This invention relates to methods for identifying optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response to a pathogen, to methods for identifying a nucleic acid sequence encoding such optimized antigenic pathogen polypeptides, and to methods for determining whether a broadly neutralizing immune response is induced in a subject following immunization with an optimized antigenic pathogen polypeptide or a nucleic acid encoding the optimized pathogen polypeptide. The invention also relates to nucleic acid molecules, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, and their use as vaccines against pathogens, especially against emerging or re-emerging pathogens (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; iii) in an evolving or unanticipated viral epidemic, developing new vaccine candidates is time-consuming 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 Filovi ruses 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.

Influenza virus is a member of the Orthomyxoviridae family. There are three types of influenza viruses, designated influenza A, influenza B, and influenza C. Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A strains, such as H5N1, cause systemic infections in poultry in which mortality may reach 100%. In 2009, H1N1 influenza was the most common cause of human influenza. A new strain of swine-origin H1N1 emerged in 2009 and was declared pandemic by the World Health Organization. This strain was referred to as "swine flu". H1N1 influenza A viruses were also responsible for the Spanish flu pandemic in 1918, the Fort Dix outbreak in 1976, and the Russian flu epidemic in 1977-1978. There are currently two influenza vaccine approaches licensed in the United States - the inactivated, split vaccine and the live-attenuated virus vaccine. The inactivated vaccines can efficiently induce humoral immune responses but generally only poor cellular immune responses. Live virus vaccines cannot be administered to immunocompromised or pregnant patients due to their increased risk of infection.

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 and influenza.

According to the invention there is provided a method for identifying a lead candidate optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to a pathogen, which comprises:

i) providing a polypeptide library comprising a plurality of different candidate optimized antigenic pathogen polypeptides, wherein the amino acid sequence of each different candidate has been optimized from a plurality of different amino acid sequences of a pathogen polypeptide and is different from each different amino acid sequence of the pathogen polypeptide, wherein each different amino acid sequence of the pathogen polypeptide comprises amino acid sequence of a polypeptide of a different isolate, and wherein each different isolate is an isolate of a pathogen of the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response;

ii) screening the candidate optimized antigenic pathogen polypeptides of the polypeptide library for binding by one or more broadly neutralizing antigen-binding molecules, each of which is able to bind and/or neutralize a pathogen of the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response; and

iii) identifying a candidate optimized antigenic pathogen polypeptide that is bound by one or more of the antigen-binding molecules in step (ii) as being a lead candidate optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to the pathogen.

Optionally each different isolate, or each of a plurality of different isolates, of the pathogen is of the same subtype or type as the pathogen to which it is desired to induce a broadly neutralizing immune response.

Optionally each different isolate, or each of a plurality of different isolates, of the pathogen is of the same species or genus as the pathogen to which it is desired to induce a broadly neutralizing immune response.

Optionally the different isolates include isolates of different subtypes or types within the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response.

Optionally the different isolates include isolates of different species or genera within the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response.

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, Bunya virus, 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 W-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 (TYR03) 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 b13-b14 loop. Cathepsin cleavage removes -60% of the amino acids from GP1, including the mucin-like domain, the glycan cap, and the outmost b strand of the proposed receptor binding region, resulting in a primed form of GP (named GPcl, the 19 kDa GP1 plus GP2). Unlike the full-length GP, the primed GPcl 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 GPcl have been determined (Wang et ai., 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 mucin-like domain, but shares 295 amino acids of GP1.

The influenza virion contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1 ), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and non-structural protein 2 (NS2). The HA, NA, M I, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and NS2 are nucleocapsid associated proteins. The M I protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell, and the sources of the major immunodominant epitopes for virus neutralization and protective immunity. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines.

For bacteria or fungi, suitable pathogen polypeptides include polypeptides that are essential for the propagation of a bacterium or fungus, or for the ability of a bacterium or fungus to infect or cause disease in a human. Suitable examples include surface-expressed polypeptides or proteins (see, for example, Hu et at., Front. Microbiol.8:82. doi: 10.3389/fmicb.2017.00082; Santos and Levitz, Cold Spring Harb Perspect Med. 2014; 4(11): a019711).

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.

Claims

1. A method for identifying a lead candidate optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to a pathogen, which comprises:

i) providing a polypeptide library comprising a plurality of different candidate optimized antigenic pathogen polypeptides, wherein the amino acid sequence of each different candidate has been optimized from a plurality of different amino acid sequences of a pathogen polypeptide and is different from each different amino acid sequence of the pathogen polypeptide, wherein each different amino acid sequence of the pathogen polypeptide comprises amino acid sequence of a polypeptide of a different isolate, and wherein each different isolate is an isolate of a pathogen of the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response;

ii) screening the candidate optimized antigenic pathogen polypeptides of the polypeptide library for binding by one or more broadly neutralizing antigen-binding molecules, each of which is able to bind and/or neutralize a pathogen of the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response; and

iii) identifying a candidate optimized antigenic pathogen polypeptide that is bound by one or more of the antigen-binding molecules in step (ii) as being a lead candidate optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to the pathogen.

2. A method according to claim 1, wherein the one or more broadly neutralizing antigen-binding molecules include an antibody that has been obtained, or derived from an antibody that has been obtained, from a subject that has been exposed to a pathogen of the same family as the pathogen to which it is desired to induce a broadly neutralizing immune response.

3. A method according to claim 1 or 2, wherein the one or more broadly neutralizing antigen-binding molecules include non-antibody antigen-binding proteins.

4. A method according to claim 3, wherein the one or more broadly neutralizing antigen-binding molecules include a designed ankyrin repeat protein (DARPin), an anticalin, an aptamer, or a T-cell receptor molecule.

5. A method according to any preceding claim, wherein the candidate optimized antigenic pathogen polypeptides of the polypeptide library have been expressed in, or on the surface of, mammalian cells.

6. A method according to any of claims 1 to 4, wherein the candidate optimized antigenic pathogen polypeptides of the polypeptide library have been expressed in, or on the surface of, bacterial, yeast, or insect cells.

7. A method according to any preceding claim, wherein the pathogen is a virus, the candidate optimized antigenic pathogen polypeptides are candidate optimized antigenic virus polypeptides, and the pathogen peptides are virus polypeptides.

8. A method according to claim 7, wherein the polypeptide library is a viral pseudotype library comprising a plurality of different viral pseudotypes, each different viral pseudotype comprising a different candidate optimized virus polypeptide.

9. A method according to claim 8, wherein in step (ii) the candidate optimized antigenic virus polypeptides are screened for binding by one or more of the antigen-binding molecules by screening the viral pseudotypes for binding and/or neutralization by one or more of the antigen-binding molecules.

10. A method according to any of claims 1 to 7, wherein the candidate optimized antigenic pathogen polypeptides are screened for binding by the one or more antigen-binding molecules by a flow cytometric assay.

11. A method according to any preceding claim, which further comprises generating the polypeptide library.

12. A method according to claim 11, wherein the polypeptide library is generated by expressing the different candidate optimized antigenic pathogen polypeptides from a nucleic acid library comprising a plurality of different nucleic acids, each different nucleic acid comprising a nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide of the polypeptide library.

13. A method according to claim 12, wherein the different candidate optimized pathogen polypeptides are expressed in, or on the surface of, mammalian cells.

14. A method according to claim 12 or 13, wherein the nucleotide sequence of each different nucleic acid of the nucleic acid library is codon-optimized, optionally gene-optimized, for expression of the encoded polypeptide in a mammalian cell.

15. A method according to any of claims 12 to 14, wherein each different nucleic acid of the nucleic acid library is part of an expression vector for expression of the nucleic acid in a mammalian cell.

16. A method according to any of claims 12 to 15, wherein the pathogen is a virus, the candidate optimized antigenic pathogen polypeptides are candidate optimized antigenic virus polypeptides, and the pathogen peptides are virus polypeptides.

17. A method according to claim 16, wherein the nucleic acid library is a viral pseudotype vector library, and each different nucleic acid of the library is part of an expression vector for production of a viral pseudotype comprising the encoded virus polypeptide, and the polypeptide library is a viral pseudotype library generated by producing viral pseudotypes from the expression vectors of the viral pseudotype vector library, wherein the viral pseudotype library comprises a plurality of different viral pseudotypes, each different viral pseudotype comprising a different candidate optimized virus polypeptide encoded by a different nucleic acid sequence of the viral pseudotype vector library.

18. A method according to any of claims 15 to 17, wherein the expression vector is also a vaccine vector.

19. A method according to claim 18, wherein the vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector.

20. A method according to claim 18 or 19, wherein the vaccine vector is based on a viral delivery vector, such as a poxvirus (e.g. 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 (e.g. Salmonella, E.coli), an RNA expression vector, or a DNA expression vector.

21. A method according to any of claims 15 to 20, wherein the vector is a pEVAC-based expression vector.

22. A method according to claim 12, wherein the different candidate optimized antigenic pathogen polypeptides are expressed in, or on the surface of, bacterial, yeast, or insect cells.

23. A method according to any of claims 12 to 22, which further comprises generating the nucleic acid library by synthesising a plurality of different nucleic acids, each different nucleic acid comprising a different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide.

24. A method according to claim 23, which further comprises:

i) obtaining amino acid sequences of the pathogen polypeptide, and/or nucleotide sequences encoding the pathogen polypeptide, of the different pathogen isolates; and

ii) generating a plurality of different nucleotide sequences, each different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide, wherein the encoded amino acid sequence of each different candidate optimized antigenic pathogen polypeptide is optimized from the obtained amino acid sequences or encoded amino acid sequences of the pathogen polypeptide, and is different from each of the obtained amino acid sequences or encoded amino acid sequences.

25. A method according to claim 24, wherein generation of the plurality of different nucleotide sequences in step (ii) of claim 24 comprises:

carrying out a multiple sequence alignment of the amino acid or nucleotide sequences obtained in step (i) of claim 24;

identifying from the multiple sequence alignment amino acid sequence or encoded amino acid sequence that is highly conserved between the polypeptides of the different pathogen isolates; and

generating a plurality of different nucleotide sequences, each different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide, wherein one or more of the different nucleotide sequences includes sequence encoding a highly conserved amino acid sequence or encoded amino acid sequence identified from the multiple sequence alignment.

26. A method according to claim 25, which further comprises:

identifying from the multiple sequence alignment amino acid sequence or encoded amino acid sequence that is ancestral amino acid sequence; and

including in one or more of the different generated nucleotide sequences sequence encoding an ancestral amino acid sequence identified from the multiple sequence alignment.

27. A method according to any of claims 24 to 26, which includes codon-optimization, optionally gene-optimization codons of the different generated nucleotide sequences for optimal expression of the encoded candidate optimized antigenic pathogen polypeptides in an expression system.

28. A method according to claim 27, wherein the expression system comprises a mammalian cell.

29. A method according to claim 27, wherein the expression system comprises a yeast, bacterial, or insect cell.

30. A method according to any of claims 24 to 29, which includes optimizing the different nucleotide sequences for antigenicity of the encoded candidate optimized antigenic pathogen polypeptides.

31. A method according to claim 30, wherein the antigenicity optimization includes any of the following:

deletion or modification of nucleic acid sequence encoding amino acid sequence that inhibits production and/or function of anti-pathogen polypeptide antibody (for example, deletion or modification of a mucin-like domain);

region swapping to recover one or more potential lost encoded epitopes;

site-specific mutation, for example of N-linked glycosylation sites;

changes to enhance stability (e.g. disulphide bond formation, reduce degradation of the encoded polypeptide by a serine protease);

removal of glycans;

insertion of nucleic acid sequence, for example to insert nucleic acid sequence encoding a desired epitope.

32. A method according to any preceding claim, wherein the one or more broadly neutralizing antigen-binding molecules recited in step (ii) of claim 1 include a broadly neutralizing antibody, preferably a broadly neutralizing monoclonal antibody (BNmAb).

33. A method according to any preceding claim, wherein the one or more antigenbinding molecules recited in step (ii) of claim 1 include an antibody obtained, or derived from an antibody obtained, from a subject that has survived an outbreak of a pathogen of the same family, optionally of the same subtype or type, as the pathogen to which it is desired to induce a broadly neutralizing immune response.

34. A method according to claim 33, wherein the subject from which the antibody has been obtained or derived is a human or non-human mammalian subject.

35. A method according to claim 33 or 34, wherein the one or more antigen-binding molecules include a broadly neutralizing monoclonal antibody (BNmAb).

36. A method according to any preceding claim, wherein the different pathogen isolates include different pathogen isolates from an outbreak of a pathogen of the same subtype as the pathogen to which it is desired to induce a broadly neutralizing immune response.

37. A method according to any preceding claim, wherein the different pathogen isolates include different pathogen isolates from an outbreak of a pathogen of a different subtype, but the same type, as the pathogen to which it is desired to induce a broadly neutralizing immune response.

38. A method according to any preceding claim, wherein the different pathogen isolates include different pathogen isolates from an outbreak of a pathogen of a different group, but the same family, as the pathogen to which it is desired to induce a broadly neutralizing immune response.

39. A method according to any preceding claim, wherein the different pathogen isolates include different prior pathogen isolates of a pathogen of the same subtype, type, or family as the pathogen to which it is desired to induce a broadly neutralizing immune response.

40. A method according to any preceding claim, wherein each candidate optimized antigenic pathogen polypeptide comprises at least 20 amino acid residues.

41. A method according to any preceding claim, wherein the pathogen is a virus.

42. A method according to claim 41, wherein the virus is an RNA virus.

43. A method according to claim 41 or 42, wherein the virus is an emerging or re-emerging RNA virus.

44. A method according to any of claims 41 to 43, wherein the virus is a Filovirus, an Arenavirus, or an Orthomyxovirus.

45. A method according to any of claims 41 to 43, wherein the virus is Ebola virus or Marburg virus.

46. A method according to any of claims 41 to 43, wherein the virus is Lassa virus.

47. A method according to any preceding claim, wherein the pathogen polypeptide is a viral glycoprotein.

48. A method according to any preceding claim, which is an in vitro method.

49. A method of identifying a nucleic acid sequence encoding an optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to a pathogen, which comprises:

i) immunizing a human, or a non-human animal, with a nucleic acid comprising a nucleic acid sequence encoding a lead candidate optimized antigenic pathogen polypeptide identified by a method according to any preceding claim;

ii) determining whether a broadly neutralizing immune response is induced in the human or non-human animal following the immunization in step (i); and

iii) identifying the nucleic acid sequence as a nucleic acid sequence encoding an optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response to the pathogen if it is determined from step (ii) that a broadly neutralizing immune response is induced in the human or non-human animal.

50. A method according to claim 49, which comprises determining whether a broadly neutralizing immune response is induced in the human or non-human animal by determining whether antibody in serum obtained from the human or non-human animal binds to and/or neutralizes more than one pathogen subtype.

51. A method according to claim 49 or 50, wherein the non-human animal is a mammal.

52. A method according to claim 51, wherein the mammal is a guinea pig, or a mouse.

53. A method according to claim 49 or 50, wherein the non-human animal is avian.

54. 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;

iii) 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.

55. 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: 10, or identical with SEQ ID NO: 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

iii) 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.

56. 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.

57. 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;

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.

58. 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; or

iii) 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.

59. 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 NO:20, or identical with SEQ ID NO:20;

iii) 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 NO:30, or identical with SEQ ID NO:30.

60. An isolated nucleic acid encoding an amino acid sequence encoded by a nucleic acid of claim 54, 55, or 56, wherein the nucleic acid is codon-optimized, optionally gene-optimized, for expression in mammalian cells.

61. An isolated nucleic acid encoding a polypeptide of claim 57, 58, or 59, wherein the nucleic acid is codon-optimized, optionally gene-optimized, for expression in mammalian cells.

62. A vector comprising a nucleic acid of claim 54, 55, 56, 60, or 61.

63. A vector according to claim 62, which further comprises a promoter operably linked to the nucleic acid.

64. A vector according to claim 63, wherein the promoter is for expression of a polypeptide encoded by the nucleic acid in mammalian cells.

65. A vector according to claim 63, wherein the promoter is for expression of a polypeptide encoded by the nucleic acid in yeast or insect cells.

66. A vector according to any of claims 62 to 65, which is a vaccine vector.

67. A vector according to claim 66, which is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector.

68. An isolated cell comprising a vector of any of claims 62 to 65.

69. A pseudotyped virus particle comprising the polypeptide of claim 57, 58, or 59.

70. A method of producing a pseudotyped virus particle of claim 69, which includes transfecting a host cell with a vector according to any of claims 62 to 64.

71. A fusion protein comprising a polypeptide according to claim 57, 58, or 59.

72. A pharmaceutical composition comprising a nucleic acid according to claim 54, 55, 56, 60, or 61, and a pharmaceutically acceptable carrier, excipient, or diluent.

73. A pharmaceutical composition comprising a vector according to any of claims 62 to 64, 66, or 67, and a pharmaceutically acceptable carrier, excipient, or diluent.

74. A pharmaceutical composition comprising a polypeptide according to claim 57, 58, or 59, and a pharmaceutically acceptable carrier, excipient, or diluent.

75. A pharmaceutical composition according to any of claims 72 to 74, 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.

76. 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 according to any of claims 54, 55, 60, or 61, a polypeptide according to claim 57 or 58, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75.

77. A method of immunizing a subject against a virus of the Filoviridae family, which comprises administering to the subject a nucleic acid according to any of claims 54, 55, 60, or 61, a polypeptide according to claim 57 or 58, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75.

78. 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 according to any of claims 56, 60, or 61, a polypeptide according to claim 59, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75.

79. A method of immunizing a subject against a virus of the Arenaviridae family, which comprises administering to the subject a nucleic acid according to any of claims 56, 60, or 61, a polypeptide according to claim 59, a vector according to any of claims 62 to 64, 66, or

67, or a pharmaceutical composition according to any of claims 72 to 75.

80. A method according to any of claims 76 to 79, wherein the composition is administered intramuscularly.

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

82. A vector according to claim 81, wherein the multiple cloning site comprises a nucleic acid sequence of SEQ ID NO: 16.

83. A vector according to claim 81 or 82, which is an expression vector, and a viral pseudotype vector.

84. A vector according to any of claims 81 to 83, which is a vaccine vector.

85. A vector according to any of claims 81 to 84, 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.

86. A vector according to claim 85, 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.

87. A vector according to any of claims 81 to 86, which further comprises an origin of replication, and nucleic acid encoding resistance to an antibiotic.

88. A vector according to claim 87, wherein the origin of replication comprises a pUC-plasmid origin of replication and/or the nucleic acid encodes resistance to kanamycin.

89. A vector according to any of claims 81 to 88, which comprises a nucleic acid sequence of SEQ ID NO: 17.

90. An isolated nucleic acid molecule 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.

91. 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.

92. 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.

93. 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.

94. 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.

95. 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.

96. 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.

97. 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.

98. 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.

99. 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.

100. 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.

101. 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.

102. A nucleic acid according to any of claims 54, 55, 60, or 61, a polypeptide according to claim 57 or 58, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, for use as a medicament.

103. A nucleic acid according to any of claims 54, 55, 60, or 61, a polypeptide according to claim 57 or 58, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, 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.

104. Use of a nucleic acid according to any of claims 54, 55, 60, or 61, a polypeptide according to claim 57 or 58, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, 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.

105. A nucleic acid according to any of claims 56, 60, or 61, a polypeptide according to claim 59, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, for use as a medicament.

106. A nucleic acid according to any of claims 56, 60, or 61, a polypeptide according to claim 59, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, 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.

107. Use of a nucleic acid according to any of claims 56, 60, or 61, a polypeptide according to claim 59, a vector according to any of claims 62 to 64, 66, or 67, or a pharmaceutical composition according to any of claims 72 to 75, 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 Arenaviridae family.

108. A nucleic acid according to claim 90 or 91, a composition according to claim 92, 93, 96, or 97, a combined preparation according to claim 94, 95, 100, or 101, or a fusion protein according to claim 98 or 99, for use as a medicament.

109. A nucleic acid according to claim 90 or 91, a composition according to claim 92, 93, 96, or 97, a combined preparation according to claim 94, 95, 100, or 101, or a fusion protein according to claim 98 or 99, 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.

110. Use of a nucleic acid according to claim 90 or 91, a composition according to claim 92, 93, 96, or 97, a combined preparation according to claim 94, 95, 100, or 101, or a fusion protein according to claim 98 or 99, 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.