblings (for example, Ebola Mayinga and Ebola Kikwit), close cousins (Tai Forest and Bundibugyo), distant cousins (Sudan), and distant relatives (Marburg virus). The 2014 Ebola outbreak in West Africa was the largest since the viral disease was first recognised. Arenaviruses are divided into two groups: the Old World and the New World viruses. The differences between these groups are distinguished geographically and genetically. At least eight arenaviruses are known to cause human disease ranging in severity. Aseptic meningitis, a severe human disease that causes inflammation covering the brain and spinal cord, can arise from the Lymphocytic choriomeningitis virus (LCMV) infection. Hemorrhagic fever syndromes are derived from infections such as Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV). Lassa Fever virus (LASV), Ebola (EBOV) and Marburg (MARV) viruses are the most important haemorrhagic fevers in West and Central Africa. Lassa fever is endemic to Western Africa with estimates ranging between 300,000 to a million infections, with 5,000 deaths per year. Lassa Fever virus (LASV), Ebola (EBOV) and Marburg (MARV) viruses are all containment level 4 pathogens with high human morbidity and mortality for which there are no established cures, and currently there are no licensed vaccines for infections caused by these viruses. There is a need, therefore, to provide effective vaccines that induce a broadly neutralising immune response to protect against emerging and re-emerging diseases, especially those caused by viruses such as RNA viruses, including VHFs. The term “pathogen” is used herein to refer to anything that can cause disease, and in particular to an infectious agent, such as a virus, bacterium, fungus, or parasite, that can cause disease. The term “polypeptide” is used herein to refer to a polymer comprising a plurality of amino acid residues linked together by peptide bonds to form a chain. All proteins are polypeptides. The term “polypeptide” is used interchangeably with the term “protein”. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced. Optionally the polypeptide is a modified polypeptide, such as co-translationally or post-translationally modified polypeptide, for example a glycosylated polypeptide or a glycosylated protein (a “glycoprotein”). Glycoproteins are proteins which contain oligosaccharide chains (glycans) covalently attached to amino acid side-chains. The carbohydrate is attached to the protein by co-translational or post-translational glycosylation. A “pathogen polypeptide” refers to any polypeptide forming part of a pathogen. Optionally the pathogen polypeptide is a structural protein (or portion thereof) of the pathogen. Optionally the pathogen polypeptide is a structural protein (or portion thereof) that is exposed on the surface of the pathogen. Optionally the pathogen polypeptide is a viral protein (or portion thereof). Optionally the pathogen polypeptide is a viral envelope protein (or portion thereof). Optionally the pathogen polypeptide is a glycoprotein (or portion thereof). Optionally the pathogen polypeptide is a viral glycoprotein (or portion thereof). Optionally the pathogen polypeptide is a viral envelope glycoprotein (or portion thereof). Optionally the pathogen polypeptide is an external viral envelope glycoprotein (or portion thereof). Optionally a pathogen polypeptide comprises an amino acid sequence of at least 20 amino acid residues. Optionally a pathogen polypeptide comprises an amino acid sequence of upto 1000, 900, 800, 700, or 600 amino acid residues. A fully assembled infectious virus is known as a virion. The simplest virions consist of nucleic acid (single- or double-stranded RNA or DNA) and a capsid protein coat. Capsids are formed as single or double protein shells and consist of only one or a few structural protein species. Enveloped viruses have envelopes covering their protective protein capsids. The envelopes are typically derived from portions of the host cell membranes (phospholipids and proteins), but include virus-encoded glycoproteins. Glycoproteins on the surface of the envelope serve to identify and bind to receptor sites on the host's membrane. The viral envelope then fuses with the host's membrane, allowing the capsid and viral genome to enter and infect the host. Virus-cell membrane fusion is the means by which all enveloped viruses, including human pathogens such as filovirus, influenza virus, and human immunodeficiency virus (HIV), enter cells and initiate virus infection. This membrane fusion process is executed by one or more viral envelope glycoproteins. The fusion can occur on the cell plasma membrane or endosomal membrane. Glycoproteins may help viruses avoid the host immune system. Enveloped viruses possess great adaptability, and can change in a short time to evade the host immune system. Enveloped viruses can cause persistent infections. Enveloped RNA viruses include, for example, Flavivirus, Togavirus, Coronavirus, Hepatitis D, Orthomyxovirus, Paramyxovirus, Rhabdovirus, Bunyavirus, Filovirus. Retroviruses are enveloped viruses. Enveloped DNA viruses include Herpesviruses, Poxviruses, Hepadnaviruses. Most external viral envelope proteins are glycoproteins, occurring as membrane-anchored spikes, often assembled as dimers or trimers. The trimeric glycoprotein (GP) spike on the envelope of filoviruses mediates all stages of virus entry, including attachment, entry, and fusion. Recognition sites for cellular receptors are often located at the furthest domain from the viral envelope (distal end) whereas proximal domains interact with the lipid bilayer of the envelope. Oligosaccharide side-chains (glycans) are attached by A / -glycosidic, or more rarely O-glycosidic, linkages. Since these are synthesized by cellular glycosyl transferases, the sugar composition of these glycans is analogous to that of host cell membrane glycoproteins. Entry of filoviruses on the cell surface has been shown to be mediated by host cell attachment factors such as C-type lectins, including DC-SIGN (dendritic-cell-specific ICAM3-grabbing non-integrin; also known as CD209) and L-SIGN (liver and lymph node SIGN; also known as CLEC4M) and several cell-surface proteins such as integrins, T cell immunoglobulin and mucin domain-containing (TIM) proteins, and tyrosine protein kinase receptor 3 (TYRO3) 4 family members. Following binding to the cell surface, filoviruses are internalized by a macropinocytosis-like process and subsequently trafficked through early and late endosomes. The viral genome then penetrates into the cytoplasm after fusion of the viral envelope with the membrane of the late endosome. In the cytoplasm, the viral genome is replicated and transcribed, and new viral proteins are synthesized to assemble progeny virions, which bud from the cell surface. The surface glycoprotein, GP, of Ebola virus (EBOV) is a key component of many vaccines and a target of neutralizing antibodies. The EBOV GP is synthesized as a single polypeptide that is subsequently cleaved by furin-like proteases into GP1 and GP2 subunits, which remain together through an inter-subunit disulfide bond and non-covalent interactions, and form a trimer of GP1-GP2 heterodimers on the viral surface. Furin cleavage, however, is not sufficient to prime EBOV GP. After entering the cell, the virus is eventually trafficked to late endosomes, where GP is further primed to remove some “cap” components, thereby triggering the induction of the crucial membrane fusion event, which leads to viral penetration. EBOV GP priming is mediated by the cysteine proteases cathepsin B and cathepsin L, which cleave GP1 within the 013-014 loop. Cathepsin cleavage removes -60% of the amino acids from GP1, including the mucin-like domain, the glycan cap, and the outmost 0 strand of the proposed receptor binding region, resulting in a primed form of GP (named GPcI, the 19 kDa GP1 plus GP2). Unlike the full-length GP, the primed GPcI cannot bind to endosomal membrane protein Niemann-Pick C1 (NPC1), which is an indispensable host entry factor for EBOV infection. The crystal structures of free NPC1-C and its complex with GPcI have been determined (Wang et al., Cell, 2016, 164, 258-268). During Ebola virus infection the primary product of the GP gene is secreted GP (sGP), a soluble dimer that lacks GP2 and the mucinlike domain, but shares 295 amino acids of GP1. The term “antigenic” is used herein to refer to a substance that is capable of inducing an immune response in a host organism. The immune response may be humoral and / or a cellular immune response. A cellular immune response is a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defence response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection or disease (i.e. prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, or antibody production. Optionally an optimized antigenic pathogen polypeptide is able to induce the production of antibodies and / or a T-cell response in a human or non-human animal to which the polypeptide has been administered (either as a polypeptide or, for example, expressed from an administered nucleic acid expression vector). The term “antibody” is used herein to refer to an immunoglobulin molecule produced by B lymphoid cells with a specific amino acid sequence. Antibodies are evoked in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable way, antibody and antigen each being defined in terms of the other. “Eliciting an antibody response” refers to the ability of an antigen or other molecule to induce the production of antibodies. “Neutralizing" antibodies or antigen-binding molecules not only bind to a pathogen, such as a virus, they bind in a manner that inhibits (i.e. reduces) or blocks infection, or progression of infection. A neutralizing antibody or antigen-binding molecule may block interactions with the receptor, or may bind to a viral capsid in a manner that inhibits uncoating of the genome. The term “neutralizing antibodies” or “neutralizing antigen-binding molecules” also includes antibodies or antigen-binding molecules that are able to prevent infection of a pathogen, such as a virus, by facilitating a cytokine response or by facilitating uptake and removal by an immune cell. In particular, the term “neutralizing antibodies” includes antibodies (or fragments or derivatives thereof) capable of inhibiting or blocking infection (or progression of infection) of a pathogen by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Only a small subset of the many antibodies that bind a virus are capable of neutralization. The term “broadly neutralizing antigen-binding molecule" is used herein to include an antigen-binding molecule, such as an antibody or fragment or derivative thereof, that is able to inhibit (i.e. reduce), neutralize or prevent infection of at least two different subtypes or species of a virus,. Optionally a broadly neutralizing antigen-binding molecule is able to inhibit (i.e. reduce), neutralize or prevent infection of most or all different subtypes or species of a virus. Optionally a broadly neutralizing antibody is able to inhibit (i.e. reduce), neutralize or prevent infection of members of at least two different types of a virus within the same family. The term “broadly neutralizing immune response" is used herein to mean an immune response elicited in a subject that is sufficient to inhibit (i.e. reduce), neutralize or prevent infection, and / or progress of infection, of at least two different subtypes or species of a virus. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and / or progress of infection, of most or all different subtypes or species of a virus. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and / or progress of infection, of members of at least two different types of a virus within the same family. Optionally a broadly neutralizing immune response is sufficient to inhibit, neutralize or prevent infection, and / or progress of infection, of members of at least two different genera of a virus within the same family. Several broadly neutralizing antibodies to pathogens are known. For example, some antibodies have been demonstrated to be capable of neutralizing viral isolates of diverse subtypes across the Filovirus family. A systematic analysis of monoclonal antibodies against Ebola virus glycoprotein is described by Saphire et al. (Cell, 2018; 174(4): 938-952). An example of a broadly neutralizing antibody to Ebolavirus is immune-elicited macaque antibody CA45, described by Zhao et al., 2017 (Cell 169, 891-904). Broadly neutralizing monoclonal antibodies against the HIV-1 envelope protein are referenced in Bruun et al. (PLoS ONE 9(10): e109196. doi:10.1371 / journal.pone.0109196) Corti etal., (Curr Opin Virol. 2017 Jun;24:60-69) provide an overview of the specificity, antiviral and immunological mechanisms of action and development into the clinic of broadly reactive monoclonal antibodies against influenza A and B viruses. Optionally the pathogen is a virus. Viruses are mainly classified by phenotypic characteristics, such as morphology, nucleic acid type, mode of replication, host organisms, and the type of disease they cause. One scheme for the classification of viruses, the Baltimore classification system, places viruses into one of seven groups depending on a combination of their nucleic acid (DNA or RNA), strandedness (single-stranded or double-stranded), sense, and method of replication: • I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses); • II: ssDNA viruses (+ strand or "sense") DNA (e.g. Parvoviruses); • III; dsRNA viruses (e.g. Reoviruses); •  IV: (+)ssRNA viruses (+ strand or sense) RNA (e.g. Picornaviruses, Togaviruses); • V: (-)ssRNA viruses (- strand or antisense) RNA (e.g. Orthomyxoviruses, Filoviruses, Arenaviruses, Rhabdoviruses); • VI: ssRNA-RT viruses (+ strand or sense) RNA with DNA intermediate in life-cycle (e.g. Retroviruses); • VII: dsDNA-RT viruses DNA with RNA intermediate in life-cycle (e.g. Hepadnavi ruses). Optionally the virus is an RNA virus. RNA viruses comprise: Group III: viruses possess double-stranded RNA genomes; Group IV: viruses possess positive-sense single-stranded RNA genomes. Many well known viruses are found in this group, including the picornaviruses (which is a family of 5 viruses that includes well-known viruses like Hepatitis A virus, enteroviruses, rhinoviruses, poliovirus, and foot-and-mouth disease virus), SARS virus, hepatitis C virus, yellow fever virus, and rubella virus; Group V: viruses possess negative-sense single-stranded RNA genomes. Ebola and Marburg viruses are well known members of this group, along with influenza virus, Lassa 10 virus, measles, mumps and rabies. Grouping of different RNA virus families under the Baltimore classification is set out in the Table below: RNA Virus Family Examples (common names) Capsid Capsid Symmetry Nucleic acid type Group Reoviridae Reovirus, rotavirus naked / enveloped Icosahedral ds III Picomaviridae Enterovirus, rhinovirus, hepatovirus, cardiovirus, aphthovirus, poliovirus, parechovirus, erbovirus, kobuvirus, teschovirus, coxsackie Naked Icosahedral ss IV Caliciviridae Norwalk virus Naked Icosahedral ss IV Togaviridae Rubella           virus, alphavirus Naked Icosahedral ss IV Arenaviridae Lymphocytic choriomeningitis virus, Lassa virus Enveloped Complex ss(-) V Flaviviridae Dengue virus, hepatitis C virus, yellow fever virus, Zika virus Enveloped Icosahedral ss IV Orthomyxoviridae Influenzavirus       A, influenzavirus        B, influenzavirus       C, isavirus, thogotovirus Enveloped Helical ss(-) V Paramyxoviridae Measles virus, mumps virus,        respiratory syncytial          virus, Rinderpest       virus, canine distemper virus Enveloped Helical ss(-) V Bunyaviridae California encephalitis virus, hantavirus Enveloped Helical ss(-) V Rhabdoviridae Rabies virus Enveloped Helical ss(-) V Filoviridae Ebola virus, Marburg virus Enveloped Helical ss(-) V Coronaviridae Corona virus Enveloped Helical ss IV Astroviridae Astrovirus Enveloped Icosahedral ss IV Bomaviridae Borna disease virus Naked Helical ss(-) V Arteriviridae Arterivirus,      equine arteritis virus Enveloped Icosahedral ss IV Hepeviridae Hepatitis E virus Enveloped Icosahedral ss IV Optionally the virus is an emerging or re-emerging RNA virus. Examples of emerging or reemerging RNA viruses include Ebola virus, Marburg virus, Lassa virus, Influenza virus, MERS coronavirus, Hendra virus, Nipah virus. Optionally the virus is a Filovirus or an Arenavirus. Optionally the virus is Ebola virus or 5 Marburg virus. Optionally the virus is Lassa virus. Optionally the pathogen is a DNA virus. Optionally the pathogen is a member of the Poxviridae family, for example monkey pox virus. DNA viruses comprise: Group I: viruses possess double-stranded DNA. Viruses that cause chickenpox and 10 herpes are found here. Group II: viruses possess single-stranded DNA. Grouping of different DNA virus families under the Baltimore classification is set out in the Table below: DNA    Virus family Examples (common names) Capsid: naked / enveloped Capsid symmetry Nucleic acid type Group Adenoviridae Adenovirus, infectious canine hepatitis virus Naked Icosahedral ds I Papovaviridae Papillomavirus, polyomaviridae, simian vacuolating virus Naked Icosahedral ds circular I Parvoviridae Parvovirus B19, canine parvovirus Naked Icosahedral ss II Herpesviridae Herpes simplex virus,    varicellazoster       virus, cytomegalovirus, Epstein-Barr virus Enveloped Icosahedral ds I Poxviridae Smallpox virus, cow pox virus, sheep pox virus, orf virus, monkey pox virus, vaccinia virus Complex coats Complex ds I Hepadnaviridae Asfarviridae Hepatitis B virus African     swine fever virus Enveloped Envelopes Icosahedral Icosahedral circular, partially ds ds VII I Optionally the pathogen is a reverse transcribing virus. Reverse transcribing viruses comprise: Group VI: viruses possess single-stranded RNA viruses that replicate through a DNA intermediate. The retroviruses are included in this group, of which HIV is a member. Group VII: viruses possess double-stranded DNA genomes and replicate using reverse transcriptase. The hepatitis B virus can be found in this group. The term “subtype” is used herein to refer to a genetic variant, or strain, of a pathogen (for example, a virus). For example, the genus Ebolavirus is a virological taxon included in the family Filoviridae. The members of this genus are called ebolaviruses. The six known ebolavirus subtypes are named for the region where each was originally identified: Bundibugyo, Reston, Sudan, Tai Forest, Zaire, and Bombali. Influenza A viruses are divided into subtypes on the basis of two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). There are 18 known HA subtypes and 11 known NA subtypes. Many different combinations of HA and NA proteins are possible. For example, an “H7N2 virus” designates an influenza A virus subtype that has an HA 7 protein and an NA 2 protein. Similarly an “H5N1” virus has an HA 5 protein and an NA 1 protein. Virus nomenclature for natural variants of the family Filoviridae is discussed in Kuhn et al. (Arch Virol. 2013 Jan; 158(1): 301-311). According to the authors a (natural) virus strain is a “variant of a given virus that is recognizable because it possesses some unique phenotypic characteristics that remain stable under natural conditions”. Such “unique phenotypic characteristics” are biological properties different from the compared reference virus, such as unique antigenic properties, host range or the signs of disease it causes. A virus variant with a simple “difference in genome sequence...is not given the status of a separate strain since there is no recognizable distinct viral phenotype”. A strain is therefore a genetically stable virus variant that differs from a natural reference virus (type variant) in that it causes a significantly different, observable, phenotype of infection (different kind of disease, infecting a different kind of host, being transmitted by different means etc.). “Genetically stable” means that the genomic changes associated with the phenotypic change are largely preserved over time through natural selection. The extent of genomic sequence variation is irrelevant for the classification of a variant as a strain since a distinct phenotype sometimes arises from few mutations. “Observable phenotype” means, for instance, that within a comparative animal experiment, it would be possible for the researcher to distinguish between the reference control virus-infected animal and the animal infected with the alleged new strain, without knowing which animal received which virus and without having any information about the differences between the two viruses. The designation of a virus variant as a virus strain is the responsibility of international expert groups. Thus far, natural filovirus strains according to this definition have not been reported. All described genetic variants of EBOV, for instance, cause a similar haemorrhagic fever in humans and even experimental animals and are transmitted similarly. None of the known EBOV genetic variants can be distinguished from others on clinical grounds alone. In fact, their variety seems to be limited to subtle differences in growth kinetics and plaque formation in vitro or subtle changes in the duration of disease in experimental animals, and ultimately derives from limited, but often stable, differences in genomic sequence. This also holds true for the different genetic variants of MARV, RAW, BDBV, RESTV, and SUDV (currently, there is only one isolate of TAFV and none of LLOV). According to Kuhn etal., a natural genetic filovirus variant is a natural filovirus that differs in its genomic consensus sequence from that of a reference filovirus (the type virus of a particular filovirus species) by <10% but is not identical to the reference filovirus and does not cause an observable different phenotype of disease (filovirus strains would be genetic filovirus variants, but most genetic filovirus variants would not be filovirus strains if a strain definition would be brought forward). Another scheme for classification of viruses is the International Committee on Taxonomy of Viruses (ICTV) system. The system shares many features with the classification system of cellular organisms, such as taxon structure. However, this system of nomenclature differs from other taxonomic codes on several points. Viral classification starts at the level of order and continues as follows, with the taxon suffixes given in italics: Order (-virales) Family (-viridae) Subfamily (-virinae) Genus (-virus) Species Species names often take the form of [Disease] virus, particularly for higher plants and animals. The establishment of an order is based on the inference that the virus families it contains have most likely evolved from a common ancestor. The majority of virus families remain unplaced. As of 2017, 9 orders, 131 families, 46 subfamilies, 803 genera, and 4,853 species of viruses have been defined by the ICTV. The orders are the Caudovirales, Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Ortervirales, Picornavirales, Bunyavirales and Tymovirales. These orders span viruses with varying host ranges. • Caudovirales are tailed dsDNA (group I) bacteriophages. • Herpesvirales contain large eukaryotic dsDNA viruses. • Ligamenvirales contains linear, dsDNA (group I) archaean viruses. • Mononegavirales include nonsegmented (-) strand ssRNA (Group V) plant and animal viruses. • Nidovirales are composed of (+) strand ssRNA (Group IV) viruses with vertebrate hosts. • Ortervirales contain single-stranded RNA and DNA viruses that replicate through a DNA intermediate (Groups VI and VII). • Picornavirales contains small (+) strand ssRNA viruses that infect a variety of plant, insect and animal hosts. • Tymovirales contain monopartite (+) ssRNA viruses that infect plants. • Bunyavirales contain tripartite (-) ssRNA viruses (Group V). According to the ICTV, a virus species is "a monophyletic group of viruses whose properties can be distinguished from those of other species by multiple criteria." The term “isolate” is used herein to refer to a pure pathogen sample that has been obtained from an infected individual. A virus-infected cell will, after only one round of replication, already contain a population of genomes, and virions derived from these genomes will vary slightly from each other. Likewise, a sample taken from an infected individual will contain numerous virions, many of which vary slightly. Consequently, an “isolate” refers to a population, and “the sequence” of an “isolate" is a consensus sequence of the population of genomes present in the analyzed sample. A virus isolate may be defined as “an instance of a particular virus”. A natural filovirus isolate is an instance of a particular natural filovirus or of a particular genetic variant. Isolates can be identical or slightly different in consensus or individual sequence from each other. The term “outbreak” is used herein to refer to the occurrence of more cases of a disease than would normally be expected in a defined institution (e.g. a hospital or a medical treatment centre), community, geographical area, or period of time. An outbreak may occur in a restricted geographical area, or may extend over several countries. It may last for a few days or weeks, or for several years. The number of cases indicating presence of an outbreak will vary according to the pathogen, size and type of population exposed, previous experience or lack of exposure to the disease, and time and place of occurrence. Therefore, the status of an outbreak is relative to the usual frequency of the disease in the same area, 12 among the same community, at the same season of the year. The existence of an outbreak may be established by comparing current information with previous incidence in the population or community during the same time of year to determine if the observed number of cases exceeds the expected number. Optionally an outbreak of a pathogen may refer to the occurrence of more cases of a disease caused by the pathogen than would normally be expected in a region (for example a continental region) or country, or in a population or community, over one or more seasons or over a year. Optionally an outbreak of a pathogen (such as a virus) is the occurrence of more cases of a disease caused by the pathogen than would normally be expected in a region (for example a continental region) over a season. Optionally an outbreak of a pathogen (such as a virus) is the occurrence of more cases of a disease caused by the pathogen than would normally be expected in a population over a season. Examples of continental regions include regions of Africa: • Northern Africa: Algeria; Canary Islands; Ceuta; Egypt; Libya; Madeira; Melilla; Morocco; Sudan; Tunisia; Western Sahara; • Eastern Africa: Burundi; Comoros; Djibouti; Eritrea; Ethiopia; Kenya; Madagascar; Malawi; Mauritius; Mayotte; Mozambique; Reunion; Rwanda; Seychelles; Somalia; South Sudan; Tanzania; Uganda; Zambia; Zimbabwe; • Central Africa: Angola; Cameroon; Central African Republic; Chad; Democratic Republic of the Congo; Republic of the Congo; Equatorial Guinea; Gabon; Sao Tome and Principe; • Western Africa: Benin; Burkina Faso; Cape Verde; Ivory Coast; Gambia; Ghana; Guinea; Guinea-Bissau; Liberia; Mali; Mauritania; Niger; Nigeria; Saint Helena; Senegal; Sierra Leone; Togo; • Southern Africa: Botswana; Lesotho; Namibia; South Africa; Swaziland. Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle. Pseudotyped particles do not carry the genetic material to produce additional viral envelope proteins, so the phenotypic changes cannot be passed on to progeny viral particles. A “pseudotype” may be defined as a hybrid virus particle comprising a protein nucleocapsid (‘core’) encasing a nucleic acid (RNA or DNA) genome, with the core itself being encapsulated in a lipid ‘envelope’ 13 membrane derived from the host cell. This envelope gained when cores exit from the cell by ‘budding’ includes proteins derived from other viruses. Many of these heterologous envelope proteins are antigenic targets for the host immune system. In pseudotypes, one or more of these envelope proteins may derive from study viruses. Many pseudotypes also carry foreign genes, called ‘transfer’ genes, engineered into their genome. When in the presence of susceptible cells, the envelope proteins bind to cell receptors permitting cellular entry, eventually resulting in transfer gene expression. Rhabdoviruses (e.g. Vesticular Stomatitis Virus, VSV) and Retroviruses (e.g. Lentiviruses) have been extensively exploited as cores for pseudotyping. In the case of retroviruses, their key characteristic is the ability to reverse transcribe their dimeric single-stranded RNA genome into a double-stranded deoxyribonucleic acid (dsDNA) copy, which is subsequently integrated into the cell genome via the use of viral and cellular enzymes. For retroviral pseudotypes, this usually leads to expression of the transfer / reporter gene, the latter being readily quantifiable. Reporter gene expression directly correlates with efficiency of viral envelope / receptor interaction, and conversely whether individual antibody responses or antiviral agents could interfere with the entry and replication process of the native virus. Binding of viral pseudotypes to broadly neutralizing antigen-binding molecules may be measured using any suitable technique known to the skilled person, for example by haemagglutination inhibition (HI) assay, or by enzyme-linked immunosorbent assay (ELISA). ELISA analysis of antibody binding to glycoprotein (GP) is described in Saphire etal., 2018 (Cell 174(4): 938-952) in relation to analysis of monoclonal antibodies against Ebola virus GP. Production of retroviral pseudotypes, and their use in pseudotype neutralisation assays and immunogenicity testing, is reviewed in detail in Temperton et al., 2015 (Retroviral Pseudotypes - From Scientific Tools to Clinical Utility. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002 / 9780470015902.a0021549.pub2). Representatives of all seven genera of retroviruses have been employed in pseudotyping studies but to date only gammaretroviral or lentiviral pseudotypes are widely used. Lentiviruses are a genus of the Retroviridae family, which unlike gammaretroviruses, can infect non-proliferating cells, which makes them amenable for gene therapy applications involving highly differentiated or quiescent cells (e.g. in Go cell cycle phase) including muscle or neurons. The most common lentivirus vector used for pseudotyping is HIV-type 1 (HIV-1), although simian immunodeficiency virus has also been employed. Generation of retroviral pseudotypes is achieved through the introduction of cloned versions of foreign envelope protein gene(s), core retroviral genes and transfer gene (e.g. reporter or therapeutic gene) concurrently into producer cells, normally highly transfectable cell lines such as human embryo kidney (HEK)293 clone 17 T cells (American Type Culture Collection #CRL-11268) (Pear et aL, 1993, PNAS USA 90: 8392-8396). 1. The envelope plasmid. Envelope gene(s) of the study virus are cloned into an appropriate expression plasmid. Genes are usually derived via polymerase chain reaction amplification of viral cDNA using specific primers or from custom gene synthesis. Some expression vectors are commercially available and utilise different, usually strong constitutive gene promoters (e.g. from the human cytomegalovirus (CMV) immediate early gene), which can influence the efficacy of pseudotype generation. 2. The retroviral gag-pol plasmid. The gag and pol genes encode polyproteins which are subsequently cleaved to release structural proteins (including matrix, capsid and nucleocapsid) found within the core, and proteins involved in viral replication (protease, reverse transcriptase and integrase) responsible for processing the structural proteins, converting the ssRNA viral genome into dsDNA and ensuring integration (of the transfer gene) into the host cell genome. In addition, in a lentiviral gag-pol construct, the rev gene is included. The Rev protein is involved in the export of viral mRNAs from nucleus to cytosol for translation. 3. The transfer / reporter plasmid. This is the gene that is stably integrated into the host cell DNA, from where the gene is expressed via various c / s-acting transcriptional elements. The transfer plasmid contains a packaging signal upstream of the gene to ensure incorporation of viral RNA containing the gene into the viral core during pseudotype generation. Once the cellular machinery has transcribed and translated the transfected genes, an RNA dimer of the transfer gene (region between the long terminal repeats; LTR) is incorporated into the pseudotype via the packaging signal. As the transfer plasmid is the only one engineered to contain a packaging signal, no other nucleic acids are incorporated into the mature pseudotype particle. A domain at the N-terminus of Gag targets the nucelocapsid to the cell plasma membrane, into which the envelope protein(s) has been inserted. The pseudotype particles budding from the cell are encapsulated in the cell membrane, which forms the viral envelope. Pseudotyped viruses are released into the producer cell culture medium. This supernatant can be titrated onto target cells to measure the concentration of functional particles. These attach to the cells via envelope protein-receptor interaction, followed by membrane fusion 15 and internalisation. The pseudotype genome, bearing the transfer / reporter gene is integrated into the host cell DNA, from where it is expressed. The level of reporter gene expression correlates with the level of transduction by viable particles. As only the transfer gene is present in the pseudotype, no viral proteins are produced in target cells, so further pseudotype production and propagation does not occur. This provides safety in working with pseudotypes compared to working with the wildtype virus. Green fluorescent protein (GFP)-based pseudotypes are readily titrated using fluorescence microscopy or flow cytometry, luciferase pseudotypes by luminometry, and beta-galactosidase (3-gal) pseudotypes by colour reaction. Many standard serological assays measure only antibody binding (hemagglutination inhibition (HI) and ELISA), rather than the inhibition of viral infectivity. Neutralisation assays allow for sensitive detection of functional antibody responses. For high-containment viruses (such as Ebola), however, these assays are not widely applicable owing to the requirement for high biosafety laboratory facilities and specially trained personnel. Using retroviral and lentiviral particles pseudotyped with the envelopes of such pathogens as ‘surrogate viruses’ for use in neutralisation assays is one way of circumventing this issue. Using a pseudotype strategy, only the envelope protein(s) of the virus is required, with no possibility of recombination or native virus escape. These pseudotypes undergo abortive replication and are unable to give rise to replication-competent progeny. Pseudotypes are excellent serological reagents for virus neutralisation assays as the virions can contain a reporter gene and bear heterologous viral envelope proteins on the surface. The transfer of these reporter genes to target cells depends on the function of the viral envelope protein; therefore, the titre of neutralising antibodies against the envelope can be measured by a reduction in reporter gene transfer and expression. PV neutralisation assays have now been developed for a wide range of RNA viruses, from numerous virus families (see Table 1 of Temperton et al., supra). Pseudotype-based influenza neutralisation assays have been shown to be highly efficient for the measurement of broadly-neutralising antibodies making them ideal serological tools for the study of cross-reactive responses against multiple subtypes with pandemic potential (Corti et a / ., 2011, Science 333 (6044): 850-856). Production of lentiviral vectors pseudotyped with filoviral glycoproteins is described in Sinn eta!., 2017 (Methods Mol Biol. 2017;1628:65-78). An example of a suitable general method for production of viral pseudotypes is as follows: For transfection, 5x106 HEK-293T cells are plated 24 h prior to addition of a complex comprising plasmid DNA and PEI, which facilitates DNA transport into the cells. A retroviral gag-pol plasmid and a reporter plasmid are transfected concurrently with the required envelope plasmid. An example of a suitable neutralization assay is as follows: In a 96-well plate, ~100xTCID50 pseudotyped virus that resulted in an output of 1x105 relative light units (RLU) is incubated with dilutions of sera for 1 h at 37% (5% CO2) before the addition of 1x104 target cells. These are incubated fora further 48 h, after which the media is removed and replaced with a 50:50 mix of fresh media and luciferase reagent. Luciferase activity is detected 2.5 min later by reading the plates on a luminometer. For all results, background RLU (virus alone or DEnv) is deducted before analysis. Saphire et al. (supra) describes three independent assays for evaluation of mAb neutralization in relation to analysis of monoclonal antibodies against Ebola virus GP: i) biologically contained EBOV (AVP30) (Halfmann et al., 2008, Proc Natl Acad Sci USA. 2008; 105:1129-1133); and ii) authentic EBOV performed under BSL-2+, BSL-3 and BSL-4 containment; and ill) replication-competent vesicular stomatitis virus bearing EBOV GP (rVSV). Neutralization of EbolaAVP30-RenLuc virus An Ebola virus in which the reporter gene Renilla luciferase is substituted for the viral transcription factor VP30 (EbolaAVP30-RenLuc virus) is used to complement a Vero cell line that stably expresses VP30 in trans (Vero VP30), thus allowing analysis at BSL-3 (Halfmann et aL, 2008). A total of 5 x 103 focus forming units of EbolaAVP30-RenLuc virus diluted in 2% fetal calf serum in minimal essential medium is incubated with 50 pg / ml monoclonal antibody for 3 hours at 37 °C. The virus / antibody mixture at a multiplicity of infection (MOI) of 0.001 is then added to Vero VP30 cells, seeded the previous day in 96-well plates at 9 x 103 cells / well and incubated for three days at 37 °C and 5% CO2. If used, guinea pig complement (Cedarlane) is added to the minimal essential medium at a final concentration of 10%. Then a live cell luciferase substrate, EnduRen (Promega), is incubated with the cells for three hours before luciferase values are measured as relative light units (RLU) using a Tecan M1000 plate reader (Tecan). Assays are performed in duplicate and a known neutralizing (GP 133 / 3.16) and non-neutralizing monoclonal (VP35 5 / 69.3.2) is used as a positive and negative control, respectively. Antibodies that neutralized luciferase signals by >95% are defined as strong neutralizers, whereas inhibition of luciferase signals by 50%-17 94% are considered moderate neutralizers and those that have 49% or lower inhibition are categorized as weak / non-neutralizers. Neutralization of authentic EBOV Assays to assess neutralization of authentic EBOV are performed according to the method described in Holtsberg et al. (Holtsberg et aL, 2015, J Virol. 2015; 90:266-278). Vero E6 cells are seeded 2.5 x 1O“4 cells / well in the inner 60 wells of black 96-well plates 24 hours prior to virus infection. Antibodies are serially diluted in Vero growth medium (Eagle minimum essential medium with Earle's salts and L-glutamine, 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin) at two times the desired final concentration (50 pg / ml), mixed with an equal volume of live EBOV, and incubated for 1 hour at 37 °C with mixing every 15 min. The antibody / virus mixture at a MOI of 0.2 is then added to the Vero cells and incubated for 1 hr at 37 °C, washed with PBS, and growth medium alone is added to all wells and the plates are incubated for an additional 48 hr at 37 °C. The cells are then fixed with 10% neutral buffered formalin and the percentage of infected cells is determined by an indirect immunofluorescence assay using the EBOV-specific human mAb KZ52 and goat anti-human IgG conjugated to Alexa Fluor 488 (Molecular Probes) as a secondary antibody. Images are acquired at 20 fields / well with a 20x objective lens on an Operetta High Content Imaging System (Perkin-Elmer). Operetta images are analyzed with a customized algorithm built from image analysis functions available in Harmony software (Perkin-Elmer). The percentage of inhibition for each antibody is determined relative to control cells incubated with media alone. Antibodies that reduced the percentage of infected cells by >80% are categorized as strong neutralizers, whereas those that reduced infection by between 50% and 79% and less than 50% are considered as moderate neutralizers and weak / non-neutralizers, respectively. Neutralization of rVSV-EBOV GP Recombinant vesicular stomatitis virus (VSV) expressing both eGFP and recombinant surface GP (rVSV-EBOV) in place of VSV G was described previously (Wee et aL, 2016, Science; 354:350-354; Wong et al., 2010, Virol.; 84:163-175). For neutralization assays, Vero cells are seeded at 6.0 x 104 cells / well and cultured overnight in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 100 LU. / ml penicillin and 100 pg / ml streptomycin at 37 °C and 5% CO2. The next day, virus is incubated with serial 3-fold antibody dilutions beginning at 330 nM (~50 pg / ml) in serum-free EMEM for one hour at room temperature before infecting Vero cell monolayers in 96-well plates. The amount of virus used for infection is determined based on titration of viral stock to achieve 35-50% final infection in control wells without antibody (MOI ~ 0.1 infectious units per cell). The virus is incubated with the cells in 50% v / v / EMEM supplemented with 2% FBS, 100 I.U. / ml penicillin and 100 pg / ml streptomycin at 37 °C and 5% CO2 for 14-16 hours before the cells are fixed and the nuclei stained with Hoescht. rVSV infectivity is measured by counting EGFP-positive cells in comparison to the total number of cells indicated by nuclear staining using a Cellinsight CX5 automated microscope and accompanying software (Thermo Scientific). The infection level in control wells lacking antibody is set to 100% and the infection is normalized to that value for each antibody dilution, which are tested in triplicate. The mean value is determined and the full 9-point dilution curve is used to determine the half-maximal inhibitor concentration, IC50 using GraphPad Prism version 6. Antibodies having IC50 5 nM are considered strong neutralizers whereas antibodies having 5 nM < IC50 < 50 nM and < 50 nM are considered moderate neutralizers and weak / non-neutralizers, respectively. The un-neutralized fraction, an indicator of antibody potency, is also determined using antibodies at the highest concentration tested, 330 nM, and measuring the GFP signal relative to that of untreated control cells. Those that reduce the signal by >98%, 50-98%, and less than 50% are considered strong, moderate, and weak / non-neutralizers, respectively. Optionally the expression vector is also a vaccine vector. Examples of vaccine vector include a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector. Viral vaccine vectors use live viruses to carry nucleic acid (for example, DNA or RNA) into human or non-human animal cells. The nucleic acid contained in the virus encodes one or more antigens that, once expressed in the infected human or non-human animal cells, elicit an immune response. Both humoral and cell-mediated immune responses can be induced by viral vaccine vectors. Viral vaccine vectors combine many of the positive qualities of nucleic acid vaccines with those of live attenuated vaccines. Like nucleic acid vaccines, viral vaccine vectors carry nucleic acid into a host cell for production of antigenic proteins that can be tailored to stimulate a range of immune responses, including antibody, T helper cell (CD4+ T cell), and cytotoxic T lymphocyte (CTL, CD8+ T cell) mediated immunity. Viral vaccine vectors, unlike nucleic acid vaccines, also have the potential to actively invade host cells and replicate, much like a live attenuated vaccine, further activating the immune system like an adjuvant. A viral vaccine vector therefore generally comprises a live attenuated virus that is genetically engineered to carry nucleic acid (for example, DNA or RNA) encoding protein antigens from an unrelated organism. Although viral vaccine vectors are generally able to produce stronger immune responses than nucleic acid vaccines, for some diseases viral vectors are used in combination with other vaccine technologies in a strategy called 19 heterologous prime-boost. In this system, one vaccine is given as a priming step, followed by vaccination using an alternative vaccine as a booster. The heterologous prime-boost strategy aims to provide a stronger overall immune response. Viral vaccine vectors may be used as both prime and boost vaccines as part of this strategy. Viral vaccine vectors are reviewed by Ura etal., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013;2:97-105). Optionally the viral vaccine vector is based on a viral delivery vector, such as a Poxvirus (for example, Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g. HSV, CMV, Adenovirus of any host species), Morbillivirus (e.g. measles), Alphavirus (e.g. SFV, Sendai), Flavivirus (e.g. Yellow Fever), or Rhabdovirus (e.g. VSV)-based viral delivery vector, a bacterial delivery vector (for example, Salmonella, E.coli), an RNA expression vector, or a DNA expression vector. Optionally the vector is a pEVAC-based expression vector. A pEVAC expression vector is described in more detail in Example 7 below. Inclusion of one or more nucleotide sequences encoding ancestral amino acid sequence may be advantageous because ancestral amino acid sequence that is highly conserved with extant amino acid sequence is expected to be of structural and / or functional importance for the survival and / or propagation of the pathogen. Also, as pathogen isolates can be extremely diverse (especially, for example, isolates of emerging or re-emerging pathogens, such as emerging or re-emerging RNA viruses), a vaccine designed to work on one patient's pathogen population might not work for a different patient, because the evolutionary distance between these two pathogen populations may be large. However, their most recent common ancestor is closer to each of the two pathogen populations than they are to each other. Thus, a vaccine designed for a common ancestor could have a better chance of being effective for a larger proportion of circulating strains. Ancestral sequence reconstruction (ASR) is discussed in Randall et a! (Nat. Commun. 7:12847 doi: 10.1038 / ncomms 12847 (2016)). The authors reference a definition of ASR as “the process of analyzing modern sequences within an evolutionary / phylogenetic context to infer the ancestral sequences at particular nodes of a tree". Ancestral sequence reconstruction (ASR) is used in the study of molecular evolution. Unlike conventional evolutionary approaches to studying proteins, by horizontal comparison of related protein homologues from different branch ends of a phylogenetic tree, ASR probes the statistically inferred ancestral proteins within the nodes of the tree in a vertical manner (see Figure 1). A phylogenetic tree is a branching diagram showing the evolutionary relationships among various biological species or other entities based upon similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of those descendants. In ASR, several related homologues of a protein of interest are selected and aligned in a multiple sequence alignment (MSA), a phylogenetic tree is constructed with statistically inferred sequences at the nodes of the branches. These sequences are the so-called 'ancestors'. The process of synthesising the corresponding DNA, transforming it into a cell and producing a protein is the so-called 'reconstruction'. Ancestral sequences are typically calculated by maximum likelihood, however Bayesian methods are also implemented. Because the ancestors are inferred from a phylogeny, the topology and composition of the phylogeny plays a major role in the output ASR sequences. ASR does not claim to recreate the actual sequence of the ancient protein / DNA, but rather a sequence that is likely to be similar to the one that was at the node. Maximum likelihood (ML) methods work by generating a sequence where the residue at each position is predicted to be the most likely to occupy that position by the method of inference used. Typically, this is a scoring matrix (similar to those used in BLASTS or MSAs) calculated from extant sequences. Alternate methods include maximum parsimony (MP) that construct a sequence based on a model of sequence evolution, usually the idea that the minimum number of nucleotide sequence changes represents the most efficient route for evolution to take and the most likely. MP is often considered the least reliable method for reconstruction as it arguably oversimplifies evolution to a degree that is not applicable on the billion year scale. Other methods include Bayesian methods, which involve the consideration of residue uncertainty. Such methods are sometimes used to compliment ML methods, but typically produce more ambiguous sequences (i.e. sequences which include residue positions where no clear substitution can be predicted). Often in such cases, several ASR sequences are produced, encompassing most of the ambiguities, and compared to one-another. Methods and algorithms for ASR are described in more detail below, based on description in Joy et al., 2016, PLOS Computational Biology 12(7): DOI:10.1371 / journal.pcbi.1004763. Optionally ASR is conducted with at least 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600 , 700 , 800 , 900, 1000, 106, 109, or 1012 different sequences. In some instances, the greater the number of sequences that are used, the better. Optionally each of the sequences used for the multiple sequence alignment is a full length sequence of a pathogen polypeptide of a pathogen isolate. Any attempt at ancestral reconstruction begins with a phylogeny. In general, a phylogeny is a tree-based hypothesis about the order in which populations (referred to as taxa) are related by descent from common ancestors. Observed taxa are represented by the tips or terminal nodes of the tree that are progressively connected by branches to their common ancestors, which are represented by the branching points of the tree that are usually referred to as the ancestral or internal nodes. Eventually, all lineages converge to the most recent common ancestor of the entire sample of taxa. In the context of ancestral reconstruction, a phylogeny is often treated as though it were a known quantity (with Bayesian approaches being an important exception). Because there can be an enormous number of phylogenies that are nearly equally effective at explaining the data, reducing the subset of phylogenies supported by the data to a single representative, or point estimate, can be a convenient and sometimes necessary simplifying assumption. Ancestral reconstruction can be thought of as the direct result of applying a hypothetical model of evolution to a given phylogeny. When the model contains one or more free parameters, the overall objective is to estimate these parameters on the basis of measured characteristics among the observed taxa (sequences) that descended from common ancestors. Parsimony is an important exception to this paradigm. It is based on the heuristic that changes in character state are rare, without attempting to quantify that rarity. Maximum Parsimony Parsimony refers to the principle of selecting the simplest of competing hypotheses. In the context of ancestral reconstruction, parsimony endeavours to find the distribution of ancestral states within a given tree that minimizes the total number of character state changes that would be necessary to explain the states observed at the tips of the tree. This method of maximum parsimony) is one of the earliest formalized algorithms for reconstructing ancestral states. Maximum parsimony can be implemented by one of several algorithms. One of the earliest examples is Fitch's method (Fitch WM. Toward defining the course of evolution: minimum change for a specific tree topology. Systematic Biology. 1971 ;20(4):406-16), which assigns ancestral character states by parsimony via two traversals of a rooted binary tree. The first stage is a post-order traversal that proceeds from the tips toward the root of a tree by visiting descendant (child) nodes before their parents. Initially, the set of possible character states are determined, Si for the i-th ancestor based on the observed character states of its descendants. Each assignment is the set intersection of the character states of the ancestor's descendants; if the intersection is the empty set, then it is the set union. In the latter case, it is implied that a character state change has occurred between the ancestor and one of its two immediate descendants. Each such event counts towards the algorithm's cost function, which may be used to discriminate among alternative trees on the basis of maximum parsimony. Next, a preorder traversal of the tree is performed, proceeding from the root towards the tips. Character states are then assigned to each descendant based on which character states it shares with its parent. Since the root has no parent node, one may be required to select a character state arbitrarily, specifically when more than one possible state has been reconstructed at the root. Parsimony methods are intuitively appealing and highly efficient, such that they are still used in some cases to seed ML optimization algorithms with an initial phylogeny (Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006;22:2688-90. pmid: 16928733). However, they suffer from several issues: 1. Variation in rates of evolution. Fitch's method assumes that changes between all character states are equally likely to occur; thus, any change incurs the same cost for a given tree. This assumption is often unrealistic and can limit the accuracy of such methods. For example, transitions tend to occur more often than transversions in the evolution of nucleic acids. This assumption can be relaxed by assigning differential costs to specific character state changes, resulting in a weighted parsimony algorithm (Sankoff D. Minimal mutation trees of sequences. SIAM Journal on Applied Mathematics. 1975;28(1 ):35-42). 2. Rapid evolution. The upshot of the "minimum evolution" heuristic underlying such methods is that such methods assume that changes are rare and thus are inappropriate in cases where change is the norm rather than the exception (Schluter D, Price T, Mooers AO, Ludwig D. Likelihood of ancestor states in adaptive radiation. Evolution. 1997;51 (6):1699-711; Felsenstein J. Maximum likelihood and minimum-steps methods for estimating evolutionary trees from data on discrete characters. Systematic Biology. 1973;22(3):240-9). 3. Variation in time among lineages. Parsimony methods implicitly assume that the same amount of evolutionary time has passed along every branch of the tree. Thus, they do not account for variation in branch lengths in the tree, which are often used to quantify the passage of evolutionary or chronological time. This limitation makes the technique liable to infer that one change occurred on a very short branch rather than multiple changes occurring on a very long branch, for example. This shortcoming is addressed by model-based methods (both ML and Bayesian methods) that infer the stochastic process of evolution as it unfolds along each branch of a tree (Li G, Steel M, Zhang L. More taxa are not necessarily better for the reconstruction of ancestral character states. Systematic biology. 2008;57(4):647-53). 4.Statistical justification. Without a statistical model underlying the method, its estimates do not have well-defined uncertainties. Maximum Likelihood (ML) ML methods of ancestral sequence reconstruction treat the character states at internal nodes of the tree as parameters and attempt to find the parameter values that maximize the probability of the data (the observed character states) given the hypothesis (a model of evolution and a phylogeny relating the observed sequences or taxa). Some of the earliest ML approaches to ancestral reconstruction were developed in the context of genetic sequence evolution (Yang Z, Kumar S, Nei M. A new method of inference of ancestral nucleotide and amino acid sequences. Genetics. 1995;141(4):1641-50; Koshi JM, Goldstein RA. Probabilistic reconstruction of ancestral protein sequences. Journal of Molecular Evolution. 1996;42(2):313-20); similar models were also developed for the analogous case of discrete character evolution (Pagel M. The maximum likelihood approach to reconstructing ancestral character states of discrete characters on phylogenies. Systematic biology. 1999;48(3):612-22). These approaches employ the same probabilistic framework as used to infer the phylogenetic tree (Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of molecular evolution. 1981:17(6):368-76). In brief, the evolution of a genetic sequence is modelled by a time-reversible continuous time Markov process. In the simplest of these, all characters undergo independent state transitions (such as nucleotide substitutions) at a constant rate over time. This basic model is frequently extended to allow different rates on each branch of the tree. In reality, mutation rates may also vary over time (due, for example, to environmental changes); this can be modelled by allowing the rate parameters to evolve along the tree, at the expense of having an increased number of parameters. A model defines transition probabilities from states i to j along a branch of length t (in units of evolutionary time). The likelihood of a phylogeny is computed from a nested sum of transition probabilities that corresponds to the hierarchical structure of the proposed tree. At each node, the likelihood of its descendants is summed over all possible ancestral character states at that node: MS                MS         / where the likelihood of the subtree rooted at node x with direct descendants y and z is computed, S, denotes the character state of the / -th node, tjjis the branch length (evolutionary time) between nodes i and j, and Q is the set of all possible character states (for example, the nucleotides A, C, G, and T). Thus, the objective of ancestral reconstruction is to find the assignment to Sx for all x internal nodes that maximizes the likelihood of the observed data for a given tree. Rather than compute the overall likelihood for alternative trees, the problem for ancestral reconstruction is to find the combination of character states at each ancestral node with the highest marginal ML. Generally speaking, there are two approaches to this problem. First, one may work upwards from the descendants of a tree to progressively assign the most likely character state to each ancestor taking into consideration only its immediate descendants. This approach is referred to as marginal reconstruction. It is akin to a greedy algorithm that makes the locally optimal choice at each stage of the optimization problem. While it can be highly efficient, it is not guaranteed to attain a globally optimal solution to the problem. Second, one may instead attempt to find the joint combination of ancestral character states throughout the tree that jointly maximizes the likelihood of the data. Thus, this approach is referred to as joint reconstruction. While it is not as rapid as marginal reconstruction, it is also less likely to be caught in the local optima in nonconvex objective functions that modern optimization methods and heuristics are designed to avoid. In the context of ancestral reconstruction, this means that a marginal reconstruction may assign a character state to the immediate ancestor that is locally optimal but deflects the joint distribution of ancestral character states away from the global optimum. Joint reconstruction is more computationally complex than marginal reconstruction. Nevertheless, efficient algorithms for joint reconstruction have been developed with a time complexity that is generally linear with the number of observed taxa or sequences. ML-based methods of ancestral reconstruction tend to provide greater accuracy than MP methods in the presence of variation in rates of evolution among characters (or across sites in a genome). However, these methods are not yet able to accommodate variation in rates of evolution over time, otherwise known as heterotachy. If the rate of evolution for a specific character accelerates on a branch of the phylogeny, then the amount of evolution that has occurred on that branch will be underestimated fora given length of the branch and assuming a constant rate of evolution for that character. In addition to that, it is difficult to distinguish heterotachy from variation among characters in rates of evolution. Since ML (unlike maximum parsimony) requires the investigator to specify a model of evolution, its accuracy may be affected by the use of a grossly incorrect model (model misspecification). Furthermore, ML can only provide a single reconstruction of character states (what is often referred to as a "point estimate") - when the likelihood surface is highly nonconvex, comprising multiple peaks (local optima), then a single point estimate cannot provide an adequate representation, and a Bayesian approach may be more suitable. Bayesian Inference Bayesian inference uses the likelihood of observed data to update the investigator's belief, or prior distribution, to yield the posterior distribution. In the context of ancestral reconstruction, the objective is to infer the posterior probabilities of ancestral character states at each internal node of a given tree. Moreover, one can integrate these probabilities over the posterior distributions over the parameters of the evolutionary model and the space of all possible trees. This can be expressed as an application of Bayes' theorem: where S represents the ancestral states, D corresponds to the observed data, and 0 represents both the evolutionary model and the phylogenetic tree. P(D\S,0) is the likelihood of the observed data that can be computed by Felsenstein's pruning algorithm as given above. P(S|0) is the prior probability of the ancestral states for a given model and tree. Finally, P(D\0) is the probability of the data for a given model and tree, integrated over all possible ancestral states. Two formulations are given to emphasize the two different applications of Bayes' theorem, discussed below. One of the first implementations of a Bayesian approach to ancestral sequence reconstruction was developed by Yang and colleagues, where the ML estimates of the evolutionary model and tree, respectively, were used to define the prior distributions. Thus, their approach is an example of an empirical Bayes method to compute the posterior probabilities of ancestral character states; this method was first implemented in the software package PAML (Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Molecular biology and evolution. 2007;24(8):1586-91). In terms of the above Bayesian rule formulation, the empirical Bayes method fixes to the empirical estimates of the model and tree obtained from the data, effectively dropping from the posterior likelihood and prior terms of the formula. Moreover, Yang and colleagues (Yang Z, Kumar S, Nei M. A new method of inference of ancestral nucleotide and amino acid sequences. Genetics. 1995;141(4):1641-50) used the empirical distribution of site patterns (i.e., assignments of nucleotides to tips of the tree) in their alignment of observed nucleotide sequences in the denominator in place of exhaustively computing P(D) over all possible values of S, given 0. Computationally, the empirical Bayes method is akin to the ML reconstruction of ancestral states except that, rather than searching for the ML assignment of states based on their respective probability distributions at each internal node, the probability distributions themselves are reported directly. Empirical Bayes methods for ancestral reconstruction require the investigator to assume that the evolutionary model parameters and tree are known without error. When the size or 26 complexity of the data makes this an unrealistic assumption, it may be more prudent to adopt the fully hierarchical Bayesian approach and infer the joint posterior distribution over the ancestral character states, model, and tree (Huelsenbeck JP, Bollback JP. Empirical and hierarchical Bayesian estimation of ancestral states. Systematic Biology. 2001:50(3):351-66). Huelsenbeck and Bollback first proposed a hierarchical Bayes method to ancestral reconstruction by using Markov chain Monte Carlo (MCMC) methods to sample ancestral sequences from this joint posterior distribution. A similar approach was also used to reconstruct the evolution of symbiosis with algae in fungal species (lichenization) (Lutzoni F, Pagel M, Reeb V. Major fungal lineages are derived from lichen symbiotic ancestors. Nature. 2001 ;411(6840):937-40). For example, the Metropolis-Hastings algorithm for MCMC explores the joint posterior distribution by accepting or rejecting parameter assignments on the basis of the ratio of posterior probabilities. Thus, the empirical Bayes approach calculates the probabilities of various ancestral states for a specific tree and model of evolution. By expressing the reconstruction of ancestral states as a set of probabilities, one can directly quantify the uncertainty for assigning any particular state to an ancestor. On the other hand, the hierarchical Bayes approach averages these probabilities over all possible trees and models of evolution, in proportion to how likely these trees and models are, given the data that has been observed. The fully Bayesian approach is limited to analyzing relatively small numbers of sequences or taxa because the space of all possible trees rapidly becomes too vast, making it computationally infeasible for chain samples to converge in a reasonable amount of time. Pathogens, especially emerging or re-emerging pathogens, such as emerging or reemerging RNA viruses, evolve at an extremely rapid rate, orders of magnitude faster than mammals or birds. For these organisms, ancestral reconstruction can be applied on a much shorter time scale, for example, to reconstruct the global or regional progenitor of an epidemic that has spanned decades rather than millions of years. It has been proposed that such reconstructed strains be used as targets for vaccine design efforts as opposed to sequences isolated from patients in the present day (Gaschen et al., Science. 2002;296(5577):2354-60). There are many software packages available that perform ancestral sequence reconstruction. The following table (taken from Joy etal., 2016, PLOS Computational Biology 12(7): DOI:10.1371 / journal.pcbi.1004763) provides a representative sample of the extensive variety of packages that implement methods of ancestral reconstruction with different strengths and features: jssswSr: Csijssrigi Tysw C pgj: isifsos {Cj >:» 5M<SsW S&> etatwers :              kKSPOSi i w MtM. $ : OwSS®. W; Q.© ass <wsa s<$a's> :             SSfSSSS W s® sm. mu, earn ®«WsSS. : IMSte S® SEWS .^•WSSWS as s^sasmssSssa i L^iW. WWS § * SSSA    WA rWW s SSS • :®WSS 5 S w' 'W'W) Kv-wt w ss-®w <w®s wws OsSSSSSSMm’ia as :         ap'ip-spw i       ass w Ws ® xWSV <S    WWS ::®VS S'» $f«WSSW -JiTS ......... * : Sw Osss® as® i :,Ssw®, a® W l-a®\ SW- vs'W''* !WU® €WST^.. w as ia®S®a®:s<sS i ®sSssS®SS j.SiSSX® j              ...... ..... as w> S $ W&::..... s :: :: as i as®sw s i Mil. s ?«;«:<■.•:•■ s : ® S, te., Mi V. ' W ' talSK^S rk i SSs:Us®ss i J-Wax; $ w :j i xk\ X ■* '!'>a XXs 's \X X s : Os®®:: MM W <>J-WS s i : aw®® MX; w s ew j Fawssiw MX; Ste. W                 i^iSS ■:<< k x           X\ s ; ®s W: s --5       <» S sWES :: S; iJ-ixy Vk::$>SY< s : $ Mtytetwi tosiy s The majority of these software packages are designed for analyzing genetic sequence data. For example, PAML (Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Molecular biology and evolution. 2007;24(8): 1586-91) is a collection of programs for the 5 phylogenetic analysis of DNA and protein sequence alignments by ML. Ancestral reconstruction can be performed using the codeml program. HyPhy, Mesquite, and MEGA are also software packages for the phylogenetic analysis of sequence data, but are designed to be more modular and customizable. HyPhy (Pond SLK, Muse SV. HyPhy: hypothesis testing using phylogenies. Statistical methods in molecular evolution: Springer; 2005. p. 12510   81) implements a joint ML method of ancestral sequence reconstruction (Pupko T, Pe I, Shamir R, Graur D. A fast algorithm for joint reconstruction of ancestral amino acid sequences. Molecular Biology and Evolution. 2000;17(6):890-6) that can be readily adapted to reconstructing a more generalized range of discrete ancestral character states such as geographic locations by specifying a customized model in its batch language. Mesquite (Maddison W, Maddison D. Mesquite: a modular system for evolutionary analysis. 2.75 ed20011) provides ancestral state reconstruction methods for both discrete and continuous characters using both maximum parsimony and ML methods. It also provides several visualization tools for interpreting the results of ancestral reconstruction. MEGA (Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution. 2007;24(8):1596-9) is a modular system, too, but places greater emphasis on ease-of-use than customization of analyses. As of version 5, MEGA allows the user to reconstruct ancestral states using maximum parsimony, ML, and empirical Bayes methods. The Bayesian analysis of genetic sequences may confer greater robustness to model misspecification. MrBayes (Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001 ;17(8):754-5) allows inference of ancestral states at ancestral nodes using the full hierarchical Bayesian approach. The PREQUEL program distributed in the PHAST package performs comparative evolutionary genomics using ancestral sequence reconstruction (Hubisz MJ, Pollard KS, Siepel A. PHAST and RPHAST: phylogenetic analysis with space / time models. Briefings in bioinformatics. 2011;12(1 ):41-51). SIMMAP stochastically maps mutations on phylogenies (Bollback JP. SIMMAP: stochastic character mapping of discrete traits on phylogenies. BMC bioinformatics. 2006;7(1 ):88). BayesTraits (Pagel M. The maximum likelihood approach to reconstructing ancestral character states of discrete characters on phylogenies. Systematic biology. 1999;48(3):612-22) analyses discrete or continuous characters in a Bayesian framework to evaluate models of evolution, reconstruct ancestral states, and detect correlated evolution between pairs of traits. Other software packages are more oriented towards the analysis of qualitative and quantitative traits (phenotypes). For example, the ape package (Paradis E. Analysis of phylogenetics and evolution with R. New York: Springer; 2006) in the statistical computing environment R also provides methods for ancestral state reconstruction for both discrete and continuous characters through the ace function, including ML. Note that ace performs reconstruction by computing scaled conditional likelihoods instead of the marginal or joint likelihoods used by other ML-based methods for ancestral reconstruction, which may adversely affect the accuracy of reconstruction at nodes other than the root. Phyrex implements a maximum parsimony-based algorithm to reconstruct ancestral gene 29 expression profiles in addition to a ML method for reconstructing ancestral genetic sequences (by wrapping around the baseml function in PAML) (Rossnes R, Eidhammer I, Liberies DA. Phylogenetic reconstruction of ancestral character states for gene expression and mRNA splicing data. BMC bioinformatics. 2005;6(1):127). Several software packages also reconstruct phylogeography. BEAST (Bayesian Evolutionary Analysis by Sampling Trees (Bouckaert R, Heled J, Kuhnert D, Vaughan T, Wu C- H, Xie D, et al. BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol. 2014; 10(4):e1003537)) provides tools for reconstructing ancestral geographic locations from observed sequences annotated with location data using Bayesian MCMC sampling methods. Diversitree (FitzJohn RG. Diversitree: comparative phylogenetic analyses of diversification in R. Methods in Ecology and Evolution. 2012;3(6):1084-92) is an R package providing methods for ancestral state reconstruction under Mk2 (a continuous time Markov model of binary character evolution (Pagel M. Detecting Correlated Evolution on Phylogenies—a General- Method for the Comparative-Analysis of Discrete Characters. Proceedings of the Royal Society of London Series B-Biological Sciences. 1994;255(1342):37-45)) and BiSSE models. Lagrange performs analyses on reconstruction of geographic range evolution on phylogenetic trees (Ree RH, Smith SA. Maximum likelihood inference of geographic range evolution by dispersal, local extinction, and cladogenesis. Systematic Biology. 2008;57(1 ):4-14). Phylomapper (Lemmon AR, Lemmon EM. A likelihood framework for estimating phylogeographic history on a continuous landscape. Systematic Biology. 2008;57(4):544-61) is a statistical framework for estimating historical patterns of gene flow and ancestral geographic locations. RASP (Yu Y, Harris AJ, Blair C, He X. RASP (Reconstruct Ancestral State in Phylogenies): a tool for historical biogeography. Molecular Phylogenetics and Evolution. 2015;87:46-9) infers ancestral state using statistical DIVA, Lagrange, Bayes-Lagrange, BayArea, and BBM methods. VIP (Arias JS, Szumik CA, Goloboff PA. Spatial analysis of vicariance: a method for using direct geographical information in historical biogeography. Cladistics. 2011 ;27(6):617-28) infers historical biogeography by examining disjunct geographic distributions. Genome rearrangements provide valuable information in comparative genomics between species. ANGES (Jones BR, Rajaraman A, Tannier E, Chauve C. ANGES: reconstructing ANcestral GEnomeS maps. Bioinformatics. 2012;28(18):2388-90) compares extant-related genomes through ancestral reconstruction of genetic markers. BADGER (Larget B, Kadane JB, Simon DL. A Bayesian approach to the estimation of ancestral genome arrangements. Molecular phylogenetics and evolution. 2005;36(2):214-23) uses a Bayesian approach to examining the history of gene rearrangement. Count (Csuds M. Count: evolutionary analysis of phylogenetic profiles with parsimony and likelihood. Bioinformatics. 2010;26(15): 1910-2) 30 reconstructs the evolution of the size of gene families. EREM (Affre L, Thompson JD, Debussche M. Genetic structure of continental and island populations of the Mediterranean endemic Cyclamen balearicum (Primulaceae). American Journal of Botany. 1997;84(4):437-51) analyses the gain and loss of genetic features encoded by binary characters. PARANA (Patro R, Sefer E, Malin J, Margais G, Navlakha S, Kingsford C. Parsimonious reconstruction of network evolution. Algorithms for Molecular Biology. 2012;7(1 ):1) performs parsimonybased inference of ancestral biological networks that represent gene loss and duplication. There are also several web server-based applications that allow investigators to use ML methods for ancestral reconstruction of different character types without having to install any software. For example, Ancestors (Diallo AB, Makarenkov V, Blanchette M. Ancestors 1.0: a web server for ancestral sequence reconstruction. Bioinformatics. 2010;26(1 ):130-1) is a web server for ancestral genome reconstruction by the identification and arrangement of syntenic regions. FastML (Ashkenazy H, Penn O, Doron-Faigenboim A, Cohen O, Cannarozzi G, Zomer O, et al. FastML: a web server for probabilistic reconstruction of ancestral sequences. Nucleic acids research. 2012;40(W1):W580-W4) is a web server for probabilistic reconstruction of ancestral sequences by ML that uses a gap character model for reconstructing indel variation. MLGO (Hu F, Lin Y, Tang J. MLGO: phylogeny reconstruction and ancestral inference from gene-order data. BMC bioinformatics. 2014; 15(1): 1) is a web server for ML gene order analysis. Codon optimization takes advantage of the degeneracy of the genetic code, and does not alter the amino acid sequence of the encoded polypeptide. Because of degeneracy, one protein can be encoded by many alternative nucleic acid sequences. Codon preference (codon usage bias) differs in each organism, and this can create challenges for expressing recombinant proteins in heterologous expression systems, resulting in low and unreliable expression. Any suitable expression system may be used. Several suitable examples are well known to the skill person, including expression in a mammalian, yeast, insect, or bacterial cell. Optionally the expression system comprises a mammalian cell. Optionally the expression system comprises a yeast, an insect, or a bacterial cell. Methods of codon-optimization are well known to those of ordinary skill in the art. A codon optimization algorithm may be used to design a codon-optimized nucleotide sequence encoding an amino acid sequence. Such algorithms are aimed at providing codon-optimized sequences which maximise expression of a polypeptide or protein in a desired expression system. Examples of suitable codon optimization algorithms include GeneOptimizer™ algorithm (ThermoFisher), OptimumGene™ algorithm (GenScript), and GeneGPS® (ATUM). Other sequence optimization can be used to maximise protein expression in a desired expression system. Such gene optimization takes account of codon usage bias, as well as other sequence-related parameters involved in gene expression, such as transcription, splicing, translation, and mRNA degradation. Examples of such sequence-related parameters are given below (the parameters are classed below as affecting transcriptional efficiency, translational efficiency, or protein refolding, but several of the parameters may influence more than one of these steps): Transcriptional Efficacy: ® GO content « CpG dinucleotides content * Cryptic splicing sites * Negative CpG islands ® SD sequence * TATA boxes * Terminal signal ® Artificial recombination sites Translational Efficiency: *  Codon usage bias ®  GC content *  mRNA secondary structure ®  Premature PolyA sites Protein Refolding: *  Codon usage bias ®   interaction of codon and anti-codon ® RNA instability rnotif (ARE) « Stable free energy of mRNA ® Internal chi sites and ribosomal binding sites « Repetitive sequences « Codon-context » RNA secondary structures Gene optimization algorithms, such as GeneOptimizer™ and OptimumGene™, take account of several of these parameters. Gene optimization for expression of human proteins in E.coli is discussed by Maertens et al. (Protein Science 2010 Vol. 19:1312—1326). According to the invention there is provided an isolated nucleic acid molecule, comprising a nucleic acid sequence that is: i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:1, or identical with SEQ ID NO:1; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:2, or identical with SEQ ID NO:2; ill) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:4, or identical with SEQ ID NO:4; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:5, or identical with SEQ ID NO:5; v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:7, or identical with SEQ ID NO:7; or vi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:8, or identical with SEQ ID NO:8; or the complement thereof. There is also provided according to the invention an isolated nucleic acid molecule, comprising a nucleic acid sequence that is: i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:10, or identical with SEQ ID NQ:10; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:12, or identical with SEQ ID NO:12; or ill) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:14, or identical with SEQ ID NO:14; or the complement thereof. There is also provided according to the invention an isolated nucleic acid molecule, comprising a nucleic acid sequence that is: i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:19, or identical with SEQ ID NO:19; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:21, or identical with SEQ ID NO:21; iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:23, or identical with SEQ ID NO:23; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:25, or identical with SEQ ID NO:25; v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:27, or identical with SEQ ID NO:27; vi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:29, or identical with SEQ ID NO:29; or vii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:31, or identical with SEQ ID NO:31; or the complement thereof. According to the invention there is further provided an isolated polypeptide, comprising an amino acid sequence that is: i) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:1, or identical with the amino acid sequence encoded by SEQ ID NO:1; ii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:2, or identical with the amino acid sequence encoded by SEQ ID NO:2; iii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:4, or identical with the amino acid sequence encoded by SEQ ID NO:4; iv) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:5, or identical with the amino acid sequence encoded by SEQ ID NO:5; v) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:7, or identical with the amino acid sequence encoded by SEQ ID NO:7; vi) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:8, or identical with the amino acid sequence encoded by SEQ ID NO:8; vii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:10, or identical with the amino acid sequence encoded by SEQ ID NQ:10; viii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:12, or identical with the amino acid sequence encoded by SEQ ID NO:12; or ix) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:14, or identical with the amino acid sequence encoded by SEQ ID NO:14. There is also provided according to the invention an isolated polypeptide, comprising an amino acid sequence that is: i) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:3, or identical with SEQ ID NO:3; ii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:6, or identical with SEQ ID NO:6; ill) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:9, or identical with SEQ ID NO:9; iv) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:11, or identical with SEQ ID NO:11; v) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO: 13, or identical with SEQ ID NO:13; or vi) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO: 15, or identical with SEQ ID NO:15. There is also provided according to the invention an isolated polypeptide, comprising an amino acid sequence that is: i) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:18, or identical with SEQ ID NO:18; ii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:20, or identical with SEQ ID NQ:20; ill) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:22, or identical with SEQ ID NO:22; iv) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:24, or identical with SEQ ID NO:24; v) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:26, or identical with SEQ ID NO:26; vi) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:28, or identical with SEQ ID NO:28; or vii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:30, or identical with SEQ ID NQ:30. We have developed a trivalent vaccine (Tri-LEMvac) that generates combined vaccine efficacy against future outbreaks of variants of the haemorrhagic fever Lassa, Ebola and Marburg viruses. We have bioinformatically designed synthetic glycoprotein sequences from the GPC open reading frames of LASV (L) as well as EBOV (E) and MARV (M) from all available Arenavirus and Filovirus databases. These conserved sequences consist of neutralising antibody and T-cell rich epitopes for each of these viruses. To ensure that these synthetically designed LASV, EBOV and MARV envelopes were functional and antigenic, they were expressed as pseudotypes and quality controlled for both binding and neutralisation against a panel of broadly neutralising antibodies. We have also developed a bivalent vaccine comprising designed antigens for Lassa nucleoprotein (NP) and Lassa glycoprotein (GP). Vaccines may be provided, for example, as nucleic acid vaccines, either as separate polynucleotides, each encoding a different subunit (for administration together or separately) or pieced together in a string as a single polynucleotide encoding all of the subunits. The separate polynucleotides may be administered as a mixture together (for example, as a 36 pharmaceutical composition comprising the separate polynucleotides), or co-administered or administered sequentially in any order (in which case, the separate polynucleotides may be provided as a combined preparation for co-administration or sequential administration). Nucleic acid vaccines may be provided as DNA, RNA, or mRNA vaccines. Strategies for multigene co-expression include introduction of multiple vectors, use of multiple promoters in a single vector, fusion proteins, proteolytic cleavage sites between genes, internal ribosome entry sites (IRES), and “self-cleaving” 2A peptides. Multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides are reviewed in Shaimardanova et al. (Pharmaceutics 2019, 11,580; doi:10.3390 / pharmaceutics11110580). Vaccine constructs of the invention may also be provided, for example, either as separate polypeptides, each comprising a different designed subunit or pieced together in a string as a single polypeptide comprising all of the subunits. The separate polypeptides may be administered as a mixture together (for example, as a pharmaceutical composition comprising the separate polypeptides), or co-administered or administered sequentially in any order (in which case, the separate polypeptides may be provided as a combined preparation for co-administration or sequential administration). According to the invention there is provided an isolated polynucleotide which comprises: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3, or the complement thereof; (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9, or the complement thereof; and (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO:28, or the complement thereof. According to the invention there is further provided an isolated polynucleotide which comprises: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof; (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof. According to the invention there is further provided an isolated polynucleotide which comprises: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18, or the complement thereof; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28, or the complement thereof. According to the invention there is further provided an isolated polynucleotide which comprises: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3, or the complement thereof; (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9, or the complement thereof; and (c) a third an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO:28, or the complement thereof; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof; (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and (c) a third an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18, or the complement thereof; and (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28, or the complement thereof; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a combined preparation, which comprises: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3, or the complement thereof; (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9, or the complement thereof; and (c) a third an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO:28, or the complement thereof. According to the invention there is further provided a combined preparation, which comprises: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof; (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and (c) a third an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof. According to the invention there is further provided a combined preparation, which comprises: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18, or the complement thereof; and (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28, or the complement thereof. According to the invention there is further provided a combined preparation, which comprises: (a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and (b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof. Optionally a polynucleotide of the invention, or each polynucleotide of a pharmaceutical composition, or a combined preparation, of the invention is provided as part of a vector. According to the invention there is provided a vector which comprises a polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3, or the complement thereof; (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9, or the complement thereof; and (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO:28, or the complement thereof. According to the invention there is further provided a vector which comprises a polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof; (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and (c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof. According to the invention there is further provided a vector which comprises a polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18, or the complement thereof; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28, or the complement thereof. According to the invention there is further provided a vector which comprises a polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and (b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof. Optionally, the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, comprises the nucleotide sequence of SEQ ID NO:2, or the complement thereof. Optionally, the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, comprises the nucleotide sequence of SEQ ID NO:8, or the complement thereof. Optionally, the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, comprises the nucleotide sequence of SEQ ID NO:29, or the complement thereof. Optionally, the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, comprises the nucleotide sequence of SEQ ID NO:19, or the complement thereof. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3; (b) a second isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9; and (c) a third an isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 3; (b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 9; and (c) a third an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18; and (b) a second isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a pharmaceutical composition comprising: (a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 18; and (b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28; and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is further provided a combined preparation comprising: (a) a first isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 3; (b) a second isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 9; and (c) a third isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28. According to the invention there is further provided a combined preparation comprising: (a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 3; (b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 9; and (c) a third isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28. According to the invention there is further provided a combined preparation comprising: (a) a first isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 18; and (b) a second isolated polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, SEQ ID NO: 28. According to the invention there is further provided a combined preparation comprising: (a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 18; and (b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28. There is also provided according to the invention a polynucleotide of the invention, a vector of the invention, a polypeptide of the invention, a pharmaceutical composition of the invention, or a combined preparation of the invention, for use as a medicament. There is also provided according to the invention a polynucleotide of the invention, a vector of the invention, a polypeptide of the invention, a pharmaceutical composition of the invention, or a combined preparation of the invention, for use in the treatment of a viral infection. There is also provided according to the invention use of a polynucleotide of the invention, a vector of the invention, a polypeptide of the invention, a pharmaceutical composition of the invention, or a combined preparation of the invention, in the manufacture of a medicament for the treatment of a viral infection. Optionally the viral infection is caused by an emerging or re-emerging virus. Optionally the viral infection is caused by a virus of the Filoviridae family. Optionally the viral infection is caused by Ebola virus or Marburg virus. Optioanlly the viral infection is caused by a virus of the Arenaviridae family. Optionally the viral infection is caused by Lassa virus. The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. AppL Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244,1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet etal., Nucleic Acids’ Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et aL, Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et aL, J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Sequence identity between nucleic acid sequences, or between amino acid sequences, can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same nucleotide, or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties. Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et aL, 2003, BMC Bioinformatics 4: 29; program available from http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et aL, 1990, J. Mol. Biol. 215: 403-410; program available from http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et aL, 2007, Bioinformatics 23: 2947-2948; program available from http: / / www.ebi.ac.uk / tools / clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs may be run using default parameters. For example, sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62. The sequence comparison may be performed over the full length of the reference sequence. There is also provided according to the invention an isolated nucleic acid molecule which comprises a nucleotide sequence encoding a polypeptide comprising an amino acid 46 sequence of SEQ ID NO: 6, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention an isolated nucleic acid molecule which comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 15. There is also provided according to the invention a composition comprising a first nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention a composition comprising a first nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 15. There is also provided according to the invention a combined preparation comprising: (i) a first nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 6; and (ii) a second nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention a combined preparation comprising: (i) a first nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13; and (ii) a second nucleic acid which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 15. There is also provided according to the invention a composition comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention a composition comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15. There is also provided according to the invention a fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention a fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15. There is also provided according to the invention a combined preparation comprising: (i) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6; and (ii) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9. There is also provided according to the invention a combined preparation comprising: (i) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13; and (ii) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15. The term "combined preparation" as used herein refers to a "kit of parts" in the sense that the combination components (i) and (ii) (or (a) and (b), or (a), (b) and (c)) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination components (i) and (ii) (or (a) and (b), or (a), (b) and (c)). The components can be administered simultaneously or one after the other. If the components are administered one after the other, preferably the time interval between administration is chosen such that the therapeutic effect of the combined use of the components is greater than the effect which would be obtained by use of only any one of the combination components (i) and (ii) (or (a) and (b), or (a), (b) and (c)). The components of the combined preparation may be present in one combined unit dosage form, or as a first unit dosage form of component (i) and a separate, second unit dosage form of component (ii) (or as a first unit dosage form of component (a) and a separate, second unit dosage form of componen (b), or as a first unit dosage form of component (a) a separate, second unit dosage form of componen (b), and a separate, third unit dosage form of component (c)). The ratio of the total amounts of the combination component (i) to the combination component (ii) (or of the combination component (a) to (b), or of the combination components (a) to (b) to (c)) to be administered in the combined preparation can be varied, for example in order to cope with the needs of a patient sub-population to be treated, or the needs of the single patient, which can be due, for example, to the particular disease, age, sex, or body weight of the patient. Preferably, there is at least one beneficial effect, for example an enhancing of the effect of component (i), or component (ii) (or of component (a) or (b), or of component (a), (b), or (c)), or a mutual enhancing of the effect of the combination components (i) and (ii) (or of component (a) and (b), or of component (a) and (b) and / or (c)), for example a more than additive effect, additional advantageous effects, fewer side effects, less toxicity, or a combined therapeutic effect compared with an effective dosage of one or both of the combination components (i) and (ii) (or of component (a) and (b), or of components (a) and (b) and (c)), and very preferably a synergism of the combination components (i) and (ii) (or of component (a) and (b), or of components (a) and (b) and / or (c)). A combined preparation of the invention may be provided as a pharmaceutical combined preparation for administration to a mammal, preferably a human. Component (i) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (ii) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent. Component (a) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (b) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent. Component (a) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (b) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (c) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent. There is further provided according to the invention an isolated nucleic acid molecule encoding an amino acid sequence encoded by a nucleic acid of the invention. There is further provided according to the invention an isolated nucleic acid molecule encoding an amino acid sequence encoded by a nucleic acid of the invention, wherein the nucleic acid is codon-optimized for expression in mammalian cells. There is further provided according to the invention an isolated nucleic acid molecule encoding an amino acid sequence encoded by a nucleic acid of the invention, wherein the nucleic acid is gene-optimized for expression in mammalian cells. There is also provided according to the invention an isolated nucleic acid molecule encoding a polypeptide of the invention. There is also provided according to the invention an isolated nucleic acid molecule encoding a polypeptide of the invention, wherein the nucleic acid is codon-optimized for expression in mammalian cells. There is also provided according to the invention an isolated nucleic acid molecule encoding a polypeptide of the invention, wherein the nucleic acid is gene-optimized for expression in mammalian cells. There is also provided according to the invention a vector comprising a nucleic acid of the invention. Optionally the vector further comprises a promoter operably linked to the nucleic acid. Optionally the promoter is for expression of a polypeptide encoded by the nucleic acid in mammalian cells. Optionally the promoter is for expression of a polypeptide encoded by the nucleic acid in yeast, bacterial, or insect cells. Optionally the vector is a vaccine vector. Optionally the vaccine vector is a viral vaccine vector, a bacterial vaccine vector, or a nucleic acid vector (for example an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector).A nucleic acid molecule of the invention may comprise a DNA or an RNA molecule. For embodiments in which the nucleic acid molecule comprises an RNA molecule, it will be appreciated that the molecule may comprise an RNA sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs: 1,2, 4, 5, 7, 8, 10, 12, 14, 19, 21, 23, 25, 27, 29, or 31, in which each T nucleotide is replaced by 'U', or the complement thereof. For example, it will be appreciated that where an RNA vaccine vector comprising a nucleic acid of the invention is provided, the nucleic acid sequence of the nucleic acid of the invention will be an RNA sequence, so may comprise for example an RNA nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 12, 14, 19, 21, 23, 25, 27, 29, or 31 in which each T nucleotide is replaced by ‘U’, or the complement thereof. There is also provided according to the invention an isolated cell comprising or transfected with a vector of the invention. There is also provided according to the invention a virus pseudotype particle comprising a polypeptide of the invention. According to the invention there is also provided a method of producing a virus pseudotype particle which includes transfecting a host cell with a vector comprising a nucleic acid of the invention. There is also provided according to the invention a fusion protein comprising a polypeptide of the invention. There is further provided according to the invention a pharmaceutical composition comprising a nucleic acid of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. There is also provided according to the invention a pharmaceutical composition comprising a vector of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. There is also provided according to the invention a pharmaceutical composition comprising a polypeptide of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. Optionally a pharmaceutical composition of the invention further comprises an adjuvant for enhancing an immune response in a subject to the polypeptide, or to a polypeptide encoded by the nucleic acid, of the composition. There is also provided according to the invention a method of inducing an immune response to a pathogen in a subject, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, a pharmaceutical composition of the invention, ora combined preparation of the invention. Optionally the pathogen is a virus. Optionally the virus is a member of the Filoviridae, Arenaviridae, or Orthomyxoviridae family.There is also provided according to the invention a method of inducing an immune response to a virus of the Filoviridae or Arenaviridae family in a subject, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, a combined preparation of the invention, ora pharmaceutical composition of the invention. Optionally the virus is a member of the Filoviridae family. Optionally the virus is an Ebola virus or a Marburg virus. Optionally the virus is a member of the Arenaviridae family. Optionally the virus is a Lassa virus. There is also provided according to the invention a method of immunizing a subject against a pathogen, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, a combined preparation of the invention, ora pharmaceutical composition of the invention. Optionally the pathogen is a virus. Optionally the virus is a member of the Filoviridae, Arenaviridae, or Orthomyxoviridae family.Optionally the virus is a member of the Filoviridae family. Optionally the virus is an Ebola virus or a Marburg virus. Optionally the virus is a member of the Arenaviridae family. Optionally the virus is a Lassa virus. There is further provided according to the invention a method of immunizing a subject against a virus of the Filoviridae family, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, or a pharmaceutical composition of the invention. Optionally the virus is an Ebola virus or a Marburg virus. There is also provided according to the invention a method of inducing an immune response to a virus of the Filoviridae family in a subject, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, or a pharmaceutical composition of the invention. Optionally the virus is an Ebola virus or a Marburg virus. Optionally the nucleic acid, vector, or pharmaceutical composition of the invention comprises a nucleic acid comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs:1, 2, 4, 5, 7, 8, 10, 12, or 14, or comprises a nucleic acid encoding an amino acid sequence encoded by a nucleic acid comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs:1,2, 4, 5, 7, 8, 10, 12, or 14. Optionally the polypeptide, vector, or pharmaceutical composition of the invention comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical with, or identical with, an amino acid sequence encoded by any of SEQ ID NOs:1, 2, 4, 5, 7, 8, 10, 12, or 14, or comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs: 3, 6, 9, 11, 13, or 15. There is further provided according to the invention a method of immunizing a subject against a virus of the Arenaviridae family, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, or a pharmaceutical composition of the invention. There is also provided according to the invention a method of inducing an immune response to a virus of the Arenaviridae family in a subject, which comprises administering to the subject a nucleic acid of the invention, a polypeptide of the invention, a vector of the invention, or a pharmaceutical composition of the invention. Optionally the nucleic acid, vector, or pharmaceutical composition of the invention comprises a nucleic acid comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs:19, 21,23, 25, 27, 29, or 31, or comprises a nucleic acid encoding an amino acid sequence encoded by a nucleic acid comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs: 19, 21,23, 25, 27, 29, or 31. Optionally the polypeptide, vector, or pharmaceutical composition of the invention comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical with, or identical with, an amino acid sequence encoded by any of SEQ ID NOs: 19, 21, 23, 25, 27, 29, or 31, or comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical with, or identical with, any of SEQ ID NOs: 18, 20, 22, 24, 26, 28, or 30. Any suitable route of administration may be used. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Administration can be systemic or local. Compositions may be administered in any suitable manner, such as with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. Administration can be accomplished by single or multiple doses. The dose administered to a subject in the context of the present disclosure should be sufficient to induce a beneficial therapeutic response in a subject over time, or to inhibit or prevent infection. The dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier and / or an adjuvant. For example, the adjuvant can be alum, Freund's complete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides). The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as one or more influenza vaccines, and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Optionally a composition of the invention is administered intramuscularly. Optionally the composition is administered intramuscularly, intradermaly, subcutaneously by needle or by gene gun, or electroporation. There is also provided according to the invention a nucleic acid expression vector, which comprises a multiple cloning site, comprising Kpnl and Notl endonuclease sites. Optionally the multiple cloning site comprises a nucleic acid sequence of SEQ ID NO:16. Optionally the nucleic acid expression vector is a nucleic acid expression vector, and a viral pseudotype vector. Optionally the nucleic acid expression vector is a vaccine vector. Optionally the nucleic acid expression vector comprises, from a 5’ to 3’ direction: a promoter; a splice donor site (SD); a splice acceptor site (SA); and a terminator signal, wherein the multiple cloning site is located between the splice acceptor site and the terminator signal. Optionally the promoter comprises a CMV immediate early 1 enhancer / promoter (CMV-IE-E / P) and / or the terminator signal comprises a terminator signal of a bovine growth hormone gene (Tbgh) that lacks a Kpnl restriction endonuclease site. Optionally the nucleic acid expression vector further comprises an origin of replication, and nucleic acid encoding resistance to an antibiotic. Optionally the origin of replication comprises a pUC-plasmid origin of replication and / or the nucleic acid encodes resistance to kanamycin. Optionally the nucleic acid expression vector comprises a nucleic acid sequence of SEQ ID NO: 17 (pEVAC). A polypeptide of the invention may include one or more conservative amino acid substitutions. Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Examples of conservative substitutions are shown below: Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gin, His Asp Glu Cys Ser Gin Asn Glu Asp His Asn; Gin lie Leu, Vai Leu lie; Vai Lys Arg; Gin; Met Leu; lie Phe Met; Leu; Tyr Ser Thr Thr                Ser Trp Tyr Tyr Trp; Phe Vai                    lie; Leu Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine. In particular embodiments of the invention sequence alignments and ancestral sequence reconstruction (ASR) are used to identify highly conserved immune targets which the pathogens cannot change and which will invariably be present in future outbreaks of that viral family, even in the most highly variable RNA viruses. Synthetic gene technology is used to produce computer generated virus genes so that they are highly expressed and can be easily cloned into an expression vector, such as the pEVAC vector (one that has proven to be a highly versatile expression vector for generating viral pseudotypes as well as direct DNA vaccination of animals and or humans). Large panels of genes can be generated using the pEVAC vector so that viral pseudotypes are rapidly generated. This allows a library of viral pseudotypes, each with its own unique viral protein to be probed with a large panel of monoclonal antibodies. This process ensures that the inserts generate conformation correct viral surface proteins to present the most accessible target to a viral Achilles heel. The down-selection of candidates by pseudotype formation and mAb binding provides a shortlist of top candidates to test by vaccination. This may be done in Guinea pigs where the streamlined process of pEVAC-vaccine inserts are delivered. If required this enables the shuttle of vaccine inserts from the DNA pEVAC vector into a variety of viral vectors based on advanced designed convenient cloning sites. Since Chimpanzee Adenovectors (ChAd) were widely used in evaluating the majority of Ebola virus vaccine candidates in phase I for the West African outbreak, we chose to compare to use the same vector for head to head comparison in humans. For screening in Guinea pigs we used DNA priming with pEVAC-vaccine insert followed by ChAd-vaccine insert. In particular embodiments of the invention: 1) High throughput “deep” sequencing technology provides viral variation data from current and past outbreaks. By analysing this data, structural, highly conserved regions can be identified which can be used as scaffolds for designing optimal vaccine inserts, and which preserve known B and T cell epitopes. 2) Human monoclonal antibody (mAb) technology allows the generation of anti-viral mAbs to vaccine targets, such as the virus envelope protein, which identify the epitope rich regions to which broadly neutralising monoclonal antibodies (BNmAbs) target. 3) Optimal gene design and synthesis incorporates the digitally modelled conserved scaffolds of genes identified in (1), to include the broadest NmAb epitopes on these scaffolds (BN epitopes are likely not to be optimally presented on naturally conserved GPs). 4) Downstream knowledge of convenient cloning sites matching the requirements of vaccine and pseudotype vectors are taken into account during the design and synthesis of RNA and codon optimised synthetic genes as vaccine inserts to enable rapid and highly efficient cloning and shuttling into different screening (i.e. lentiviral pseudotypes; PVs) and vaccine (i.e. MVA, ChAd, VSV, DNA etc) vectors. 5) Viral pseudotypes (lentiviral) generated from digitally designed inserts are screened in vitro for functionality via transduction and infection studies. Further to this, neutralisation assays using a panel of BNmAbs and patient sera is undertaken to ensure that known epitopes are preserved. 6) Down-selection of several synthetic vaccine inserts to the best-in-class vaccine inserts are confirmed for immunogenicity in guinea pigs, using rapid DNA priming (and if required) with adenovirus boosting, a method that gives high and reproducible titres. In vivo screening confirms which are the most immunogenic and give the greatest neutralisation breadth. The central role of the viral glycoprotein in cell attachment, fusion and uncoating make it a key antigenic target for viral vaccines and monoclonal antibody therapies that have been pioneered during the West African Ebola outbreak. Analysis of the GP sequences between species of EBOV showed a high degree of diversity at the nucleotide and amino acid level (only -60-65% nt identity). For current conventional Filovirus vaccine approaches, GP targeting vaccines need to be multivalent, encoding GPs specific for each species, which are more conserved (-97-98% identity in the GP nucleotide sequence). Although it has been suggested that vaccines using older strains of EBOV (rVSV.ZEBOV = Kikwit) may provide cross-protection (Henao-Restrepo AM, Lancet 2015), there is concern that this may have limited efficacy against future outbreaks of other diverse highly pathogenic Filoviruses. We can achieve dramatic improvements in vaccine efficacy against new viral variants based on sequence data (optionally including, for example, outbreak sequence data) to generate synthetic optimised vaccine inserts to give the broadest possible vaccine protection against future outbreaks of variable RNA viruses. In particular embodiments, our new vaccine technology merges: (1) Sequences of outbreak pathogens (2) Broadly anti-viral neutralising monoclonal antibodies (BNmAb) derived from outbreak survivors (3) Computational modelling methodologies (4) Synthetic gene technology and antigen display technology (5) High-throughput viral binding and neutralisation screens (6) In vivo immune selection and vaccine efficacy readouts The end products are novel immunogens used to trigger the broadest spectrum of protective immune responses. We have provided proof of concept that the next generation single vaccine inserts do induce broad neutralisation profiles against the Ebolavirus genus (Zaire, Sudan, Bundibugyo), additionally targeting the more distant filovirus, Marburg virus. Embodiments of the invention are described, by way of illustration only, in the Examples below, with reference to the accompanying drawings in which: Figure 1 shows an illustration of a phylogenetic tree and its relation to ancestral sequence reconstruction; Figure 2 shows a phylogenetic tree comparing ebolaviruses and Marburg viruses. Numbers indicate percent confidence of branches; Figure 3 shows a plasmid map for pEVAC; Figure 4 shows challenge study results for an Ebola challenge model. Ebola challenge model was lethal for non-vaccinated guinea pigs (Group 1, lower line) whereas all vaccinated guinea pigs (Group 2, upper line) were protected (left) and continued to gain weight (right); Figure 5 shows the results of a pseudotype virus neutralisation assay illustrating the strength of neutralising antibody responses to target antigens expressed on the surface of a pseudotyped virus, representative of all Ebola virus species and Marburg viruses. Strength of neutralisation is indicated by the heat-map where red (darkest shading) is very strong neutralisation, decreasing through orange to yellow (progressively lighter shading) and no neutralising / equal to negative control values are white. T2-4 and T2-6 are nucleic acid vaccines encoding lead candidate optimized antigenic Ebola polypeptide, combined with T2-11 a Marburg candidate, at pre-clinical stage testing with serum samples taken from immunised guinea pigs; Figure 6 shows a schematic of GMP-compatible DNA vaccine candidates used in the challenge study (top line), and recombinant MVAs (Tri-LEMvac and Bivalent Lassa) used in the challenge study (bottom line); Figure 7 shows phylogenetic trees for Lassa virus nucleoprotein (top line), Marburg virus glycoprotein (middle line), Sudan ebolavirus glycoprotein (bottom line), and Lassa virus glycoprotein (figure 7 continued) immunogen designs. Ancestral sequences are shown unhighlighted, and RNA- and codon-optimised sequences shown in highlight; Figure 8 shows construction of recombinant trivalent MVA, Tri-LEMvac, (top line) and bivalent Lassa virus MVA vaccine; Figure 9 shows in vitro characterisation of Tri-LEMvac MVA, using (a) Western blot analysis and (b) flow cytometry to study expression of the individual antigens; Figure 10 shows in vitro characterisation of bivalent Lassa MVA, using (a) Western blot analysis and (b) flow cytometry to study expression of the individual antigens; Figure 11 shows immunogenicity of Tri-LEMvac MVA in mice. The figure shows vaccination and bleed schedule for mice, wherein 50 pg DNA (s.c) and / or 1x107 pfu MVA (i.m) was administered (top line), and binding antibody, neutralising antibody, and T cell response to Lassa virus, Marburg virus, and Ebola virus antigens (bottom line); Figure 12 shows immunogenicity of Tri-LEMvac MVA in guinea pigs. The figure shows (a) vaccination and bleed schedule for the guinea pigs, wherein 150 pg DNA (s.c) and / or 2x107 pfu MVA (i.m) was administered, and (b) binding antibody response to Sudan ebolavirus, Ebola, Marburg, and Lassa virus antigens; Figure 13 shows neutralising antibody response in guinea pigs vaccinated according to vaccination schedule in figure 12(a); Figure 14 shows vaccination and bleed schedule for the bivalent Lassa vaccine and Tri-LEMvac, in DNA and MVA, for the guinea pig challenge models; Figure 15 shows challenge study results for an Ebola virus challenge model. Hartley guinea pigs were immunized with either Tri-LEMvac DNA followed by two booster immunizations using Tri-LEMvac MVA, or with an empty vector followed by two boosts of MVAWT (schedule as in figure 14). Animals were subsequently challenged with lethal doses of guinea-pig adapted Sudan Ebola virus. All guinea pigs were monitored for survival, weight, and fluctuations in body temperature, as well as viral load in the blood, lung, heart, spleen, liver and kidneys. In Figure 15(a), the guinea pigs immunised with Tri-LEMvac are represented by the line showing 100% survival. In Figure 15(b) the guinea pigs immunised with Tri-LEMvac are represented by the data points on the graph on the right-hand side for each of the organs shown on the x-axis. In Figure 15(c), the guinea pigs immunised with Tri-LEMvac are represented by the data points showing stable % initial weight and temperature; Figure 16 shows challenge study results for a Marburg virus challenge model. Hartley guinea pigs were immunized with either Tri-LEMvac DNA followed by two booster immunizations using Tri-LEMvac MVA, or with an empty vector followed by two boosts of MVAWT (schedule as in figure 14). Animals were subsequently challenged with lethal doses of guinea-pig adapted Marburg virus. All guinea pigs were monitored for survival, weight, and fluctuations in body temperature, as well as viral load in the blood, lung, heart, spleen, liver and kidneys. In Figure 16(a), the guinea pigs immunised with Tri-LEMvac are represented by the line showing 100% survival. In Figure 16(b) the guinea pigs immunised with Tri-LEMvac are represented by the data points on the graph on the right-hand side for each of the organs shown on the x-axis. In Figure 16(c), the guinea pigs immunised with Tri-LEMvac are represented by the data points showing stable % initial weight and temperature; Figure 17 shows challenge study results for a Lassa virus challenge model. Hartley guinea pigs were immunized with either Tri-LEMvac DNA followed by two booster immunizations using Tri-LEMvac MVA, or with an empty vector followed by two boosts of MVAWT (schedule as in figure 14). Animals were subsequently challenged with lethal doses of guinea-pig adapted Lassa virus. All guinea pigs were monitored for survival, weight, and fluctuations in body temperature, as well as viral load in the blood, lung, heart, spleen, liver and kidneys. In Figure 17(a), the guinea pigs immunised with Tri-LEMvac and Bivalent LASV are represented by the lines showing 100% survival. In Figure 17(b) and (c), the guinea pigs immunised with Tri-LEMvac and Bivalent LASV are represented by the data points showing stable % initial weight and temperature. Figure 17(D), the guinea pigs immunised with Tri-LEMvac and Bivalent LASV are represented by the lines showing disease score of 0; Figure 18 shows kinetics of cross-binding filovirus-specific antibodies for Tri-LEMvac (lines showing higher AUC), following immunisation schedule as in Figure 14 (prior to challenge). Filoviruses tested include Sudan ebolavirus, Ebola virus, Marburg virus, Bundibugyo ebolavirus, Reston virus, Ravn virus; Figure 19 shows binding and neutralising filovirus-specific antibody data for guinea pigs immunised with Tri-LEMvac (prior to challenge). In figure 19(a), guinea pigs immunised with Trri-LEMvac DNA + MVA are the top data points shown in the figure. In Figure 19(b), guinea pigs immunised with Trri-LEMvac DNA + MVA (n=21) are the left-hand data points on the figure; Figure 20 shows kinetics of binding antibodies specific for Lassa nucleoprotein and Monkeypox H3 antigens, after immunisation with Tri-LEMvac (lines showing higher AUC), against empty vector control prior to challenge (prior to challenge); and Figure 21 shows binding antibodies specific for Lassa nucleoprotein and Lassa glycoprotein antigens after vaccination with bivalent Lassa vaccine (lines showing higher AUC), (prior to challenge). Examples of unoptimized Ebola and Marburg viral ancestral nucleic acid sequences (i.e. sequences which have not been codon-optimized or gene-optimized) are given below, as well as gene-optimized nucleic acid sequences encoding candidate antigenic pathogen polypeptides. Methodology For a given virus species, candidate primary sequences are downloaded, for example, from GenBank (and from any other available sources, such as outbreak data), and are filtered to remove identical sequences, sequences that do not span the protein of interest, and sequences that have a high number of ambiguous nucleotides. A multiple sequence alignment of the filtered sequences is generated (typically using MAFFT), and checked manually to ensure that sequences are in the correct open reading frame. A maximum likelihood phylogeny is generated using IQTREE, with automated model selection, and rooted using one of several methods; an outgroup sequence, midpoint rooting, centre-of-the-tree, or a tree that maximises the association between root-to-tip distance and sampling time. Ancestral sequences are generated using HyPhy assuming a MG94 by F3x4 model of codon substitution, and are checked to ensure that known epitopes have been preserved. A phylogenetic tree with both primary and ancestral sequences is generated using IQTREE to check the placement of the ancestral strains. Ancestral sequences are then modified in a number of ways: deletion of regions (e.g. removal of the mucin-like domain); region swapping (to recover potential lost epitopes); mutation of specific sites (e.g. in the fusion domain of the filoviruses), including editing of N-linked glycosylation sites and introduction of mutations to enhance stability. Example 1 Ebola Sudan ancestor (T2-4) Unoptimised (SEQ ID NO: 1) ATGGGGGGTCTTAGCCTACTCCAATTGCCCAGGGACAAATTTCGGAAAAGCTCTTTCTTTGTTTGGG TCATCATCTTATTCCAAAAGGCCTTTTCCATGCCTTTGGGTGTTGTGACTAACAGCACTTTAGAAGT AACAGAGATTGACCAGCTAGTCTGCAAGGATCATCTTGCATCCACTGACCAGCTGAAATCAGTTGGT CTCAACCTCGAGGGGAGCGGAGTATCTACTGATATCCCATCTGCAACAAAGCGTTGGGGCTTCAGAT CTGGTGTTCCTCCCAAGGTGGTCAGCTATGAAGCGGGAGAATGGGCTGAAAATTGCTACAATCTTGA AATAAAGAAGCCGGACGGGAGCGAATGCTTACCCCCACCGCCAGATGGTGTCAGAGGCTTTCCAAGG TGCCGCTATGTTCACAAAGCCCAAGGAACCGGGCCCTGCCCAGGTGACTACGCCTTTCACAAGGATG GAGCTTTCTTCCTCTATGACAGGCTGGCTTCAACTGTAATTTACAGAGGAGTCAATTTTGCTGAGGG GGTAATTGCATTCTTGATATTGGCTAAACCAAAAGAAACGTTCCTTCAGTCACCCCCCATTCGAGAG GCAGTAAACTACACTGAAAATACATCAAGTTATTATGCCACATCCTACTTGGAGTATGAAATCGAAA ATTTTGGTGCTCAACACTCCACGACCCTTTTCAAAATTGACAATAATACTTTTGTTCGTCTGGACAG GCCCCACACGCCTCAGTTCCTTTTCCAGCTGAATGATACCATTCACCTTCACCAACAGTTGAGCAAC ACAACTGGGAGACTAATTTGGACACTAGATGCTAATATCAATGCTGATATTGGTGAATGGGCTTTTT GGGAAAATAAAAAAAATCTCTCCGAACAACTACGTGGAGAAGAGCTGTCTTTCGAAGCTTTATCGCT CACAACAGCGGTTAAAACTGTCTTGCCACAGGAGTCCACAAGCAACGGTCTAATAACTTCAACAGTA ACAGGGATTCTTGGGAGTCTTGGGCTTCGAAAACGCAGCAGAAGACAAGTTAACACCAAAGCCACGG GTAAATGCAATCCCAACTTACACTACTGGACTGCACAAGAACAACATAATGCTGCTGGGATTGCCTG GATCCCGTACTTTGGACCGGGTGCGGAAGGCATATACACTGAAGGCCTGATGCATAACCAAAATGCC TTAGTCTGTGGACTTAGGCAACTTGCAAATGAAACAACTCAAGCTCTGCAGCTTTTCTTAAGAGCCA CAACGGAGCTGCGGACATATACCATACTCAATAGGAAGGCCATAGATTTCCTTCTGCGACGATGGGG CGGGACATGCAGGATCCTGGGACCAGATTGTTGCATTGAGCCACATGATTGGACAAAAAACATCACT GATAAAATCAACCAAATCATCCATGATTTCATCGACAACCCCTTACCTAATCAGGATAATGATGATA ATTGGTGGACGGGCTGGAGACAGTGGATCCCTGCAGGAATAGGCATTACTGGAATTATTATTGCAAT TATTGCTCTTCTTTGCGTTTGCAAGCTGCTTTGCTAG Gene-optimised (SEQ ID NO:2) ATGGGAGGACTGTCTCTGCTGCAACTGCCCCGGGACAAGTTCCGGAAGTCCAGCTTCTTCGTGTGGG TCATCATCCTGTTCCAGAAAGCCTTCAGCATGCCCCTGGGCGTCGTGACCAATAGCACACTGGAAGT GACCGAGATCGACCAGCTCGTGTGCAAGGATCACCTGGCCAGCACCGATCAGCTGAAGTCTGTGGGA CTGAATCTGGAAGGCAGCGGCGTGTCCACAGATATCCCTAGCGCCACCAAGAGATGGGGCTTTAGAA GCGGAGTGCCTCCTAAGGTGGTGTCTTATGAAGCCGGCGAGTGGGCCGAGAACTGCTACAACCTGGA AATCAAGAAGCCCGACGGCAGCGAGTGTCTGCCTCCTCCACCTGATGGCGTCAGAGGCTTCCCTAGA TGCAGATACGTGCACAAGGCCCAAGGCACAGGACCCTGTCCTGGCGATTACGCCTTTCACAAGGACG GCGCCTTTTTCCTGTACGATCGGCTGGCCTCCACCGTGATCTACAGAGGCGTTAACTTTGCCGAGGG CGTGATCGCCTTCCTGATCCTGGCCAAGCCTAAAGAGACATTCCTGCAAAGCCCTCCAATCCGCGAG GCCGTGAACTACACAGAGAACACCAGCAGCTACTACGCCACCAGCTACCTGGAATACGAGATCGAGA ATTTCGGCGCCCAGCACAGCACCACACTGTTCAAGATCGACAACAACACCTTCGTGCGGCTGGACAG ACCCCACACACCTCAGTTTCTGTTCCAGCTGAACGACACCATCCATCTGCATCAGCAGCTGAGCAAC ACCACCGGCAGACTGATTTGGACCCTGGACGCCAACATCAACGCCGACATTGGAGAGTGGGCCTTTT GGGAGAACAAGAAGAACCTGAGCGAACAGCTGAGAGGCGAGGAACTGAGCTTTGAGGCCCTGTCTCT GACCACCGCCGTGAAAACAGTGCTGCCTCAAGAGTCCACCAGCAACGGCCTGATCACAAGCACAGTG ACAGGCATCCTGGGCAGCCTGGGCCTGAGAAAAAGGTCCAGACGGCAAGTGAATACCAAGGCCACCG GCAAGTGCAACCCCAACCTGCACTATTGGACAGCCCAAGAGCAGCACAATGCCGCCGGAATCGCCTG GATTCCTTATTTTGGACCTGGCGCCGAGGGCATCTATACCGAGGGACTGATGCACAACCAGAACGCC CTCGTGTGTGGACTGAGACAGCTGGCCAATGAGACAACACAGGCCCTCCAGCTGTTTCTGAGAGCCA CCACCGAGCTGAGAACCTACACCATCCTGAACCGGAAGGCCATCGACTTTCTGCTGAGAAGATGGGG CGGCACCTGTAGAATCCTGGGACCTGATTGCTGCATCGAGCCCCACGACTGGACCAAGAACATCACC GACAAGATCAACCAGATCATCCACGACTTCATCGACAACCCTCTGCCTAACCAGGACAACGACGACA ATTGGTGGACAGGCTGGCGGCAGTGGATTCCTGCCGGAATTGGCATCACCGGCATCATCATTGCCAT TATCGCCCTGCTGTGTGTGTGCAAGCTGCTGTGTTGA Amino acid sequence encoded by unoptimised and gene-optimised sequences (SEQ ID NO: 3): MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGWTNSTLEVTEIDQLVCKDHLASTDQLKSVGLNLEGSG VSTDIPSATKRWGFRSGVPPKWSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKAQGTGPCP GDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIREAVNYTENTSSYYATSYLEYEI ENFGAQHSTTLFKIDNNTFVRLDRPHTPQFLFQLNDTIHLHQQLSNTTGRLIWTLDANINADIGEWAFWENKKN LSEQLRGEELSFEALSLTTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRRQVNTKATGKCNPNLHYWTAQ EQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQLANETTQALQLFLRATTELRTYTILNRKAIDFLLR RWGGTCRILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQWIPAGIGITGIIIAIIALL CVCKLLC Example 2 Ebolavirus global ancestor (T2-6) Unoptimised (SEQ ID NO:4) ATGGGGGGTGGATCCAGACTTCTCCAATTGCCCCGGGAACGCTTTCGGAAAACCTCATTCTTTGTTT GGGTAATCATCCTATTCCAAAAAGCCTTTTCCATGCCATTGGGTGTTGTAACCAACAGCACTCTAAA AGTAACAGAAATTGACCAATTGGTTTGCCGGGACAAACTTTCATCCACAAGTCAGCTGAAATCAGTT GGGCTGAATCTGGAAGGGAATGGAGTTGCAACTGATGTCCCATCAGCAACAAAACGATGGGGCTTCC GATCTGGTGTTCCTCCCAAGGTGGTCAGCTATGAAGCTGGAGAATGGGCTGAAAATTGCTACAATCT GGAAATCAAGAAGCCAGACGGGAGTGAATGCCTACCTCCACCGCCAGACGGTGTAAGAGGCTTCCCC AGGTGCCGCTATGTCCACAAAGTTCAAGGAACAGGGCCGTGTCCTGGTGACTTCGCCTTCCACAAAG ATGGAGCTTTCTTCCTGTATGATAGACTGGCTTCAACTGTCATTTACCGAGGGACAACTTTTGCTGA AGGTGTCGTTGCATTTTTGATCCTGCCCAAACCTAAAAAGGACTTTTTCCAATCACCCCCAATACGT GAGCCGGTAAACACCACAGAAGATCCATCAAGTTACTACACCACATCAACACTTAGCTATGAGATTG ACAATTTTGGGGCCAATAAAACTAAAACTCTTTTCAAAGTTGACAATCACACTTATGTGCAACTAGA CCGACCACACACACCACAGTTCCTTGTCCAGCTCAATGAAACCATTCATACAAATAACCGTCTAAGC AACACCACAGGGAGACTAATTTGGACATTAGATCCTAAAATTGATACCGACATTGGTGAGTGGGCCT TCTGGGAAAATAAAAAAAACTTCTCCAAACAACTTCGTGGAGAAGAGTTGTCTTTCAAAGCTCTATC AACAAAAACTGGAGCTAACGCAGTAGACACTGACGAATCAAGCAAACCTGGCCTAATTACCAACACA GTAAGAGGGGTTGCTGATTTACTGAGCCCTTGGAGAAGAAAAAGAAGACAAGTCAACCCAAACACAA CAAATAAATGCAACCCAAACCTACACTATTGGACAGCCCAAGATGAAGGTGCTGCCGTTGGATTAGC CTGGATCCCATACTTCGGACCAGCAGCAGAAGGCATTTACACTGAAGGAATAATGCATAATCAAAAT GGGTTAATCTGTGGGCTGAGGCAGCTGGCCAATGAAACGACTCAAGCTCTTCAATTATTCTTGAGGG CCACAACGGAGCTGCGGACTTACTCTATACTCAATAGAAAAGCCATTGATTTCCTTCTCCAACGATG GGGAGGAACATGCCGCATCTTAGGACCAGATTGTTGCATTGAGCCACATGATTGGACAAAAAACATT ACTGATAAAATTAACCAAATCATACATGATTTTATTGACAACCCTCTACCAGATCAGGACGATGATG ACAATTGGTGGACAGGCTGGAGACAATGGATCCCTGCTGGAATTGGAATTACTGGAGTTATAATTGC AATTATAGCTCTACTTTGTATTTGCAAGTTTCTGTGTTAG Gene-optimised (SEQ ID NO:5) ATGGGCGGAGGATCTAGACTGCTGCAACTGCCCAGAGAGCGGTTCAGAAAGACCAGCTTCTTCGTGT GGGTCATCATCCTGTTCCAGAAAGCCTTCAGCATGCCCCTGGGCGTCGTGACCAATAGCACCCTGAA AGTGACCGAGATCGACCAGCTCGTGTGCAGAGATAAGCTGAGCAGCACCAGCCAGCTGAAGTCCGTG GGACTGAATCTGGAAGGCAATGGCGTGGCCACAGATGTGCCTAGCGCCACCAAAAGATGGGGCTTTA GAAGCGGCGTGCCACCTAAGGTGGTGTCTTATGAAGCCGGCGAGTGGGCCGAGAACTGCTACAACCT GGAAATCAAGAAGCCCGACGGCAGCGAGTGTCTGCCTCCTCCACCTGATGGCGTCAGAGGCTTCCCT AGATGCAGATACGTGCACAAGGTGCAAGGCACAGGCCCCTGTCCTGGCGATTTCGCCTTTCACAAGG ACGGCGCCTTTTTCCTGTACGATCGGCTGGCCTCCACCGTGATCTACAGAGGCACAACATTTGCCGA AGGCGTGGTGGCCTTCCTGATCCTGCCTAAGCCTAAGAAGGACTTCTTTCAGAGCCCTCCTATCCGC GAGCCTGTGAACACAAC^GAGGACCCCAGCAGCTACTACACCACCAGCACACTGAGCTACGAGATCG ATAACTTCGGCGCCAAC^GACCAAGACACTGTTCAAGGTGGACAACCACACCTACGTGCAGCTGGA C^GACCCCACACACCTCAGTTTCTGGTGCAGCTGAACGAGACAATCCACACCAACAACAGACTGAGC AACACCACCGGCAGGCTGATCTGGACCCTGGATCCTAAGATCGACACCGACATCGGAGAGTGGGCCT TTTGGGAGAACAAGAAGAACTTCAGCAAGCAGCTGAGAGGCGAGGAACTGAGCTTTAAGGCCCTGAG CACCAAGACAGGCGCCAACGCTGTGGATACCGATGAGTCTAGCAAGCCCGGCCTGATCACCAACACA GTTAGAGGCGTTGCCGACCTGCTGAGCCCTTGGAGAAGAAAGCGGAGACAAGTGAACCCCAATACCA CCAACAAGTGCAACCCTAACCTGCACTACTGGACAGCCCAGGATGAAGGCGCTGCTGTTGGACTGGC CTGGATTCCTTATTTTGGACCTGCCGCCGAGGGCATCTACACAGAGGGAATCATGCACAACCAGAAT GGCCTGATCTGCGGCCTGAGACAGCTGGCCAATGAGACAACACAGGCCCTCCAGCTGTTTCTGAGAG CCACC^CCGAGCTGAGAACCTACAGCATCCTGAACCGGAAGGCCATCGACTTTCTGCTGCAAAGATG GGGAGGCACCTGTAGAATCCTGGGACCTGATTGCTGCATCGAGCCCCACGACTGGACCAAGAACATC ACCGACAAGATCAACCAGATCATCCACGACTTCATCGACAACCCTCTGCCTGACCAGGACGACGACG ATAATTGGTGGACAGGATGGCGGCAGTGGATTCCTGCCGGAATCGGAATCACAGGCGTGATCATTGC CATTATCGCCCTGCTGTGCATCTGCAAGTTTCTGTGCTGA Amino acid sequence encoded by unoptimised and gene-optimised sequences (SEQ ID NO: 6): MGGGSRLLQLPRERFRKTSFFVWVIILFQKAFSMPLGWTNSTLKVTEIDQLVCRDKLSSTSQLKSV GLNLEGNGVATDVPSATKRWGFRSGVPPKWSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFP RCRYVHKVQGTGPCPGDFAFHKDGAFFLYDRLASTVIYRGTTFAEGWAFLILPKPKKDFFQSPPIR EPVNTTEDPSSYYTTSTLSYEIDNFGANKTKTLFKVDNHTYVQLDRPHTPQFLVQLNETIHTNNRLS NTTGRLIWTLDPKIDTDIGEWAFWENKKNFSKQLRGEELSFKALSTKTGANAVDTDESSKPGLITNT VRGVADLLSPWRRKRRQVNPNTTNKCNPNLHYWTAQDEGAAVGLAWIPYFGPAAEGIYTEGIMHNQN GLICGLRQLANETTQALQLFLRATTELRTYSILNRKAIDFLLQRWGGTCRILGPDCCIE PHDWTKNI TDKINQIIHDFIDNPLPDQDDDDNWWTGWRQWIPAGIGITGVIIAIIALLCICKFLC Example 3 Marburqvirus ancestor (T2-11) Unoptimised (SEQ ID NO:7) ATGAAGACCATATATTTTCTGATTAGTCTCATTTTAATCCAAAGTATAAAAACTCTCCCTGTTTTAG AAATTGCTAGTAACAGCCAACCTCAAGATGTAGATTCAGTGTGCTCCGGAACCCTCCAAAAGACAGA AGATGTTCATCTGATGGGATTTACACTGAGTGGGCAAAAAGTTGCTGATTCCCCTTTGGAAGCATCT AAACGATGGGCTTTCAGGACAGGTGTTCCTCCCAAGAACGTTGAGTATACGGAAGGAGAAGAAGCCA AAACATGTTACAATATAAGTGTAACAGACCCTTCTGGAAAATCCTTGCTGCTGGATCCTCCCAGTAA TATCCGCGATTACCCTAAATGTAAAACTGTTCATCATATTCAAGGTCAAAACCCTCATGCACAGGGG ATTGCCCTCCATTTGTGGGGGGCATTTTTCCTGTATGATCGCATTGCCTCCACAACAATGTACCGAG GCAAAGTCTTCACTGAAGGGAACATAGCAGCTATGATTGTCAATAAGACAGTGCACAAAATGATTTT CTCGAGGCAAGGACAAGGGTACCGTCACATGAATCTGACTTCTACTAATAAATATTGGACAAGTAGC AACGGAACGCAAACGAATGACACTGGATGCTTCGGTGCTCTTCAAGAATACAATTCTACGAAGAACC AAACATGTGCTCCGTCCAAAATACCTCCACCACTGCCCACAGCCCGTCCGGAGATCAAACCCACAAG CACCCCAACTGATGCCACCAAACTCAACACCACAGACCCAAACAGTGATGATGAGGACCTCACAACA TCCGGCTCAGGGTCCGGAGAACAGGAACCCTACACAACTTCTGATGCGGTCACTAAGCAAGGGCTTT CATCAACAATGCCACCCACTCCCTCACCACAACCAAGCACGCCACAGCAAGGAGGAAACAACACAAA CCATTCCCAAGGTGCTGTGACTGAACCCGACAAAACCAACACAACTGCACAACCGTCCATGCCCCCC CACAACACTACTACAATCTCTACTAACAACACCTCCAAGCACAACTTCAGCACTCTCTCTGCACCAC TACAAAACACCACCAATTACAACACACAGAGCACGGCCACTGAAAATGAGCAAACCAGTGCCCCCTC GAAAACAACCCTGCCTCCAACAGGAAATCCTACCACAGCAAAGAGCACCAACAGCACAAAAGGCCCC ACCACAACGGCACCAAATACGACAAATGGGCATTTCACCAGTCCCTCCCCCACCCCCAACTCGACTA CACAACATCTTGTATATTTCAGAAGGAAACGAAGTATCCTCTGGAGGGAAGGCGACATGTTCCCTTT TTTAGATGGGTTAATAAATACTGAAATTGATTTTGATCCAATCCCAAACACAGAAACAATCTTTGAT GAATCCCCCAGCTTTAATACTTCAACTAATGAGGAACAACACACTCCCCCGAATATCAGTTTAACTT TCTCTTATTTTCCTGATAAAAATGGAGATACTGCCTACTCTGGGGAAAACGAGAATGATTGTGATGC AGAGTTGAGGATTTGGAGTGTGCAGGAGGACGATTTGGCGGCAGGGCTTAGCTGGATACCATTTTTT GGCCCTGGAATCGAAGGACTCTATACTGCCGGTTTAATCAAAAATCAGAACAATTTAGTTTGTAGGT TGAGGCGCTTAGCTAATCAAACTGCTAAATCCTTGGAGCTCTTGTTAAGGGTCACAACCGAGGAAAG GACATTTTCCTTAATCAATAGGCATGCAATTGACTTTTTGCTTACGAGGTGGGGCGGAACATGCAAG GTGCTAGGACCTGATTGTTGCATAGGAATAGAAGATCTATCTAAAAATATCTCAGAACAAATTGACA AAATCAGAAAGGATGAACAAAAGGAGGAAACTGGCTGGGGTCTAGGTGGCAAATGGTGGACATCTGA CTGGGGTGTTCTCACCAATTTGGGCATCCTGCTACTATTATCTATAGCTGTTCTGATTGCTCTGTCC TGTATCTGTCGTATCTTCACTAAATATATCGGATAG Gene-optimised (SEQ ID NO:8) ATGAAGACCATCTACTTTCTGATCAGCCTGATCCTGATCCAGAGCATCAAGACCCTGCCTGTGCTGG AAATCGCCAGCAACAGTCAGCCCCAGGATGTGGATAGCGTGTGTAGCGGCACCCTCCAGAAAACCGA GGATGTGCACCTGATGGGCTTTACCCTGAGCGGCCAGAAAGTGGCCGATTCTCCACTGGAAGCCAGC AAGAGATGGGCCTTTAGAACCGGCGTGCCACCTAAGAACGTCGAGTACACAGAGGGCGAAGAGGCCA AGACCTGCTACAACATCAGCGTGACCGATCCTAGCGGCAAGAGCCTGCTGCTGGACCCTCCTAGCAA CATCAGAGACTACCCCAAGTGCAAGACCGTGCACCACATCCAGGGACAGAATCCCCATGCTCAGGGA ATTGCCCTGCACCTGTGGGGCGCCTTTTTCCTGTATGATCGGATCGCCTCCACCACCATGTACAGAG GCAAAGTGTTCACCGAGGGCAATATCGCCGCCATGATCGTGAACAAGACAGTGCACAAGATGATCTT CAGCCGGCAAGGCCAGGGCTACAGACACATGAATCTGACCAGCACCAACAAGTACTGGACCAGCAGC AACGGCACCCAGACCAATGATACAGGCTGCTTTGGCGCCCTGCAAGAGTACAACAGCACCAAGAATC AGACATGCGCCCCTAGCAAGATCCCTCCTCCACTGCCTACTGCCAGACCTGAGATCAAGCCTACCAG CACACCTACCGACGCCACCAAGCTGAACACCACCGATCCAAACAGCGACGACGAGGATCTGACAACA AGCGGATCTGGCTCTGGCGAGCAAGAGCCATACACCACCTCTGATGCCGTGACAAAGCAGGGCCTGA GCAGCACAATGCCTCCAACACCTTCTCCACAGCCTAGCACACCTCAGCAAGGCGGCAACAACACAAA TCACTCTCAGGGCGCCGTGACCGAGCCTGACAAGACAAATACCACAGCTCAGCCCAGCATGCCTCCT CACAACACCACCACAATCTCCACCAACAACACCAGCAAGCACAACTTCAGCACACTGAGCGCCCCTC TCCAGAATACCACCAACTACAATACCCAGAGCACCGCCACCGAGAACGAGCAGACATCTGCCCCTTC TAAGACCACACTGCCACCTACCGGCAATCCTACCACCGCCAAGAGCACCAATAGCACAAAGGGCCCT ACCACCACCGCTCCTAACACCACAAATGGCCACTTCACAAGCCCAAGTCCTACACCTAACAGCACAA CCCAGCACCTGGTGTACTTCAGACGGAAGCGGAGCATCCTTTGGCGCGAGGGCGATATGTTCCCTTT CCTGGACGGCCTGATCAACACCGAGATCGACTTCGACCCCATTCCAAACACCGAAACCATCTTCGAC GAGAGCCCCAGCTTCAACACCTCCACCAATGAGGAACAGCACACCCCTCCAAACATCTCCCTGACCT TCAGCTACTTCCCCGACAAGAACGGCGATACAGCCTACAGCGGCGAGAATGAGAATGACTGCGACGC CGAGCTGCGGATTTGGAGCGTTCAAGAGGATGATCTGGCTGCCGGCCTGAGCTGGATCCCTTTTTTT GGACCTGGCATCGAGGGCCTGTACACCGCCGGACTGATCAAGAACCAGAACAACCTCGTGTGCAGAC TGCGGAGACTGGCCAATCAGACCGCCAAGTCTCTGGAACTGCTGCTGCGCGTGACCACCGAGGAAAG AACCTTCTCTCTGATCAACCGGCACGCCATCGATTTTCTGCTGACCAGATGGGGCGGCACCTGTAAA GTTCTGGGCCCTGATTGCTGCATCGGAATCGAGGACCTGAGCAAGAACATCTCCGAGCAGATCGACA AGATCCGCAAGGACGAGCAGAAAGAGGAAACAGGCTGGGGACTCGGCGGCAAGTGGTGGACATCTGA TTGGGGCGTGCTGACCAATCTGGGAATCCTGCTGCTCCTGTCTATCGCCGTGCTGATCGCCCTGAGC TGCATCTGCCGGATCTTCACCAAGTACATCGGCTGA Amino acid sequence encoded by unoptimised and gene-optimised sequences (SEQ ID NO: 9): MKTIYFLISLILIQSIKTLPVLEIASNSQPQDVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRT GVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPSNIRDYPKCKTVHHIQGQNPHAQGIALHLWGAFFLYDR IASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTK NQTCAPSKIPPPLPTARPEIKPTSTPTDATKLNTTDPNSDDEDLTTSGSGSGEQEPYTTSDAVTKQGLSSTMPP TPSPQPSTPQQGGNNTNHSQGAVTEPDKTNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNYNTQSTA TENEQTSAPSKTTLPPTGNPTTAKSTNSTKGPTTTAPNTTNGHFTSPSPTPNSTTQHLVYFRRKRSILWREGDM FPFLDGLINTEIDFDPIPNTETIFDESPSFNTSTNEEQHTPPNISLTFSYFPDKNGDTAYSGENENDCDAELRI WSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAID FLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDKIRKDEQKEETGWGLGGKWWTSDWGVLTNLGILLLLSIAVL IALSCICRIFTKYIG Example 4 Tier 2-4 (SUPV anc -MLP) Sudan ebolavirus ancestral sequences with deleted (minus mucin-like domain Nucleotide sequence (SEQ ID NO:10): atgggaggac tgtctctgct gcaactgccc cgggacaagt tccggaagtc cagcttcttc 60 gtgtgggtca tcatcctgtt ccagaaagcc ttcagcatgc ccctgggcgt cgtgaccaat 120 agcacactgg aagtgaccga gatcgaccag ctcgtgtgca aggatcacct ggccagcacc 180 gatcagctga agtctgtggg actgaatctg gaaggcagcg gcgtgtccac agatatccct 240 agcgccacca agagatgggg ctttagaagc ggagtgcctc ctaaggtggt gtcttatgaa 300 gccggcgagt gggccgagaa ctgctacaac ctggaaatca agaagcccga cggcagcgag 360 tgtctgcctc ctccacctga tggcgtcaga ggcttcccta gatgcagata cgtgcacaag 420 gcccaaggca caggaccctg tcctggcgat tacgcctttc acaaggacgg cgcctttttc 480 ctgtacgatc ggctggcctc caccgtgatc tacagaggcg ttaactttgc cgagggcgtg 540 atcgccttcc tgatcctggc caagcctaaa gagacattcc tgcaaagccc tccaatccgc 600 gaggccgtga actacacaga gaacaccagc agctactacg ccaccagcta cctggaatac 660 gagatcgaga atttcggcgc ccagcacagc accacactgt tcaagatcga caacaacacc 720 ttcgtgcggc tggacagacc ccacacacct cagtttctgt tccagctgaa cgacaccatc 780 catctgcatc agcagctgag caacaccacc ggcagactga tttggaccct ggacgccaac 840 atcaacgccg acattggaga gtgggccttt tgggagaaca agaagaacct gagcgaacag 900 ctgagaggcg aggaactgag ctttgaggcc ctgtctctga ccaccgccgt gaaaacagtg 960 ctgcctcaag agtccaccag caacggcctg atcacaagca cagtgacagg catcctgggc 1020 agcctgggcc tgagaaaaag gtccagacgg caagtgaata ccaaggccac cggcaagtgc 1080 aaccccaacc tgcactattg gacagcccaa gagcagcaca atgccgccgg aatcgcctgg 1140 attccttatt ttggacctgg cgccgagggc atctataccg agggactgat gcacaaccag 1200 aacgccctcg tgtgtggact gagacagctg gccaatgaga caacacaggc cctccagctg 1260 tttctgagag ccaccaccga gctgagaacc tacaccatcc tgaaccggaa ggccatcgac 1320 tttctgctga gaagatgggg cggcacctgt agaatcctgg gacctgattg ctgcatcgag 1380 ccccacgact ggaccaagaa catcaccgac aagatcaacc agatcatcca cgacttcatc 1440 gacaaccctc tgcctaacca ggacaacgac gacaattggt ggacaggctg gcggcagtgg 1500 attcctgccg gaattggcat caccggcatc atcattgcca ttatcgccct gctgtgtgtg 1560 tgcaagctgc tgtgttga 1578 Amino acid sequence (SEQ ID NO:11): MGGLSLLQLPRDKFRKS S FFVWVIILFQKAFSMPLGWTNS TLEVTEIDQ 50 LVCKDHLASTDQLKSVGLNLEGSGVSTDIPSATKRWGFRSGVPPKWSYE 100 AGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKAQGTGPCPGD 150 YAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIR 200 EAVNYTENTSSYYATSYLEYEIENFGAQHSTTLFKIDNNTFVRLDRPHTP 250 QFLFQLNDTIHLHQQLSNTTGRLIWTLDANINADIGEWAFWENKKNLSEQ 300 LRGEELSFEALSLTTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRR 350 QVNTKATGKCNPNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQ 400 NALVCGLRQLANETTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTC 450 RILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQW 500 IPAGIGITGIIIAIIALLCVCKLLC* Example 5 Tier 2-6 (SUDV EBOV-TAFV-BDBV anc -MLP) Ancestral sequence to the four species Sudan, Zaire, Tai Forest, and Bundibugyo ebolavirus with the mucin-like-domain deleted. Nucleotide sequence (SEQ IP NO:12): atgggcggag ttcgtgtggg aatagcaccc accagccagc cctagcgcca gaagccggcg gagtgtctgc aaggtgcaag gatctagact tcatcatcct tgaaagtgac tgaagtccgt ccaaaagatg agtgggccga ctcctccacc gcacaggccc gctgcaactg gttccagaaa cgagatcgac gggactgaat gggctttaga gaactgctac tgatggcgtc ctgtcctggc cccagagagc gccttcagca cagctcgtgt ctggaaggca agcggcgtgc aacctggaaa agaggcttcc gatttcgcct ggttcagaaa tgcccctggg gcagagataa atggcgtggc cacctaaggt tcaagaagcc ctagatgcag ttcacaagga gaccagcttc cgtcgtgacc gctgagcagc cacagatgtg ggtgtcttat cgacggcagc atacgtgcac cggcgccttt 60 120 180 240 300 360 420 ttcctgtacg gtggtggcct cgcgagcctg tacgagatcg acctacgtgc atccacacca aagatcgaca cagctgagag gtggataccg gacctgctga tgcaacccta tggattcctt cagaatggcc ctgtttctga gactttctgc gagccccacg atcgacaacc tggattcctg atctgcaagt atcggctggc tcctgatcct tgaacacaac ataacttcgg agctggacag acaacagact ccgacatcgg gcgaggaact atgagtctag gcccttggag acctgcacta attttggacc tgatctgcgg gagccaccac tgcaaagatg actggaccaa ctctgcctga ccggaatcgg ttctgtgctg ctccaccgtg gcctaagcct agaggacccc cgccaacaag accccacaca gagcaacacc agagtgggcc gagctttaag caagcccggc aagaaagcgg ctggacagcc tgccgccgag cctgagacag cgagctgaga gggaggcacc gaacatcacc ccaggacgac aatcacaggc a atctacagag aagaaggact agcagctact accaagacac cctcagtttc accggcaggc ttttgggaga gccctgagca ctgatcacca agacaagtga caggatgaag ggcatctaca ctggccaatg acctacagca tgtagaatcc gacaagatca gacgataatt gtgatcattg gcacaacatt tctttcagag acaccaccag tgttcaaggt tggtgcagct tgatctggac acaagaagaa ccaagacagg acacagttag accccaatac gcgctgctgt cagagggaat agacaacaca tcctgaaccg tgggacctga accagatcat ggtggacagg ccattatcgc tgccgaaggc ccctcctatc cacactgagc ggacaaccac gaacgagaca cctggatcct cttcagcaag cgccaacgct aggcgttgcc caccaacaag tggactggcc catgcacaac ggccctccag gaaggccatc ttgctgcatc ccacgacttc atggcggcag cctgctgtgc 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1581 Amino acid sequence (SEQ ID NO:13): MGGGSRLLQLPRERFRKTSFFVWVIILFQKAFSMPLGWTNS TLKVTEID 50 QLVCRDKLSSTSQLKSVGLNLEGNGVATDVPSATKRWGFRSGVPPKWSY 100 EAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKVQGTGPCPG 150 DFAFHKDGAFFLYDRLASTVIYRGTTFAEGWAFLILPKPKKDFFQSPPI 200 REPVNTTEDPSSYYTTSTLSYEIDNFGANKTKTLFKVDNHTYVQLDRPHT 250 PQFLVQLNETIHTNNRLSNTTGRLIWTLDPKIDTDIGEWAFWENKKNFSK 300 QLRGEELSFKALSTKTGANAVDTDESSKPGLITNTVRGVADLLSPWRRKR 350 RQVNPNTTNKCNPNLHYWTAQDEGAAVGLAWIPYFGPAAEGIYTEGIMHN 400 QNGLICGLRQLANETTQALQLFLRATTELRTYSILNRKAIDFLLQRWGGT 450 CRILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPDQDDDDNWWTGWRQ 500 WIPAGIGITGVIIAIIALLCICKFLC* Example 6 Tier 2-11 (RAW MARV anc) Ancestral sequence to the strains Marburg Virus and Ravn Virus Nucleotide sequence (SEQ ID NO:14): atgaagacca gtgctggaaa ctccagaaaa gattctccac gtcgagtaca agcggcaaga accgtgcacc ggcgcctttt accgagggca cggcaaggcc agcaacggca accaagaatc gagatcaagc agcgacgacg acctctgatg cagcctagca gagcctgaca atctccacca accaccaact aagaccacac ggccctacca cctaacagca gagggcgata attccaaaca tctactttct tcgccagcaa ccgaggatgt tggaagccag cagagggcga gcctgctgct acatccaggg tcctgtatga atatcgccgc agggctacag cccagaccaa agacatgcgc ctaccagcac aggatctgac ccgtgacaaa cacctcagca agacaaatac acaacaccag acaataccca tgccacctac ccaccgctcc caacccagca tgttcccttt ccgaaaccat gatcagcctg cagtcagccc gcacctgatg caagagatgg agaggccaag ggaccctcct acagaatccc tcggatcgcc catgatcgtg acacatgaat tgatacaggc ccctagcaag acctaccgac aacaagcgga gcagggcctg aggcggcaac cacagctcag caagcacaac gagcaccgcc cggcaatcct taacaccaca cctggtgtac cctggacggc cttcgacgag atcctgatcc caggatgtgg ggctttaccc gcctttagaa acctgctaca agcaacatca catgctcagg tccaccacca aacaagacag ctgaccagca tgctttggcg atccctcctc gccaccaagc tctggctctg agcagcacaa aacacaaatc cccagcatgc ttcagcacac accgagaacg accaccgcca aatggccact ttcagacgga ctgatcaaca agccccagct agagcatcaa atagcgtgtg tgagcggcca ccggcgtgcc acatcagcgt gagactaccc gaattgccct tgtacagagg tgcacaagat ccaacaagta ccctgcaaga cactgcctac tgaacaccac gcgagcaaga tgcctccaac actctcaggg ctcctcacaa tgagcgcccc agcagacatc agagcaccaa tcacaagccc agcggagcat ccgagatcga tcaacacctc gaccctgcct tagcggcacc gaaagtggcc acctaagaac gaccgatcct caagtgcaag gcacctgtgg caaagtgttc gatcttcagc ctggaccagc gtacaacagc tgccagacct cgatccaaac gccatacacc accttctcca cgccgtgacc caccaccaca tctccagaat tgccccttct tagcacaaag aagtcctaca cctttggcgc cttcgacccc caccaatgag 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 gaacagcaca cccctccaaa catctccctg accttcagct acttccccga caagaacggc 1500 gatacagcct acagcggcga gaatgagaat gactgcgacg ccgagctgcg gatttggagc 1560 gttcaagagg atgatetgge tgccggcctg agctggatcc ctttttttgg acctggcatc 1620 gagggcctgt acaccgccgg actgatcaag aaccagaaca acctcgtgtg cagactgcgg 1680 agactggcca atcagaccgc caagtctctg gaaetgetge tgcgcgtgac caccgaggaa 1740 agaaccttct ctctgatcaa ccggcacgcc atcgattttc tgctgaccag atggggcggc 1800 acctgtaaag ttctgggccc tgattgctgc ateggaateg aggacctgag caagaacatc 1860 tccgagcaga tcgacaagat ccgcaaggac gagcagaaag aggaaacagg ctggggactc 1920 ggcggcaagt ggtggacatc tgattggggc gtgctgacca atctgggaat cctgctgctc 1980 etgtetateg ccgtgctgat cgccctgagc tgcatctgcc ggatcttcac caagtacatc 2040 ggctga 2046 Amino acid sequence (SEQ ID NO:15): MKTIYFLISLILIQSIKTLPVLEIASNSQPQPVPSVCSGTLQKTEPVHLM 50 GFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDP 100 SGKSLLLDPPSNIRDYPKCKTVHHIQGQNPHAQGIALHLWGAFFLYDRIA 150 STTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTS 200 SNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKPTSTPTD 250 ATKLNTTDPNSDDEDLTTSGSGSGEQEPYTTSDAVTKQGLSSTMPPTPSP 300 QPSTPQQGGNNTNHSQGAVTEPDKTNTTAQPSMPPHNTTTISTNNTSKHN 350 FSTLSAPLQNTTNYNTQSTATENEQTSAPSKTTLPPTGNPTTAKSTNSTK 400 GPTTTAPNTTNGHFTSPSPTPNSTTQHLVYFRRKRSILWREGDMFPFLDG 450 LINTEIDFDPIPNTETIFDESPSFNTSTNEEQHTPPNISLTFSYFPDKNG 500 DTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIK 550 NQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGG 600 TCKVLGPDCCIGIEDLSKNISEQIDKIRKDEQKEETGWGLGGKWWTSDWG 650 VLTNLGILLLLSIAVLIALSCICRIFTKYIG* Example 7 pEVAC Expression Vector Figure 3 shows a map of the pEVAC expression vector. The sequence of the multiple cloning site of the vector is given below, followed by its entire nucleotide sequence. Sequence of pEVAC Multiple Cloning Site (MCS) (SEQ ID NO:16): PstI             Kpnl Sall pEVAC 1301 ACAGACTGTT CCTTTCCATG GGTCTTTTCT GCAGTCACCG TCGGTACCGT Bell Xbal BamHI Natl Bglll pEVAC 1351 CGACACGTGT GATCATCTAG AGGATCCGCG GCCGCAGATC T Entire Sequence of pEVAC (SEQ ID NO:17): CMV-IE-E / P: 248 -   989 CMV immediate early 1 enhancer / promoter KanR: 3445 - 4098 Kanamycin resistance SP: 990 - 1220 Splice donor SA: 1221 - 1343 Splice acceptor Tbgh: 1392 - 1942 Terminator signal from bovine growth hormone pUC-ori: 2096 - 2769 pUC-plasmid origin of replication TCGCGCGTTT GAGACGGTCA TCAGGGCGCG CGGCATCAGA CCGCACAGAT TTGCATACGT TCCAACATTA AATCAATTAC ACATAACTTA CCCATTGACG CTTTCCATTG CGGTGATGAC CAGCTTGTCT TCAGCGGGTG GCAGATTGTA GCGTAAGGAG TGTATCCATA CCGCCATGTT GGGGTCATTA CGGTAAATGG TCAATAATGA ACGTCAATGG GGTGAAAACC GTAAGCGGAT TTGGCGGGTG CTGAGAGTGC AAAATACCGC TCATAATATG GACATTGATT GTTCATAGCC CCCGCCTGGC CGTATGTTCC GTGGAGTATT TCTGACACAT GCCGGGAGCA TCGGGGCTGG ACCATATGCG ATCAGATTGG TACATTTATA ATTGACTAGT CATATATGGA TGACCGCCCA CATAGTAACG TACGGTAAAC GCAGCTCCCG GACAAGCCCG CTTAACTATG GTGTGAAATA CTATTGGCCA TTGGCTCATG TATTAATAGT GTTCCGCGTT ACGACCCCCG CCAATAGGGA TGCCCACTTG 551 GCAGTACATC 601 TGACGGTAAA 651 ACTTTCCTAC 701 GTGATGCGGT 751 ACGGGGATTT 801 GGCACCAAAA 851 TTGACGCAAA 901 AGCTCGTTTA 351 TTGACCTCCA 1001 TCTCTCCTTC 1051 TTGAGTCGCG 1101 CGCCGTCTAG .1151 CGCTCCCTTG 1201 ACCCTGCTTG 1251 AGTACTCGTT 1301 ACAGACTGTT 1351 CGACACGTGT 1401 TCTAGTTGCC 1451 CCTGGAAGGT 1501 CATCGCATTG 1551 CAGGACAGCA .1601 TGCGGTGGGC .1651 CCTGGGCCAG 1701 CCACGCCCCT 1751 CAGGAGGGCT 1801 CTCCCTCCCT 1851 AGAAATTAAA 1901 CCAACATGTG 1351 TTTAAGGCCA 2001 TGCGCTCGGT 2051 GTAATACGGT 2101 AGCAAAAGGC 2151 CGTTTTTCCA 2201 TCAAGTCAGA 2251 TCCCCCTGGA 2301 CCGGATACCT 2351 AGCTCACGCT 2401 GGGCTGTGTG 2451 GTAACTATCG 2501 GCAGCAGCCA 2551 TACAGAGTTC 2601 TATTTGGTAT 2651 GGTAGCTCTT 2701 TGTTTGCAAG 2751 CTTTGATCTT 2801 TAAGGGATTT 2 8 51 TTTAAATTAA 2901 CTTGGTCTGA 2351 ATCTGTCTAT 3001 CTGAGGTCTG 3051 TCGCCCCATC 3101 TTGTTGTAGG 3151 ACGGTCTGCG 3201 AAGTTCGATT .3251 TCTGCCAGTG 3301 AGCATCAAAT 3351 TTTTTGAAAA 3401 TCCATAGGAT 3451 ACATCAATAC 3501 TGAGAAATCA 3551 TATGCATTTC 3601 TCAAAATCAC AAGTGTATCA TGGCCCGCCT TTGGCAGTAC TTTGGCAGTA CCAAGTCTCC TCAACGGGAC TGGGCGGTAG GTGAACCGTC TAGAAGACAC ACGCGCCCGC TTCTGCCGCC GTAAGTTTAA GAGCCTACCT CTCAACTCTA GCTGCCGCGC CCTTTCCATG GATCATCTAG AGCCATCTGT GCCACTCCCA TCTGAGTAGG AGGGGGAGGA TCTATGGCTA AAAGAAGCAG GGTTCTTAGT CCGCCTTCAA CATCAGCCCA GCAAGATAGG AGGAAGTAAT TCATGGCCTT CGTTCGGCTG TATCCACAGA CAGCAAAAGG TAGGCTCCGC GGTGGCGAAA AGCTCCCTCG GTCCGCCTTT GTAGGTATCT CACGAACCCC TCTTGAGTCC CTGGTAACAG TTGAAGTGGT CTGCGCTCTG GATCCGGCAA CAGCAGATTA TTCTACGGGG TGGTCATGAG AAATGAAGTT CAGTTACCAA TTCGTTCATC CCTCGTGAAG ATCCAGCCAG TGGACCAGTT TTGTCGGGAA TATTCAACAA TTACAACCAA GAAACTGCAA AGCCGTTTCT GGCAAGATCC AACCTATTAA CCATGAGTGA TTTCCAGACT TCGCATCAAC TATGCCAAGT GGCATTATGC ATCTACGTAT CATCAATGGG ACCCCATTGA TTTCCAAAAT GCGTGTACGG AGATCGCCTG CGGGACCGAT CGCCCTACCT TCCCGCCTGT AGCTCAGGTC AGACTCAGCC GTTAACGGTG GCGCCACCAG GGTCTTTTCT AGGATCCGCG TGTTTGCCCC CTGTCCTTTC TGTCATTCTA TTGGGAAGAC CCCAGGTGCT GCACATCCCC TCCAGCCCCA TCCCACCCGC CCAAACCAAA CTATTAAGTG GAGAGAAATC AATCTTCCGC CGGCGAGCGG ATCAGGGGAT CCAGGAACCG CCCCCTGACG CCCGACAGGA TGCGCTCTCC CTCCCTTCGG CAGTTCGGTG CCGTTCAGCC AACCCGGTAA GATTAGCAGA GGCCTAACTA CTGAAGCCAG ACAAACCACC CGCGCAGAAA TCTGACGCTC ATTATCAAAA TTAAATCAAT TGCTTAATCA CATAGTTGCC AAGGTGTTGC AAAGTGAGGG GGTGATTTTG GATGCGTGAT AGCCGCCGTC TTAACCAATT TTTATTCATA GTAATGAAGG TGGTATCGGT TTTCCCCTCG CGACTGAATC TGTTCAACAG CAAACCGTTA ACGCCCCCTA CCAGTACATG TAGTCATCGC CGTGGATAGC CGTCAATGGG GTCGTAACAA TGGGAGGTCT GAGACGCCAT CCAGCCTCCA GAGGCCGCCA GGTGCCTCCT GAGACCGGGC GGCTCTCCAC GAGGGCAGTG ACATAATAGC GCAGTCACCG GCCGCAGATC TCCCCCGTGC CTAATAAAAT TTCTGGGGGG AATAGCAGGC GAAGAATTGA TTCTCTGTGA CTCATAGGAC TAAAGTACTT CCTAGCCTCC CAGAGGGAGA ATAGAATTTT TTCCTCGCTC TATCAGCTCA AACGCAGGAA TAAAAAGGCC AGCATCACAA CTATAAAGAT TGTTCCGACC GAAGCGTGGC TAGGTCGTTC CGACCGCTGC GACACGACTT GCGAGGTATG CGGCTACACT TTACCTTCGG GCTGGTAGCG AAAAGGATCT AGTGGAACGA AGGATCTTCA CTAAAGTATA GTGAGGCACC TGACTCGGGG TGACTCATAC AGCCACGGTT AACTTTTGCT CTGATCCTTC CCGTCAAGTC CTGATTAGAA TCAGGATTAT AGAAAACTCA CTGCGATTCC TCAAAAATAA CGGTGAGAAT GCCAGCCATT TTCATTCGTG TTGACGTCAA ACCTTATGGG TATTACCATG GGTTTGACTC AGTTTGTTTT CTCCGCCCCA ATATAAGCAG CCACGCTGTT TCGGCTCGCA TCCACGCCGG GAACTGCGTC CTTTGTCCGG GCTTTGCCTG TAGTCTGAGC TGACAGACTA TCGGTACCGT TGCTGTGCCT CTTCCTTGAC GAGGAAATTG TGGGGTGGGG ATGCTGGGGA CCCGGTTCCT CACACCCTGT ACTCATAGCT GGAGCGGTCT AAGAGTGGGA GAAAATGCCT AAGGCCATGA ACTGACTCGC CTCAAAGGCG AGAACATGTG GCGTTGCTGG AAATCGACGC ACCAGGCGTT CTGCCGCTTA GCTTTCTCAT GCTCCAAGCT GCCTTATCCG ATCGCCACTG TAGGCGGTGC AGAAGAACAG AAAAAGAGTT GTGGTTTTTT CAAGAAGATC AAACTCACGT CCTAGATCCT TATGAGTAAA TATCTCAGCG GGGGGGGGCG CAGGCCTGAA GATGAGAGCT TTGCCACGGA AACTCAGCAA AGCGTAATGC AAACTCATCG CAATACCATA CCGAGGCAGT GACTCGTCCA GGTTATCAAG GGCAAAAGCT ACGCTCGTCA ATTGCGCCTG 3651 AGCGAGACGA AATACGCGAT CGCTGTTAAA AGGACAATTA 3701 TCGAATGCAA CCGGCGCAGG AACACTGCCA GCGCATCAAC 3751 CCTGAATCAG GATATTCTTC TAATACCTGG AATGCTGTTT 3801 CGCAGTGGTG AGTAACCATG CATCATCAGG AGTACGGATA 5    3851 TGGTCGGAAG AGGCATAAAT TCCGTCAGCC AGTTTAGTCT 3951 TCTGTAACAT CATTGGCAAC GCTACCTTTG CCATGTTTCA 3951 TGGCGCATCG GGCTTCCCAT ACAATCGATA GATTGTCGCA 4001 CGACATTATC GCGAGCCCAT TTATACCCAT ATAAATCAGC 4051. GAATTTAATC GCGGCCTCGA GCAAGACGTT TCCCGTTGAA 10    41.01 AACACCCCTT GTATTACTGT TTATGTAAGC AGACAGTTTT 4151 ATGATATATT TTTATCTTGT GCAATGTAAC ATCAGAGATT 4201 ACGTGGCTTT CCCCCCCCCC CCATTATTGA AGCATTTATC 4251 TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT 4301 GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT 15    4351 ATTATTATCA TGACATTAAC CTATAAAAAT AGGCGTATCA 4401 TCGTC CAAACAGGAA AATATTTTCA TCCCGGGGAT AAATGCTTGA GACCATCTCA GAAACAACTC CCTGATTGCC ATCCATGTTG TATGGCTCAT ATTGTTCATG TTGAGACACA AGGGTTATTG AAACAAATAG CTAAGAAACC CGAGGCCCTT Example 8 Lead candidate optimized antigenic Ebola polypeptides able to induce a broadly neutralizing antibody response There was a significant interest to develop vaccines against Ebola followed the West African outbreak in 2014. Programmes currently in clinical development have so far taken a ‘classical’ approach to vaccine development using Ebola and / or Marburg virus surface glycoproteins (GPs) from one to three strains expressed in a viral vector backbone. Antigen specificity comes only from the included EBOV strains: for example Merck use a GP from Kikwit; GSK use Mayinga EBOV and Gulu SUDV strains; Crucell and Profectus Biosiences both use a Marburg virus together with Zaire and Sudan Ebola strains; with the Novavax approach being unigue in using the 2014 Makona EBOV strain. Table 1 below shows flow cytometric assay results illustrating the strength of antibody binding to target antigens, representative of all Ebola virus species (subtypes) and Marburg viruses. Strength of binding is indicated by the heat-map where red (the darkest shading when viewed in grayscale) is very strong binding, decreasing through orange to yellow (progressively lighter shading when viewed in grayscale) and no binding / egual to negative control values are white. Serum samples 1-22 were taken from individuals immunised with other Ebola virus vaccine candidates. T2-4 and T2-6 are nucleic acid vaccines encoding lead candidate optimized antigenic Ebola polypeptide, combined with T2-11 a Marburg candidate, at pre-clinical stage testing with serum samples taken from immunised guinea pigs. Table 1 Ebola Mayings TM............. Ebola Sudan T1-2............ Ebola Bundi 113............ Ebola Tai (Ebola Forrest [Reston T1-4     |ti-5 Marburg T1-6 Marburg I Ebol a Raw     Ki k wit T0-1      sKikwit GFP con GFP con Sample 1 40 01C     7.303     9.103    12.483     9.997     6.351     5.106    28,086    10,040 28 510     7.139     6.406    10,790     6.728     5.447    4,103    27,544     7,705 17,334     4.308     4,068     6.412     4.169     2 895     2.570    20.704     4,993 38,712     7 / 83     6,245     8,809     6.621     -1.239     3.S29    35.046     8,031 1,W,56S    6,675    11,082    12,339    6.556    5.327    4.960 1,215,877    9,983 42.743     8 351     8.319    10,038     4,622     2.803     2,488    41,380     5,212 112,821    21.100   18.551    23,329    15,623    6,557    6,373   1$?,«    10,203 103,788   42.134   28.650   54,319   13,858    6 584    4,878   58,113    7,517 '836,515   23,701    25,515    59.485    12.529     4.405     2.584   153,091     ^,617 131,342   21,760    14,868    21,924    10.612     7.508     3.682    98,488     8.455 4S2;017    17,926    25,222    38,485    12.897     5.714     3.411   356,130     6.650 73-888   12,226    11,916    28.503     7.268     3.4-5     2,626    38,323     4,556 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 102-60!    12.899    15.227    24.098    11,086     8,645     7,866    97,309    12,811 113.028    19.731    24.98g    37.834    15,667    12 576     9.206   142,277    19,049 97,»   19,349   21,949   33,013    16.803    12.244    10.926  112,7^0   26.297 57,387    8 085     9,380    14,060     5.866     3.545     3.882    70,361     5,107 30.537     3.942     5,098     6,354     -4.138     2.721     2,329    16.354     3,641 22 323     7.173     8.84 5     8,560    11.392     4,424     4,007    18,922     6,802 506,813   60-158   51,225   72,452   8T5S7   46,423   36,302  21?,«  111,316 17,428     64 95     5.918    10.031     5.386     3 589     3.386    12.405     5,454 40,444     6,791     8.479    14,824     6.680     4.636     3.774    45.013     5.568 3,510    4,424    5,254    4,206    2.858    2,725'^0^       3,654 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 T2-4 1 152.841 2,180,438  125,057  229,144    65.656    3.612     2.771   180,762     5,828 547,668 WS, 189  285,485  491,539  402.143    6 334    5,728  684,«    9,198 167,604 2,052,458  318,211   179,231    88,815     2 953     2.007   194,433     3,701 1M912 1,781,«  136,078  241,383   70.818    8 964    6 316  179, 382   10,413 69»2,2?9,«   78,»  307,786   43.840    6.328    5.096   72,613    8,829 202.425 2,«,«  179,014  321,933  119,511    9.847    6.712  1S8.O17   11,929 T2-4 2 T2-4 3 12-4 4 T2-4 5 T2-4 6 T2-6T2-11 1 T2-6 T2-11 2 238-.142  213.346   148,494  334,685  111.837 1.453,732 3,333,784  231,760     9,654 32^176  454,-652  194,068  252,923  134,786 1,«,» 1,109,521  204,227   10,497 442,535  347,»  418,041  858,331  137.388 1.571.854 15W  323,336   10,925 437,288  2MW  185,«  881,«   81.374 1,294,796  836,.«  «,»   10.945 414,571  378.811   298,297 1.«,«   88.382 1.111,93$ 1,824,34?  473,584   10,033 36 653   50.512   42,979  239,226   53,948 1.496,041  789,981    52.957    7,050 1.389     1.478     1.323     2,276     1,459 1.112     1,008     1.310     1,320 1,637     1,395     1,078     1,402     1,724 851     1,006       894     1,131 72-672-11 3 T2-6 72-11 4 T2-6T2-11 5 T2-6 72-11 6 No sera hu No sera gp Example 9 Protection achieved by a trivalent Lassa, Ebola and Marburg viral vaccine (Tri-LEMvac) in an Ebola challenge model We have developed a trivalent vaccine (Tri-LEMvac) that generates combined vaccine efficacy against future outbreaks of variants of the haemorrhagic fever Lassa, Ebola and Marburg viruses. We have bioinformatically designed synthetic glycoprotein seguences from the GPC open reading frames of LASV (L) as well as EBOV (E) and MARV (M) from all available Arenavirus and Filovirus databases. These conserved seguences consist of neutralising antibody and T-cell rich epitopes for each of these viruses. To ensure that these synthetically designed LASV, EBOV and MARV envelopes were functional and antigenic, they were expressed as pseudotypes and guality controlled for both binding and neutralisation against a panel of broadly neutralising antibodies. Herein, we chose the vaccine derived vector Modified Vaccinia Ankara (MVA) for construction of the trimeric LEM vaccine. The Modified Vaccinia Ankara (MVA) vaccine platform is a non-replicating strain (i.e. nonreplicating in human cells), third generation smallpox vaccine and one of the most advanced recombinant poxviral vaccine vectors in human clinical trials (Cottingham & Carroll, Vaccine, 2013, 31(39):4247-51). MVA is a robust vector system capable of co-expressing up to four transgenes facilitating potent promoters and stable insertion sites (Orubu et al, Pone, 2012,7(6)e0040167). MVA was chosen because: 1) its significant capacity to stably express multiple independent ORFs via compatible expression cassettes with strong and timely regulated promoters for trivalent LEM vaccination in one cost effective vaccine lot; 2) its ability to induce robust B and T-cell immune responses in animals and humans especially when primed or boosted with DNA or RNA vectors; and 3) vaccine lots can be thermally stabilised for storage and transport in developing countries in the absence of cold chain (Frey etal, Vaccine, 2015, 33(39):5225-34). Proof of principle for the Trivalent vaccine candidate has been demonstrated by: i) cassette validation for independent L, E and M GPC expression and epitope presentation; and ii) preclinical efficacy by Filovirus challenge. The challenge study results are shown in Figure 4. The Ebola challenge model was lethal for non-vaccinated guinea pigs (Group 1, lower line) whereas all vaccinated guinea pigs (Group 2, upper line) were protected (left) and continued to gain weight (right). The trivalent vaccine (Tri-LEMvac) described herein comprises polynucleotide encoding T2-4 (amino acid sequence SEQ ID NO:3), T2-11 (amino acid sequence SEQ ID NO:9), and L-NP-1 (SEQ ID NO:28) (see Figure 8). Example 10 Pseudotype virus neutralisation assay Figure 5 shows the results of a pseudotype virus neutralisation assay illustrating the strength of neutralising antibody responses to target antigens expressed on the surface of a pseudotyped virus, representative of all Ebola virus species and Marburg viruses. Strength of neutralisation is indicated by the heat-map where red (darkest shading when viewed in grayscale) is very strong neutralisation, decreasing through orange to yellow (progressively lighter shading when viewed in grayscale) and no neutralising / equal to negative control values are white. T2-4 and T2-6 are nucleic acid vaccines each encoding lead candidate optimized antigenic Ebola polypeptide, combined with T2-11 a Marburg candidate, at pre-clinical stage testing with serum samples taken from immunised guinea pigs. The results show that administering a combination of T2-6 and T2-11 vaccine inserts gave a synergistic increase in the breadth of the immune response. Example 11 Lassa virus glycoprotein This example describes Lassa virus glycoprotein ancestral sequence produced using a method according to an embodiment of the invention, and modifications to the ancestral sequence to improve its immunogenicity by stabilising the structure. Lassa fever is a hemorrhagic disease caused by an Old World (OW) arenavirus known as Lassa virus (LASV). The virus was first isolated in Nigeria in 1969 and is currently endemic in West Africa. Due to the high morbidity and mortality associated with Lassa hemorrhagic fever, LASV is classified as a category A pathogen. Lassa virus is an enveloped ambisense RNA virus with a bisegmented genome. Viral particles are covered in mature glycoprotein (GP) trimeric spikes, which mediate viral entry. Like other class 1 viral fusion proteins, the envelope glycoprotein precursor (GPC) is translated as a single polypeptide and is proteolytically cleaved into three subunits. Processing occurs first in the endoplasmic reticulum (ER) by a cellular signal peptidase. GPC is then trafficked to the cis-Golgi apparatus and processed by cellular proprotein convertase subtilisin kexin isozyme-1 / site-1 protease (SKI-1 / S1P) to produce a noncovalent stablesignal peptide (SSP) / GP1 / GP2 heterotrimer. Unlike other class I fusion proteins, the relatively long signal peptide of GPC is not degraded; it serves a chaperone-like function necessary for the correct trafficking and processing of GP. SSP interacts with the cytoplasmic 76 domain of GP2 and is involved in pH sensing. GP1 is responsible for binding to cellular receptors, while GP2 mediates membrane fusion during viral entry. Lassa virus glycoprotein ancestral sequence to lineages III and IV (L-10) (construct 1) was produced using a method according to an embodiment of the invention. Modifications were then introduced independently into the parental ancestral sequence (construct 1) to provide: (A) SOSEP (construct 2); and (B) FLEP (construct 4), as well as in combination with a glycan knock-out, called NtoK (to provide constructs 3 and 5), to stabilize the otherwise flexible heterotrimers and prevent dissociation of the external domain of the glycoprotein from the non-covalently linked transmembrane domain. (A) Two cystein residues were introduced at positions 207 and 360 to allow formation of a disulfide bridge (SOS) between the exterior and the transmembrane domains of GP. To facilitate complete cleavage of these two domains, the furin cleavage site was modified from RRLL to RRRR at position 256-259. Mutation of glutamate to proline at position 329 (EP) prevents structural rearrangements making the protein less flexible. (B) The furin cleavage site (256-RRLL-259) between the C-terminus of the external domain and the N-terminus of the transmembrane domain was replaced by a flexible linker with the sequence 256-GGGGSGGGGS-265. Additionally, the EP-mutation as in (A) was introduced at position 335. Variants of both designs were generated that additionally contain an asparagine to lysine mutation at position 272 or 278, for SOSEP-NtoK or FLEP-NtoK, respectively, to inactivate a glycosylation motif. Glycans at this position might block access of some neutralizing antibodies, such as 37.7H. Construct 1: Lassa virus glycoprotein ancestral sequence to lineages III and IV (L-10 = LASV III IV anc) Amino acid sequence (SEQ ID NO:18): MGQIVTFFQEVPHVIEEVMNIVLIALSLLAILKGLYNVATCGLIGLVTFL 50 LLCGRSCSTTLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIRVGNE 100 TGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQY 150 EAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFMRMAWGGSY 200 IALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTR 250 DIYISRRLLGTFTWTLSDSEGNETPGGYCLTRWMLIEAELKCFGNTAVAK 300 CNEKHDEEFCDMLRLFDFNKQAIRRLKAEAQMSIQLINKAVNALINDQLI 350 MKNHLRDIMGIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYLNETHFS 400 DDIEQQADNMITEMLQKEYMDRQGKTPLGLVDLFVFSTSFYLISIFLHLV 450 KIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWKR* DNA sequence (SEQ ID NO:19): 1 ATGGGCCAGA TCGTGACATT CTTCCAAGAG GTGCCCCACG TGATCGAAGA 51 AGTGATGAAC ATCGTCCTGA TCGCCCTGAG CCTGCTGGCC ATCCTGAAGG 101 GCCTGTATAA TGTGGCCACC TGTGGCCTGA TCGGCCTGGT CACATTTCTG 151 CTGCTGTGCG GCAGAAGCTG CTCCACCACA CTGTATAAGG GCGTGTACGA 201 GCTGCAAACC CTGGAACTGA ACATGGAAAC CCTGAACATG ACCATGCCTC 251 TGAGCTGCAC CAAGAACAAC AGCCACCACT ACATCAGAGT GGGCAACGAG 301 ACAGGCCTCG AGCTGACCCT GACCAACACC AGCATCATCA ACCACAAGTT 351 CTGCAACCTG AGCGACGCCC ACAAGAAGAA CCTGTACGAT CACGCCCTGA 401 TGAGCATCAT CTCCACCTTC CACCTGAGCA TCCCCAACTT CAACCAGTAC 451 GAGGCCATGA GCTGCGACTT CAACGGCGGA AAGATCAGCG TGCAGTACAA 501 TCTGAGCCAC AGCTATGCCG TGGACGCCGC CAATCATTGT GGAACAGTGG 551 CCAATGGCGT GCTCCAGACC TTCATGAGAA TGGCCTGGGG CGGCAGCTAT 601 ATCGCCCTGG ATTCTGGCAG AGGCAACTGG GACTGCATCA TGACCAGCTA 651 CCAGTACCTG ATCATCCAGA ACACCACCTG GGAAGATCAC TGCCAGTTCA 701 GCAGACCCTC TCCTATCGGA TACCTGGGCC TGCTGTCCCA GAGAACCCGG 751 GACATCTACA TCTCTAGACG GCTGCTGGGC ACCTTCACCT GGACACTGTC 801 TGATAGCGAG GGCAATGAGA CACCTGGCGG CTACTGTCTG ACCCGGTGGA 851 TGCTGATTGA GGCCGAGCTG AAGTGCTTCG GAAATACCGC CGTGGCCAAG 901 TGCAACGAGA AGCACGACGA GGAATTCTGC GACATGCTGC GGCTGTTCGA 951 TTTCAACAAG CAGGCCATCA GACGGCTGAA GGCCGAGGCT CAGATGTCCA 1001 TCCAGCTGAT CAACAAGGCC GTGAATGCCC TGATTAACGA CCAGCTCATC 1051 ATGAAGAACC ACCTCAGGGA CATCATGGGC ATCCCTTACT GCAACTACAG 1101 CAAGTACTGG TATCTGAACC ACACCATCAC CGGCAAGACC AGCCTGCCTA 1151 AGTGCTGGCT GGTGTCCAAC GGCAGCTACC TGAACGAGAC ACACTTCAGC 1201 GACGACATCG AGCAGCAGGC CGACAACATG ATCACCGAGA TGCTCCAGAA 1251 AGAGTACATG GACCGGCAGG GCAAGACACC TCTGGGCCTT GTGGATCTGT 1301 TCGTGTTCAG CACCAGCTTC TACCTGATCT CTATCTTCCT GCACCTGGTC 1351 AAGATCCCCA CACACAGACA CATCGTGGGC AAGCCCTGTC CTAAGCCTCA 1401 CAGACTGAAC CATATGGGCA TCTGTAGCTG CGGCCTGTAC AAACAGCCTG 1451 GCGTGCCAGT GCGGTGGAAG AGATAA Construct 2: SOSEP-variant of construct 1 (L-10-SOSEP) Amino acid sequence (SEQ ID NO:20): MGQIVTFFQEVPHVIEEVMNIVLIALSLLAILKGLYNVATCGLIGLVTFL 50 LLCGRSCSTTLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIRVGNE 100 TGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQY 150 EAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFMRMAWGGSY 200 IALDSGCGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTR 250 DIYISRRRRGTFTWTLSDSEGNETPGGYCLTRWMLIEAELKCFGNTAVAK 300 CNEKHDEEFCDMLRLFDFNKQAIRRLKAPAQMSIQLINKAVNALINDQLI 350 MKNHLRDIMCIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYLNETHFS 400 DDIEQQADNMITEMLQKEYMDRQGKTPLGLVDLFVFSTSFYLISIFLHLV 450 KIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWKR* DNA-sequence (SEQ ID NO:21): 1 ATGGGCCAGA TCGTGACATT CTTCCAAGAG GTGCCCCACG TGATCGAAGA 51 AGTGATGAAC ATCGTCCTGA TCGCCCTGAG CCTGCTGGCC ATCCTGAAGG 101 GCCTGTATAA TGTGGCCACC TGTGGCCTGA TCGGCCTGGT CACATTTCTG 151 CTGCTGTGCG GCAGAAGCTG CTCCACCACA CTGTATAAGG GCGTGTACGA 201 GCTGCAAACC CTGGAACTGA ACATGGAAAC CCTGAACATG ACCATGCCTC 251 TGAGCTGCAC CAAGAACAAC AGCCACCACT ACATCAGAGT GGGCAACGAG 301 ACAGGCCTCG AGCTGACCCT GACCAACACC AGCATCATCA ACCACAAGTT 351 CTGCAACCTG AGCGACGCCC ACAAGAAGAA CCTGTACGAT CACGCCCTGA 401 TGAGCATCAT CTCCACCTTC CACCTGAGCA TCCCCAACTT CAACCAGTAC 451 GAGGCCATGA GCTGCGACTT CAACGGCGGA AAGATCAGCG TGCAGTACAA 501 TCTGAGCCAC AGCTATGCCG TGGACGCCGC CAATCATTGT GGAACAGTGG 551 CCAATGGCGT GCTCCAGACC TTCATGAGAA TGGCCTGGGG CGGCAGCTAT 601 ATCGCCCTGG ATTCTGGCTG TGGCAACTGG GACTGCATCA TGACCAGCTA 651 CCAGTACCTG ATCATCCAGA ACACCACCTG GGAAGATCAC TGCCAGTTCA 701 GCAGACCCTC TCCTATCGGA TACCTGGGCC TGCTGTCCCA GAGAACCCGG 751 GACATCTACA TCTCTCGGCG GAGAAGAGGC ACCTTCACCT GGACACTGTC 801 TGATAGCGAG GGCAATGAGA CACCTGGCGG CTACTGTCTG ACCCGGTGGA 851 TGCTGATTGA GGCCGAGCTG AAGTGCTTCG GAAATACCGC CGTGGCCAAG 901 TGCAACGAGA AGCACGACGA GGAATTCTGC GACATGCTGC GGCTGTTCGA 951 TTTCAACAAG CAGGCCATCA GACGGCTGAA GGCCCCTGCT CAGATGTCCA 1001 TCCAGCTGAT CAACAAGGCC GTGAATGCCC TGATTAACGA CCAGCTCATC 1051 ATGAAGAACC ACCTCAGGGA CATCATGTGC ATCCCTTACT GCAACTACAG 1101 CAAGTACTGG TATCTGAACC ACACCATCAC CGGCAAGACC AGCCTGCCTA 1151 AGTGCTGGCT GGTGTCCAAC GGCAGCTACC TGAACGAGAC ACACTTCAGC 1201 GACGACATCG AGCAGCAGGC CGACAACATG ATCACCGAGA TGCTCCAGAA 1251 AGAGTACATG GACCGGCAGG GCAAGACACC TCTGGGCCTT GTGGATCTGT 1301 TCGTGTTCAG CACCAGCTTC TACCTGATCT CTATCTTCCT GCACCTGGTC 1351 AAGATCCCCA CACACAGACA CATCGTGGGC AAGCCCTGTC CTAAGCCTCA 1401 CAGACTGAAC CATATGGGCA TCTGTAGCTG CGGCCTGTAC AAACAGCCTG 1451 GCGTGCCAGT GCGGTGGAAG AGATAA Construct 3: SOSEP-variant of construct 1 with N-to-K-mutation (L-10-SQSEP-NtoK) Amino acid sequence (SEQ ID NO:22): MGQIVTFFQEVPHVIEEVMNIVLIALSLLAILKGLYNVATCGLIGLVTFL 50 LLCGRSCSTTLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIRVGNE 100 TGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQY 150 EAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFMRMAWGGSY 200 IALDSGCGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTR 250 DIYISRRRRGTFTWTLSDSEGKETPGGYCLTRWMLIEAELKCFGNTAVAK 300 CNEKHDEEFCDMLRLFDFNKQAIRRLKAPAQMSIQLINKAVNALINDQLI 350 MKNHLRDIMCIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYLNETHFS 400 DDIEQQADNMITEMLQKEYMDRQGKTPLGLVDLFVFSTSFYLISIFLHLV 450 KIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWKR* DNA-sequence (SEQ ID NO:23): 1 ATGGGCCAGA TCGTGACATT 51 AGTGATGAAC ATCGTCCTGA 101 GCCTGTATAA TGTGGCCACC 151 CTGCTGTGCG GCAGAAGCTG 201 GCTGCAAACC CTGGAACTGA 251 TGAGCTGCAC CAAGAACAAC 301 ACAGGCCTCG AGCTGACCCT 351 CTGCAACCTG AGCGACGCCC 401 TGAGCATCAT CTCCACCTTC 451 GAGGCCATGA GCTGCGACTT 501 TCTGAGCCAC AGCTATGCCG 551 CCAATGGCGT GCTCCAGACC 601 ATCGCCCTGG ATTCTGGCTG CTTCCAAGAG GTGCCCCACG TGATCGAAGA TCGCCCTGAG CCTGCTGGCC ATCCTGAAGG TGTGGCCTGA TCGGCCTGGT CACATTTCTG CTCCACCACA CTGTATAAGG GCGTGTACGA ACATGGAAAC CCTGAACATG ACCATGCCTC AGCCACCACT ACATCAGAGT GGGCAACGAG GACCAACACC AGCATCATCA ACCACAAGTT ACAAGAAGAA CCTGTACGAT CACGCCCTGA CACCTGAGCA TCCCCAACTT CAACCAGTAC CAACGGCGGA AAGATCAGCG TGCAGTACAA TGGACGCCGC CAATCATTGT GGAACAGTGG TTCATGAGAA TGGCCTGGGG CGGCAGCTAT TGGCAACTGG GACTGCATCA TGACCAGCTA 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 CCAGTACCTG ATCATCCAGA ACACCACCTG GGAAGATCAC TGCCAGTTCA GCAGACCCTC TCCTATCGGA TACCTGGGCC TGCTGTCCCA GAGAACCCGG GACATCTACA TCTCTCGGCG GAGAAGAGGC ACCTTCACCT GGACACTGTC TGATAGCGAG GGCAAAGAGA CACCTGGCGG CTACTGTCTG ACCCGGTGGA TGCTGATTGA GGCCGAGCTG AAGTGCTTCG GAAATACCGC CGTGGCCAAG TGCAACGAGA AGCACGACGA GGAATTCTGC GACATGCTGC GGCTGTTCGA TTTCAACAAG CAGGCCATCA GACGGCTGAA GGCCCCTGCT CAGATGTCCA TCCAGCTGAT CAACAAGGCC GTGAATGCCC TGATTAACGA CCAGCTCATC ATGAAGAACC ACCTCAGGGA CATCATGTGC ATCCCTTACT GCAACTACAG CAAGTACTGG TATCTGAACC ACACCATCAC CGGCAAGACC AGCCTGCCTA AGTGCTGGCT GGTGTCCAAC GGCAGCTACC TGAACGAGAC ACACTTCAGC GACGACATCG AGCAGCAGGC CGACAACATG ATCACCGAGA TGCTCCAGAA AGAGTACATG GACCGGCAGG GCAAGACACC TCTGGGCCTT GTGGATCTGT TCGTGTTCAG CACCAGCTTC TACCTGATCT CTATCTTCCT GCACCTGGTC AAGATCCCCA CACACAGACA CATCGTGGGC AAGCCCTGTC CTAAGCCTCA CAGACTGAAC CATATGGGCA TCTGTAGCTG CGGCCTGTAC AAACAGCCTG GCGTGCCAGT GCGGTGGAAG AGATAA Construct 4: FLEP-variant of construct 1 (L-10-FLEP) Amino acid sequence (SEQ ID NO:24): MGQIVTFFQEVPHVIEEVMNIVLIALSLLAILKGLYNVATCGLIGLVTFL 50 LLCGRSCSTTLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIRVGNE 100 TGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQY 150 EAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFMRMAWGGSY 200 IALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTR 250 DIYISGGGGSGGGGSGTFTWTLSDSEGNETPGGYCLTRWMLIEAELKCFG 300 NTAVAKCNEKHDEEFCDMLRLFDFNKQAIRRLKAPAQMSIQLINKAVNAL 350 INDQLIMKNHLRDIMGIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYL 400 NETHFSDDIEQQADNMITEMLQKEYMDRQGKTPLGLVDLFVFSTSFYLIS 450 IFLHLVKIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWKR* DNA-sequence (SEQ ID NO:25): 1 ATGGGCCAGA TCGTGACATT CTTCCAAGAG GTGCCCCACG TGATCGAAGA 51 AGTGATGAAC ATCGTCCTGA TCGCCCTGAG CCTGCTGGCC ATCCTGAAGG 101 GCCTGTATAA TGTGGCCACC TGTGGCCTGA TCGGCCTGGT CACATTTCTG 151 CTGCTGTGCG GCAGAAGCTG CTCCACCACA CTGTATAAGG GCGTGTACGA 201 GCTGCAAACC CTGGAACTGA ACATGGAAAC CCTGAACATG ACCATGCCTC 251 TGAGCTGCAC CAAGAACAAC AGCCACCACT ACATCAGAGT GGGCAACGAG 301 ACAGGCCTCG AGCTGACCCT GACCAACACC AGCATCATCA ACCACAAGTT 351 CTGCAACCTG AGCGACGCCC ACAAGAAGAA CCTGTACGAT CACGCCCTGA 401 TGAGCATCAT CTCCACCTTC CACCTGAGCA TCCCCAACTT CAACCAGTAC 451 GAGGCCATGA GCTGCGACTT CAACGGCGGA AAGATCAGCG TGCAGTACAA 501 TCTGAGCCAC AGCTATGCCG TGGACGCCGC CAATCATTGT GGAACAGTGG 551 CCAATGGCGT GCTCCAGACC TTCATGAGAA TGGCCTGGGG CGGCAGCTAT 601 ATCGCCCTGG ATTCTGGCAG AGGCAACTGG GACTGCATCA TGACCAGCTA 651 CCAGTACCTG ATCATCCAGA ACACCACCTG GGAAGATCAC TGCCAGTTCA 701 GCAGACCCTC TCCTATCGGA TACCTGGGCC TGCTGTCCCA GAGAACCCGG 751 GACATCTACA TCTCTGGCGG CGGAGGATCT GGCGGAGGTG GAAGTGGCAC 801 CTTCACCTGG ACACTGTCTG ATAGCGAGGG CAATGAGACA CCTGGCGGCT 851 ACTGTCTGAC CCGGTGGATG CTGATTGAGG CCGAGCTGAA GTGCTTCGGA 901 AATACCGCCG TGGCCAAGTG CAACGAGAAG CACGACGAGG AATTCTGCGA 951 CATGCTGCGG CTGTTCGATT TCAACAAGCA GGCCATCAGA CGGCTGAAGG 1001 1051 1101 1151 5    1201 1251 1301 1351 1401 10    1451 CCCCTGCTCA ATTAACGACC CCCTTACTGC GCAAGACCAG AACGAGACAC CACCGAGATG TGGGCCTTGT ATCTTCCTGC GCCCTGTCCT GCCTGTACAA GATGTCCATC AGCTCATCAT AACTACAGCA CCTGCCTAAG ACTTCAGCGA CTCCAGAAAG GGATCTGTTC ACCTGGTCAA AAGCCTCACA ACAGCCTGGC CAGCTGATCA GAAGAACCAC AGTACTGGTA TGCTGGCTGG CGACATCGAG AGTACATGGA GTGTTCAGCA GATCCCCACA GACTGAACCA GTGCCAGTGC ACAAGGCCGT CTCAGGGACA TCTGAACCAC TGTCCAACGG CAGCAGGCCG CCGGCAGGGC CCAGCTTCTA CACAGACACA TATGGGCATC GGTGGAAGAG GAATGCCCTG TCATGGGCAT ACCATCACCG CAGCTACCTG ACAACATGAT AAGACACCTC CCTGATCTCT TCGTGGGCAA TGTAGCTGCG ATAA Construct 5: FLEP-variant of construct 1 with N-to-K-mutation (L-10-FLEP-NtoK) Amino acid sequence (SEQ ID NO:26): MGQIVTFFQEVPHVIEEVMNIVLIALSLLAILKGLYNVATCGLIGLVTFL 50 LLCGRSCSTTLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIRVGNE 100 TGLELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQY 150 EAMSCDFNGGKISVQYNLSHSYAVDAANHCGTVANGVLQTFMRMAWGGSY 200 IALDSGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTR 250 DIYISGGGGSGGGGSGTFTWTLSDSEGKETPGGYCLTRWMLIEAELKCFG 300 NTAVAKCNEKHDEEFCDMLRLFDFNKQAIRRLKAPAQMSIQLINKAVNAL 350 INDQLIMKNHLRDIMGIPYCNYSKYWYLNHTITGKTSLPKCWLVSNGSYL 400 NETHFSDDIEQQADNMITEMLQKEYMDRQGKTPLGLVDLFVFSTSFYLIS 450 IFLHLVKIPTHRHIVGKPCPKPHRLNHMGICSCGLYKQPGVPVRWKR* DNA-sequence (SEQ ID NO:27): 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 ATGGGCCAGA AGTGATGAAC GCCTGTATAA CTGCTGTGCG GCTGCAAACC TGAGCTGCAC ACAGGCCTCG CTGCAACCTG TGAGCATCAT GAGGCCATGA TCTGAGCCAC CCAATGGCGT ATCGCCCTGG CCAGTACCTG GCAGACCCTC GACATCTACA CTTCACCTGG ACTGTCTGAC AATACCGCCG CATGCTGCGG CCCCTGCTCA ATTAACGACC CCCTTACTGC GCAAGACCAG AACGAGACAC CACCGAGATG TGGGCCTTGT ATCTTCCTGC GCCCTGTCCT GCCTGTACAA TCGTGACATT ATCGTCCTGA TGTGGCCACC GCAGAAGCTG CTGGAACTGA CAAGAACAAC AGCTGACCCT AGCGACGCCC CTCCACCTTC GCTGCGACTT AGCTATGCCG GCTCCAGACC ATTCTGGCAG ATCATCCAGA TCCTATCGGA TCTCTGGCGG ACACTGTCTG CCGGTGGATG TGGCCAAGTG CTGTTCGATT GATGTCCATC AGCTCATCAT AACTACAGCA CCTGCCTAAG ACTTCAGCGA CTCCAGAAAG GGATCTGTTC ACCTGGTCAA AAGCCTCACA ACAGCCTGGC CTTCCAAGAG TCGCCCTGAG TGTGGCCTGA CTCCACCACA ACATGGAAAC AGCCACCACT GACCAACACC ACAAGAAGAA CACCTGAGCA CAACGGCGGA TGGACGCCGC TTCATGAGAA AGGCAACTGG ACACCACCTG TACCTGGGCC CGGAGGATCT ATAGCGAGGG CTGATTGAGG CAACGAGAAG TCAACAAGCA CAGCTGATCA GAAGAACCAC AGTACTGGTA TGCTGGCTGG CGACATCGAG AGTACATGGA GTGTTCAGCA GATCCCCACA GACTGAACCA GTGCCAGTGC GTGCCCCACG CCTGCTGGCC TCGGCCTGGT CTGTATAAGG CCTGAACATG ACATCAGAGT AGCATCATCA CCTGTACGAT TCCCCAACTT AAGATCAGCG CAATCATTGT TGGCCTGGGG GACTGCATCA GGAAGATCAC TGCTGTCCCA GGCGGAGGTG CAAAGAGACA CCGAGCTGAA CACGACGAGG GGCCATCAGA ACAAGGCCGT CTCAGGGACA TCTGAACCAC TGTCCAACGG CAGCAGGCCG CCGGCAGGGC CCAGCTTCTA CACAGACACA TATGGGCATC GGTGGAAGAG TGATCGAAGA ATCCTGAAGG CACATTTCTG GCGTGTACGA ACCATGCCTC GGGCAACGAG ACCACAAGTT CACGCCCTGA CAACCAGTAC TGCAGTACAA GGAACAGTGG CGGCAGCTAT TGACCAGCTA TGCCAGTTCA GAGAACCCGG GAAGTGGCAC CCTGGCGGCT GTGCTTCGGA AATTCTGCGA CGGCTGAAGG GAATGCCCTG TCATGGGCAT ACCATCACCG CAGCTACCTG ACAACATGAT AAGACACCTC CCTGATCTCT TCGTGGGCAA TGTAGCTGCG ATAA Example 12 Lassa virus nucleoprotein This example describes Lassa virus nucleoprotein ancestral sequence produced using a method according to an embodiment of the invention. Construct 6: Lassa virus nucleoprotein ancestral sequence of Nigerian Lassa isolates (L-NP-1 = L-NP-CovAnc-1 N) Amino acid sequence (SEQ ID NO:28): MSASKEVKSFLWTQSLRRELSGYCSNIKLQWKDAQALLHGLDFSEVSNV 50 QRLMRKQKRDDSDLKRLRDLNQAVNNLVELKSTQQKSILRVGTLTSDDLL 100 TLAADLEKLKSKVIRTERPLSSGVYMGNLSTQQLEQRRALLNMIGMVGGA 150 QGTQPGRDGWRVWDVKNPDLLNNQFGTMPSLTLACLTKQGQVDLNDAVL 200 ALTDLGLIYTAKYPNSSDLDRLSQSHPILNMVDTKKSSLNISGYNFSLGA 250 AVKAGACMLDGGNMLETIKVTPQTMDGILKSILKVKKSLGMFVSDTPGER 300 NPYENILYKICLSGDGWPYIASRTSIVGRAWENTTVDLESDGKPQKVGTA 350 GSNKSLQSAGFPTGLTYSQLMTLKDSMMQLDPSAKTWIDIEGRPEDPVEI 400 ALYQPMSGCYIHFFREPTDLKQFKQDAKYSHGIDVADLFPAQPGLTSAVI 450 EALPRNMVLTCQGSDDIKRLLDSQGRRDIKLIDIALSKADSRRFENAVWD 500 QCKDLCHMHTGVWEKKKRGGKEEITPHCALMDCIMYDAAVSGGLNIPVL 550 RAVLPRDMVFRTSSPKWL* DNA-sequence (SEQ ID NO:29): 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 ATGAGCGCCA GAGAGAGCTG ACGCCCAGGC CAGCGGCTGA GAGGGATCTG AGCAGAAATC ACACTGGCCG GAGGCCACTG TGGAACAGAG CAGGGAACAC GAACCCCGAC TGGCCTGCCT GCTCTGACTG CGACCTGGAC CCAAGAAGTC GCCGTGAAAG CATCAAAGTG AAGTGAAGAA AACCCCTACG GCCCTATATC CCACCGTGGA GGCAGCAACA CAGCCAGCTG CCAAGACCTG GCTCTGTACC CACCGATCTG ACGTGGCCGA GCAAAGAAGT TCTGGCTACT TCTGCTGCAT TGAGAAAGCA AACCAGGCCG CATCCTGAGA CCGATCTGGA TCTAGCGGAG AAGGGCCCTG AGCCTGGAAG CTGCTCAACA GACAAAGCAG ATCTGGGCCT AGGCTGAGCC CAGCCTGAAC CCGGCGCTTG ACCCCTCAGA ATCCCTGGGC AGAACATCCT GCCAGCAGAA CCTGGAATCC AGAGCCTCCA ATGACCCTGA GATCGACATT AGCCTATGAG AAGCAGTTCA TCTGTTCCCA GAAAAGCTTC GCTCCAACAT GGCCTGGATT GAAGCGGGAC TGAACAACCT GTGGGCACCC AAAGCTGAAG TGTACATGGG CTGAACATGA AGATGGTGTC ACCAGTTCGG GGCCAAGTGG GATCTACACC AGTCTCACCC ATCAGCGGCT TATGCTTGAC CCATGGACGG ATGTTCGTGT GTACAAGATT CATCTATCGT GATGGCAAGC GTCTGCCGGA AGGACAGCAT GAGGGCAGAC CGGCTGCTAT AGCAGGACGC GCTCAGCCAG CTCTGGACCC CAAGCTCCAG TCAGCGAGGT GACAGCGACC GGTGGAACTG TGACCAGCGA TCCAAAGTGA CAACCTGAGC TCGGCATGGT GTCAGAGTGT CACCATGCCT ACCTGAACGA GCCAAGTATC CATCCTGAAC ACAACTTCTC GGCGGCAACA CATCCTGAAA CCGACACACC TGCCTGAGCG GGGCAGAGCT CTCAGAAAGT TTTCCTACCG GATGCAGCTG CCGAGGATCC ATCCACTTCT CAAGTACAGC GACTGACATC AGAGCCTGCG GTGGTCAAGG GTCCAACGTG TGAAGAGACT AAGTCTACCC CGATCTGCTG TCCGGACCGA ACCCAGCAGC TGGAGGCGCC GGGACGTGAA TCTCTGACCC TGCCGTGCTG CCAACAGCTC ATGGTGGACA TCTGGGCGCT TGCTGGAAAC AGTATCCTGA CGGCGAGAGA GCGACGGCTG TGGGAGAACA GGGCACAGCC GCCTGACATA GACCCTAGCG CGTGGAAATC TCAGAGAGCC CACGGAATCG CGCCGTGATT 1351 GAAGCCCTGC CTAGAAACAT GGTGCTGACC TGTCAGGGCA GCGACGACAT 1401 CAAGAGACTG CTGGACAGCC AGGGCAGAAG AGATATCAAG CTGATCGATA 1451 TCGCCCTGAG CAAGGCCGAC TCTCGGAGAT TCGAAAACGC CGTGTGGGAC 1501 CAGTGCAAGG ACCTGTGTCA CATGCACACA GGCGTGGTGG TGGAAAAGAA 5   1551 GAAGCGCGGA GGCAAAGAGG AAATCACCCC TCACTGCGCC CTGATGGACT 1601 GCATTATGTA TGACGCCGCC GTGTCTGGCG GCCTGAATAT CCCTGTTCTG 1651 AGAGCCGTGC TGCCCCGCGA CATGGTGTTT AGAACAAGCA GCCCCAAGGT 1701 GGTGCTCTGA 10 Example 13 Lassa virus nucleoprotein This example describes Lassa virus nucleoprotein ancestral sequence produced using a method according to an embodiment of the invention. Construct 7: 15 Lassa virus nucleoprotein ancestral sequence of Sierra Leone isolates (L-NP-1 = L-NP-CovAnc-2 SL) Amino acid sequence (SEQ ID NO:30): 20 MSASKEIKSFLWTQSLRRELSGYCSNIKLQWKDAQALLHGLDFSEVSNV 50 QRLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTLTSDDLL 100 ILAADLEKLKSKVTRTERPLSAGVYMGNLSSQQLDQRRALLNMIGMSGGN 150 QGARAGRDGWRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQ 200 ALTDLGLIYTAKYPNTSDLDRLTQSHPILNMIDTKKSSLNISGYNFSLGA 250 25 AVKAGACMLDGGNMLETIKVSPQTMDGILKSILKVKKALGMFISDTPGER 300 NPYENILYKICLSGDGWPYIASRTSITGRAWENTWDLESDGKPQKAGSN 350 NSNKSLQSAGFTAGLTYSQLMTLKDAMLQLDPNAKTWMDIEGRPEDPVEI 400 ALYQPSSGCYIHFFREPTDLKQFKQDAKYSHGIDVTDLFAAQPGLTSAVI 450 DALPRNMVTTCQGSDDIRKLLESQGRKDIKLIDIALSKTDSRKYENAVWD 500 30 QYKDLCHMHTGVWEKKKRGGKEEITPHCALMDCIMFDAAVSGGLNTSVL 550 RAVLPRDMVFRTSTPRWL* DNA-sequence (SEQ ID NO:31): 35        1 51 101 151 201 40     251 301 351 401 451 45     501 551 601 651 701 50     751 801 ATGAGCGCCA GAGAGAGCTG ACGCCCAGGC CAGCGGCTGA GAGGGATCTG AGCAGAAATC ATTCTGGCCG GAGGCCACTG TGGATCAGAG CAGGGCGCTA GAATGCCGAG TGGCCTGCCT GCCCTGACTG CGACCTGGAC CCAAGAAGTC GCCGTGAAAG CATCAAGGTG AAGTGAAGAA GCAAAGAGAT TCTGGCTACT TCTGCTGCAT TGCGGAAAGA AACCAGGCCG CATCCTGAGA CCGACCTGGA TCTGCTGGTG AAGGGCCCTG GAGCTGGCAG CTGCTCAACA GACAAAGCAG ATCTGGGCCT AGACTGACCC CAGCCTGAAC CCGGCGCTTG TCCCCACAGA AGCCCTGGGC CAAGAGCTTC GCTCCAACAT GGCCTGGATT GAGAAGGGAC TGAACAACCT GTGGGCACCC AAAGCTGAAG TCTACATGGG CTGAACATGA AGATGGCGTC ACCAGTTCGG GGCCAAGTGG GATCTACACC AGTCTCACCC ATCAGCGGCT TATGCTTGAC CCATGGACGG ATGTTCATCA CTGTGGACCC CAAGCTCCAG TCAGCGAGGT GACAACGACC GGTGGAACTG TGACCAGCGA TCCAAAGTGA CAACCTGAGC TCGGCATGAG GTCAGAGTGT CACCATGCCT ACCTGAACGA GCCAAGTATC CATCCTGAAT ACAACTTCTC GGCGGCAACA CATCCTGAAA GCGACACCCC AGAGCCTGCG GTGGTCAAGG GTCCAACGTG TGAAGCGGCT AAGTCTACCC CGATCTGCTG CCCGGACCGA AGCCAGCAGC CGGCGGAAAT GGGACGTGAA AGCCTGACAC TGCTGTGCAG CCAACACCAG ATGATCGACA TCTGGGCGCT TGCTGGAAAC AGTATCCTGA TGGCGAGAGA 901 AACCCCTACG AGAACATCCT GTACAAGATT TGCCTGAGCG GCGACGGCTG 951 GCCCTATATC GCCAGCAGAA CCAGCATTAC CGGCAGAGCT TGGGAGAACA 1001 CCGTGGTGGA TCTGGAAAGC GACGGCAAGC CTCAGAAGGC CGGCAGCAAC 1051 AACTCCAACA AGAGCCTCCA GTCCGCCGGC TTCACAGCCG GCCTGACATA 1101 TAGCCAGCTG ATGACCCTGA AGGACGCCAT GCTGCAACTG GACCCCAATG 1151 CCAAGACCTG GATGGACATC GAGGGCAGAC CTGAGGACCC TGTGGAAATC 1201 GCCCTGTACC AGCCTAGCTC CGGCTGCTAT ATCCACTTCT TCAGAGAGCC 1251 CACCGATCTG AAGCAGTTCA AGCAGGACGC CAAGTACAGC CACGGCATCG 1301 ACGTGACCGA TCTGTTTGCT GCTCAGCCCG GACTGACCTC CGCCGTGATT 1351 GATGCCCTGC CTCGGAACAT GGTCATCACC TGTCAGGGCA GCGACGACAT 1401 CCGGAAGCTG CTGGAATCTC AGGGCAGAAA GGATATCAAG CTGATCGATA 1451 TCGCCCTGAG CAAGACCGAC AGCCGGAAGT ACGAAAACGC CGTGTGGGAC 1501 CAGTACAAGG ACCTGTGCCA CATGCACACA GGCGTGGTGG TGGAAAAGAA 1551 GAAGCGCGGA GGCAAAGAGG AAATCACCCC TCACTGCGCT CTGATGGACT 1601 GCATCATGTT TGACGCCGCC GTGTCTGGCG GCCTGAATAC CTCTGTTCTG 1651 AGAGCCGTGC TGCCCAGAGA CATGGTGTTC AGAACAAGCA CCCCTAGAGT 1701 GGTGCTCTGA Example 14 Immunogenicity and efficacy of a trivalent hemorrhagic fever vaccine candidate Emerging or re-emerging hemorrhagic fever viruses (HFVs)such as Ebola (EBOV), Marburg (MARV) and Lassa fever virus (LASV) represent one of the most serious threats to public health with significant epidemic potential and negative economic impact in affected African countries with fragile public health infrastructure. Despite of approved vaccines against EBOV and MARV disease, a cost-effective vaccine delivering a set of antigens from a single vector to protect against all 3 HFVs is not available yet. Apart from Ebola and Marburg virus Lassa fever virus is also considered as a dangerous pathogen and listed as a blueprint priority disease by the WHO as there is no vaccine available. The changing distribution of outbreaks of MARV in previously unaffected regions of sub-Saharan Africa, with the unexpected outbreak of Sudan ebolavirus in Uganda (SUDV), compounded the burden of almost half a million of Lassa fever cases annually in West Africa. The fluctuation in distribution and overlapping clinical manifestation of these HFVs highlights the urgent need to develop a single trivalent vaccine capable of protecting against all three of these HFVs. In this study, we assessed the immunogenicity and efficacy of this trivalent vaccine (Tri-LEMvac) based on the highly attenuated modified vaccinia virus Ankara (MVA) expressing three antigen sequences from a single vector encoding for the glycoproteins of SUDV (referred to as T2-4; SEQ ID NO:3) and MARV (Referred to as T2-11; SEQ ID NO:9), as well as the nucleoprotein of LASV (referred to as L-NP-CovAnc-1; SEQ ID NO:28). In addition, a bivalent “Lassa only” MVA coding for the LASV glycoprotein (referred to as L-10; SEQ ID NOU8) and nucleoprotein (referred to as L-NP-CovAnc-1; SEQ ID NO:28) was used for comparative purposes for challenge against LASV. Figure 6 shows a schematic of the trivalent and bivalent MVA vaccines, as well as trivalent and bivalent DNA vaccines used as a prime. Figures 9 and 10 show that the bivalent Lassa and Tri-LEMvac MVA vaccine antigens were expressed correctly and stably. Hartley guinea pigs were immunized with either the bi- / trivalent DNA vaccine or empty vector followed by two booster immunizations using the bi- / trivalent MVA or MVA WT, respectively (Figure 14). Animals were subsequently challenged with lethal doses of guinea-pig adapted SUDV, MARV and LASV, respectively. All guinea pigs were monitored for survival, weight, and fluctuations in body temperature, as well as viral load in the blood, lung, heart, spleen, liver and kidneys (Figures 15 to 17). As shown in Figures 15 to 17, 100% protection against SUDV, MARV, and LASV was achieved in animals vaccinated with the trivalent vaccine. Serological antibody responses were analyzed by Luminex and virus pseudotype neutralization assays. Analysis of sera revealed that the designed Tri-LEMvac induced a potent and specific immune response against SUDV GP and MARV GP and LASV NP (Figures 18 to 20). All Tri-LEMvac vaccinated animals showed potent binding antibodies and broad neutralization responses to MARV as well as a panel of EBOV viruses. For the Lassa nucleoprotein, all guinea pigs showed high levels of binding antibodies. Figure 18 shows that Tri-LEMvac is effectively boosting DNA prime, and induces crossbinding antibodies against filoviruses. Figure 19 shows that Tri-LEMvac induces binding antibodies specific for a panel of filoviruses. In particular the figure shows that that all animals that received Tri-LEMvac developed binding antibodies against SUDV , and 70% of the animals also had neutralising antibodies against SUDV. Figure 20 shows that Tri-LEMvac induces high levels of antibodies having binding specific for LASV-NP, and high levels of antibodies having binding specific for Monkeypox H3 after 4 weeks post 1st immunisation. Figure 21 shows that the bivalent Lassa vaccine induces binding antibodies specific for LASV nucleoprotein and glycoprotein antigens. Based on these results our trivalent MVA (Tri-LEMvac) could be used as a prophylactic or booster vaccine candidate against the hemorrhagic fever viruses Sudan Ebolavirus, Marburg and Lassa fever virus. Based on preclinical findings, scaleup for clinical development and human trials has been 5 initiated. Example 15 Further immunogenicity data for Tri-LEMvac in guinea pigs The guinea pigs followed the immunisation and bleed schedule as in Figure 12(a). Figure 12(b) shows that designed Tri-LEMvac induced a potent and specific immune 10 response against SUDV GP and MARV GP and LASV NP. Figure 13 shows that Tri-LEMvac induces neutralizing antibody titres specific for a panel of ebolavruses, including EBOV, SUDAN Gulu, SUDV 2022 (Uganda 2022 strain), as well as MARV and closely related RAW (Figure 13).

Claims

1. An isolated polynucleotide, comprising a nucleic acid sequence that is:i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:1, or identical with SEQ ID NO:1;ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:2, or identical with SEQ ID NO:2;ill) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:4, or identical with SEQ ID NO:4;iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:5, or identical with SEQ ID NO:5;v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:7, or identical with SEQ ID NO:7; orvi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:8, or identical with SEQ ID NO:8;or the complement thereof.

2. An isolated polynucleotide, comprising a nucleic acid sequence that is:i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:10, or identical with SEQ ID NQ:10;ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:12, or identical with SEQ ID NO:12; orill) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:14, or identical with SEQ ID NO:14;or the complement thereof.

3. An isolated polynucleotide, comprising a nucleic acid sequence that is:i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:19, or identical with SEQ ID NO:19;ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:21, or identical with SEQ ID NO:21;ill) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:23, or identical with SEQ ID NO:23;iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:25, or identical with SEQ ID NO:25;v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:27, or identical with SEQ ID NO:27;vi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:29, or identical with SEQ ID NO:29; orvii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:31, or identical with SEQ ID NO:31;or the complement thereof.

4. An isolated polypeptide, comprising an amino acid sequence that is:i) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:1, or identical with the amino acid sequence encoded by SEQ ID NO:1;ii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:2, or identical with the amino acid sequence encoded by SEQ ID NO:2;ill) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:4, or identical with the amino acid sequence encoded by SEQ ID NO:4;iv) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:5, or identical with the amino acid sequence encoded by SEQ ID NO:5;v) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:7, or identical with the amino acid sequence encoded by SEQ ID NO:7;vi) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:8, or identical with the amino acid sequence encoded by SEQ ID NO:8;vii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NQ:10, or identical with the amino acid sequence encoded by SEQ ID NQ:10;viii) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:12, or identical with the amino acid sequence encoded by SEQ ID NO:12;ix) at least 95%, 96%, 97%, 98%, or 99% identical with an amino acid sequence encoded by SEQ ID NO:14, or identical with the amino acid sequence encoded by SEQ ID NO:14.

5. An isolated polypeptide, comprising an amino acid sequence that is:i) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:3, or identical with SEQ ID NO:3;ii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:6, or identical with SEQ ID NO:6; orill) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:9, or identical with SEQ ID NO:9;iv) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:11, or identical with SEQ ID NO:11;v) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO: 13, or identical with SEQ ID NO:13; orvi) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:15, or identical with SEQ ID NO:15.

6. An isolated polypeptide, comprising an amino acid sequence that is:i) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:18, or identical with SEQ ID NO:18;ii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:20, or identical with SEQ ID NQ:20;ill) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:22, or identical with SEQ ID NO:22;iv) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:24, or identical with SEQ ID NO:24;v) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:26, or identical with SEQ ID NO:26;vi) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NO:28, or identical with SEQ ID NO:28; orvii) at least 95%, 96%, 97%, 98%, or 99% identical with SEQ ID NQ:30, or identical with SEQ ID NQ:30.

7. An isolated polynucleotide encoding an amino acid sequence encoded by a nucleicacid of claim 1, 2, or 3, wherein the polynucleotide is codon-optimized, optionally gene-optimized, for expression in mammalian cells.

8. An isolated polynucleotide encoding a polypeptide of claim 4, 5, or 6, wherein the polynucleotide is codon-optimized, optionally gene-optimized, for expression in mammalian cells.

9. A vector comprising a polynucleotide of claim 1,2,3, 7, or 8.

10. A vector according to claim 9, which further comprises a promoter operably linked to the nucleotide sequence.

11. A vector according to claim 10, wherein the promoter is for expression of a polypeptide encoded by the nucleic acid in mammalian cells.

12. A vector according to claim 10, wherein the promoter is for expression of a polypeptide encoded by the nucleic acid in yeast or insect cells.

13. A vector according to any of claims 9 to 12, which is a vaccine vector.

14. A vector according to claim 13, which is a viral vaccine vector, a bacterial vaccinevector, an RNA vaccine vector, or a DNA vaccine vector.

15. An isolated cell comprising a vector of any of claims 9 to 12.

16. A pseudotyped virus particle comprising the polypeptide of claim 4, 5, or 6.

17. A method of producing a pseudotyped virus particle of claim 16, which includes transfecting a host cell with a vector according to any of claims 9 to 11.

18. A fusion protein comprising a polypeptide according to claim 4, 5, or 6.

19. A pharmaceutical composition comprising a polynucleotide according to claim 1, 2, 3, 7, or 8, and a pharmaceutically acceptable carrier, excipient, or diluent.

20. A pharmaceutical composition comprising a vector according to any of claims 9 to 11, 13, or 14, and a pharmaceutically acceptable carrier, excipient, or diluent.

21. A pharmaceutical composition comprising a polypeptide according to claim 4, 5, or 6, and a pharmaceutically acceptable carrier, excipient, or diluent.

22. A pharmaceutical composition according to any of claims 19 to 21, which further comprises an adjuvant for enhancing an immune response in a subject to the polypeptide, or to a polypeptide encoded by the nucleic acid, of the composition.

23. A method of inducing an immune response to a virus of the Filoviridae family in a subject, which comprises administering to the subject a polynucleotide according to any of claims 1, 2, 7, or 8, a polypeptide according to claim 4 or 5, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22.

24. A method of immunizing a subject against a virus of the Filoviridae family, which comprises administering to the subject a polynucleotide according to any of claims 1,2,7, or 8, a polypeptide according to claim 4 or 5, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22.

25. A method of inducing an immune response to a virus of the Arenaviridae family in a subject, which comprises administering to the subject a polynucleotide according to any ofclaims 3, 7, or 8, a polypeptide according to claim 6, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22.

26. A method of immunizing a subject against a virus of the Arenaviridae family, which comprises administering to the subject a polynucleotide according to any of claims 3, 7, or 8, a polypeptide according to claim 6, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22.

27. A method according to any of claims 23 to 26, wherein the composition is administered intramuscularly.

28. A nucleic acid expression vector, which comprises a multiple cloning site, comprising Kpnl and Notl endonuclease sites.

29. A vector according to claim 28, wherein the multiple cloning site comprises a nucleic acid sequence of SEQ ID NO:16.

30. A vector according to claim 28 or 29, which is an expression vector, and a viral pseudotype vector.

31. A vector according to any of claims 28 to 30, which is a vaccine vector.

32. A vector according to any of claims 28 to 31, which comprises, from a 5’ to 3' direction: a promoter; a splice donor site; a splice acceptor site; and a terminator signal, wherein the multiple cloning site is located between the splice acceptor site and the terminator signal.

33. A vector according to claim 32, wherein the promoter comprises a CMV immediate early 1 enhancer / promoter and / or the terminator signal comprises a terminator signal of a bovine growth hormone gene that lacks a Kpnl restriction endonuclease site.

34. A vector according to any of claims 28 to 33, which further comprises an origin of replication, and nucleic acid encoding resistance to an antibiotic.

35. A vector according to claim 34, wherein the origin of replication comprises a pUC-plasmid origin of replication and / or the nucleic acid encodes resistance to kanamycin.

36. A vector according to any of claims 28 to 35, which comprises a nucleic acid sequence of SEQ ID NO:17.

37. An isolated polynucleotide which comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

38. An isolated polynucleotide which comprises a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

39. A composition comprising a first polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

40. A composition comprising a first polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

41. A combined preparation comprising: (i) a first polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 6; and (ii) a second polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

42. A combined preparation comprising: (i) a first polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 13; and (ii) a second polynucleotide which includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

43. A composition comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

44. A composition comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

45. A fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

46. A fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

47. A combined preparation comprising: (i) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 6; and (ii) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9.

48. A combined preparation comprising: (i) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 13; and (ii) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 15.

49. A polynucleotide according to any of claims 1,2,7, or 8, a polypeptide according to claim 4 or 5, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, for use as a medicament.

50. A polynucleotide according to any of claims 1,2,7, or 8, a polypeptide according to claim 4 or 5, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, for use in the treatment of a viral infection, preferably a viral infection caused by an emerging or re-emerging virus, preferably a virus of the Filoviridae family.

51. Use of a polynucleotide according to any of claims 1, 2, 7, or 8, a polypeptide according to claim 4 or 5, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, in the manufacture of a medicament for the treatment of a viral infection, preferably a viral infection caused by an emerging or re-emerging virus, preferably a virus of the Filoviridae family.

52. A polynucleotide according to any of claims 3, 7, or 8, a polypeptide according to claim 6, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, for use as a medicament.

53. A polynucleotide according to any of claims 3, 7, or 8, a polypeptide according to claim 6, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, for use in the treatment of a viral infection, preferably a viral infection caused by an emerging or re-emerging virus, preferably a virus of the Arenaviridae family.

54. Use of a polynucleotide according to any of claims 3, 7, or 8, a polypeptide according to claim 6, a vector according to any of claims 9 to 11, 13, or 14, or a pharmaceutical composition according to any of claims 19 to 22, in the manufacture of a medicament for the treatment of a viral infection, preferably a viral infection caused by an emerging or reemerging virus, preferably a virus of the Arenaviridae family.

55. A polynucleotide according to claim 37 or 38, a composition according to claim 39, 40, 43, or 44, a combined preparation according to claim 41,42, 47, or 48, or a fusion protein according to claim 45 or 46, for use as a medicament.

56. A polynucleotide according to claim 37 or 38, a composition according to claim 39, 40, 43, or 44, a combined preparation according to claim 41,42, 47, or 48, or a fusion protein according to claim 45 or 46, for use in the treatment of a viral infection, preferably a viral infection caused by an emerging or re-emerging virus, preferably a virus of the Filoviridae family.

57. Use of a polynucleotide according to claim 37 or 38, a composition according to claim 39, 40, 43, or 44, a combined preparation according to claim 41, 42, 47, or 48, or a fusion protein according to claim 45 or 46, in the manufacture of a medicament for the treatment of a viral infection, preferably a viral infection caused by an emerging or re-emerging virus, preferably a virus of the Filoviridae family.

58. An isolated polynucleotide which comprises:(a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof;(b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and(c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof.

59. An isolated polynucleotide which comprises:(a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and(b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof.

60. A pharmaceutical composition comprising:(a) a first isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof;(b) a second isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and(c) a third an isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof; anda pharmaceutically acceptable carrier, excipient, or diluent.

61. A pharmaceutical composition comprising:(a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and(b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof; anda pharmaceutically acceptable carrier, excipient, or diluent.

62. A combined preparation, which comprises:(a) a first isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof;(b) a second isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and(c) a third an isolated polynucletide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof.

63. A combined preparation, which comprises:(a) a first isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and(b) a second isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof.

64. A vector which comprises a polynucleotide comprising:(a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, or the complement thereof;(b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, or the complement thereof; and(c) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:28, or the complement thereof.

65. A vector which comprises a polynucleotide comprising:(a) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, or the complement thereof; and(b) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, or the complement thereof.

66. A vector according to claim 64 or 65, which further comprises a promoter operably linked to the polynucleotide.

67. A vector according to claim 64 or 65, which further comprises, for each nucleotide sequence of the vector encoding a separate polypeptide, a separate promoter operably linked to that nucleotide sequence.

68. A pharmaceutical composition which comprises an isolated polynucleotide according to claim 58 or 59, or a vector according to any of claims 64 to 67, and a pharmaceutically acceptable carrier, excipient, or diluent.

69. An isolated polynucleotide according to claim 58, a pharmaceutical composition according to claim 60 or 68, a combined preparation according to claim 62, or a vector according to any of claims 64 to 67, wherein the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 3, comprises the nucleotide sequence of SEQ ID NO:2, or the complement thereof.

70. An isolated polynucleotide according to claim 58 or 69, a pharmaceutical composition according to claim 60 or 68, or 69, a combined preparation according to claim 62 or 69, or a vector according to any of claims 64 to 67, or 69, wherein the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 9, comprises the nucleotide sequence of SEQ ID NO:8, or the complement thereof.

71. An isolated polynucleotide according to claim 58, 59, 69, or 70, a pharmaceutical composition according to claim 60, 61,69, or 70, a combined preparation according to claim 62, 69 or 70, or a vector according to any of claims 64 to 67, 69, or 70, wherein the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 28, comprises the nucleotide sequence of SEQ ID NO:29, or the complement thereof.

72. An isolated polynucleotide according to claim 59 or 71, a pharmaceutical composition according to claim 61, 70, or 71, a combined preparation according to claim 63 or 71, or a vector according to any of claims 65 to 67, or 71, wherein the nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 18, comprises the nucleotide sequence of SEQ ID NO:19, or the complement thereof.

73. A vector according to any of claims 64 to 67, or 69 to 72, or a pharmaceutical composition according to claim 68, wherein the, or each promoter is for expression of a polypeptide encoded by the nucleotide sequence in mammalian cells.

74. A vector according to any of claims 64 to 67, or 69 to 72, or a pharmaceutical composition according to claim 68, wherein the, or each promoter is for expression of a polypeptide encoded by the nucleic acid in yeast or insect cells.

75. A vector according to any of claims 64 to 67, or 69 to 73, or a pharmaceutical composition comprising a vector according to any of claims 68 to 73, which is a vaccine vector.

76. A vector, or a pharmaceutical composition according to claim 75, wherein the vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector.

77. An isolated cell comprising a vector of any of claims to 64 to 67, or 69 to 76.

78. An isolated polypeptide which comprises an amino acid sequence of SEQ ID NO: 3; an amino acid sequence of SEQ ID NO: 9; and an amino acid sequence of SEQ ID NO:28.

79. An isolated polypeptide which comprises an amino acid sequence of SEQ ID NO: 18; and an amino acid sequence of SEQ ID NO: 28.

80. A pharmaceutical composition comprising:(a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 3;(b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 9; and(c) a third an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28; anda pharmaceutically acceptable carrier, excipient, or diluent.

81. A pharmaceutical composition comprising:(a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 18; and(b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28; anda pharmaceutically acceptable carrier, excipient, or diluent.

82. A combined preparation comprising:(a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 3;(b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 9; and(c) a third isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28.

83. A combined preparation comprising:(a) a first isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 18; and(b) a second isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 28.

84. A fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 3, a second polypeptide comprising an amino acid sequence of SEQ ID NO: 9, and a third polypeptide comprising an amino acid sequence of SEQ ID NO: 28.

85. A fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 18, and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 28.

86. A pharmaceutical composition, which comprises an isolated polypeptide according to claim 78 or 79, and a pharmaceutically acceptable carrier, excipient, or diluent.

87. A pharmaceutical composition according to any of claims 60, 61,68 to 76, 80, 81, or 86, which further comprises an adjuvant for enhancing an immune response in a subject to a polypeptide, or to a polypeptide encoded by a nucleotide, of the composition.

88. A pseudotyped virus particle comprising a polypeptide according to claim 78 or 79.

89. A polynucleotide according to any of claims 1, 2, or 69 to 72, a vector according to any of claims 64 to 67, or 69 to 76, a polypeptide according to claim 78, or 79, a pharmaceutical composition according to any of claims 60, 61,68 to 76, 80, 81,86 or 87, or a combined preparation according to any of claims 60, 61, 69-72, 82 or 83, for use as a medicament.

90. A polynucleotide according to any of claims 1, 2, or 69 to 72, a vector according to any of claims 64 to 67, or 69 to 76, a polypeptide according to claim 78, or 79, a pharmaceutical composition according to any of claims 60, 61,68 to 76, 80, 81,86 or 87, or a combined preparation according to any of claims 60, 61, 69-72, 82 or 83, for use in the treatment of a viral infection.

91. Use of a polynucleotide according to any of claims 1,2, or 69 to 72, a vector according to any of claims 64 to 67, 69 to 76, a polypeptide according to claim 78, or 79, a pharmaceutical composition according to any of claims 60, 61,68 to 76, 80, 81,86 or 87, or a combined preparation according to any of claims 60, 61, 69-72, 82 or 83, in the manufacture of a medicament for the treatment of a viral infection.

92. A polynucleotide, a vector, a polypeptide, a pharmaceutical composition, or a combined preparation, for use according to claim 90, or use according to claim 91, wherein the viral infection is caused by an emerging or re-emerging virus.

93. A polynucleotide, a vector, a polypeptide, a pharmaceutical composition, or a combined preparation, for use according to claim 90 or 92, or use according to claim 91 or 92, wherein the viral infection is caused by a virus of the Filoviridae family.

94. A polynucleotide, a vector, a polypeptide, a pharmaceutical composition, or a combined preparation, for use according to claim 93, or use according to claim 93, wherein the viral infection is caused by Ebola virus or Marburg virus.

95. A polynucleotide, a vector, a polypeptide, a pharmaceutical composition, or a combined preparation, for use according to claim 90 or 92, or use according to claim 91 or 92, wherein the viral infection is caused by a virus of the Arenaviridae family.

96. A polynucleotide, a vector, a polypeptide, a pharmaceutical composition, or a combined preparation, for use according to claim 95, or use according to claim 95, wherein the viral infection is caused by Lassa virus.

97. A method of inducing an immune response to a virus in a subject, which comprises administering to the subject an effective amount of a polynucleotide according to any of claims 1,2, or 69 to 72, a vector according to any of claims 64 to 67, or 69 to 76, a polypeptide according to claim 78, or 79, a pharmaceutical composition according to any of claims 60, 61, 68 to 76, 80, 81,86 or 87, or a combined preparation according to any of claims 60, 61, 69-72, 82 or 83.

98. A method of immunizing a subject against a virus, which comprises administering to the subject an effective amount of a polynucleotide according to any of claims 1,2, or 69 to 72, a vector according to any of claims 64 to 67, or 69 to 76, a polypeptide according to claim 78, or 79, a pharmaceutical composition according to any of claims 60, 61, 68 to 76, 80, 81, 86 or 87, or a combined preparation according to any of claims 60, 61, 69-72, 82 or 83.

99. A method according to claim 97 or 98, wherein the virus is a member of the Filoviridae family.

100. A method according to claim 99, wherein the virus is an Ebola virus or a Marburg virus.

101. A method according to claim 97 or 98, wherein the virus is a member of the Arenaviridae family.

102. A method according to claim 101, wherein the virus is a Lassa virus.

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