Vaccines and methods

Abstract

To provide vaccines and methods.SOLUTION: Described herein are methods for identifying optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response and associated T-cell responses to a pathogen, and nucleic acid sequences encoding such polypeptides. Also described are 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. Further described are 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).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for identifying optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against pathogens, methods for identifying nucleic acid sequences encoding such optimized antigenic pathogen polypeptides, and methods for determining whether a broadly neutralizing immune response is induced in a subject after immunization with an optimized antigenic pathogen polypeptide or a nucleic acid encoding the optimized pathogen polypeptide. The present invention also relates to nucleic acid molecules, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, and their use as vaccines against pathogens, particularly emerging or re-emerging pathogens (especially RNA viruses). The present invention also relates to pseudotyped virus particles.

[0002] The fundamental principle of vaccines is to prepare the immune system for an encounter with a pathogen. Vaccines trigger the immune system to produce antibodies and T-cell responses, which help fight infection. Historically, once a pathogen was isolated and grown, it was mass-produced, killed, or weakened for use as a vaccine. Later, recombinant genes from isolated pathogens were used to generate recombinant proteins, which were then mixed with adjuvants to stimulate an immune response. More recently, pathogen genes were cloned into vector systems (attenuated bacteria or viruses) to express and deliver antigens in vivo. All of these strategies rely on pathogens isolated from past outbreaks to prevent future epidemics. This traditional technique is effective for pathogens that do not change significantly or change slowly. However, some pathogens tend to mutate, and antibodies do not necessarily recognize different strains of the pathogen. Emerging and re-emerging pathogens often hide or disguise their vulnerable antigens from the immune system.

[0003] Among 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). RNA viruses are viruses that have RNA as their genetic material. This nucleic acid is usually single-stranded RNA (ssRNA) but can also be double-stranded RNA (dsRNA). RNA viruses generally have a much higher mutation rate than DNA viruses because viral RNA polymerase lacks the proofreading ability of DNA polymerase. This is one reason why it is difficult to generate effective vaccines to prevent diseases caused by RNA viruses. For the most part, current vaccine candidates against RNA viruses are limited to the virus strain used as the vaccine insert, which is often selected based on the availability of wild-type strains rather than being intelligently designed. Technical challenges in developing vaccines for enveloped RNA viruses include: i) viral variation in wild-type field isolate glycoproteins (GPs) results in limited breadth of protection as vaccine antigens; ii) selection of vaccine antigens expressed by vaccine inserts is highly empirical, and immunogen selection is a slow trial-and-error process; and iii) in emerging or unexpected virus outbreaks, development of new vaccine candidates is time-consuming, which can delay vaccine deployment.

[0004] Notable human diseases caused by RNA viruses include viral hemorrhagic fevers (VHFs), a group of illnesses caused by viruses from several distinct families. The term "viral hemorrhagic fever" is generally used to describe a severe, multisystemic syndrome (i.e., affecting multiple organ systems throughout the body). Characteristically, the vascular system is damaged throughout the body, impairing the body's ability to regulate itself. These symptoms are often accompanied by hemorrhage (blood loss), although the blood loss itself is rarely life-threatening. While some types of hemorrhagic fever viruses can cause relatively mild illness, many viruses cause severe, life-threatening disease. VHFs are caused by viruses from at least five distinct families: Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, and Paramyxoviridae. All viruses in these families are RNA viruses, and all are either lipid-coated or enveloped. Survival from VHFs depends on animal or insect hosts (natural reservoirs). Viruses are geographically restricted to areas where their host species survive, and humans become infected when they come into contact with an infected host. For some viruses, after transmission from the host, humans can transmit the virus to each other. Human cases or outbreaks of hemorrhagic fever caused by these viruses occur sporadically and irregularly. The occurrence of outbreaks cannot be easily predicted. With some exceptions, there is no cure for VHF, and no established drug treatment.

[0005] VHFs, caused by both arenaviruses and filoviruses, span a wide geographic region from West Africa to Central Africa, threatening neighboring regions where infected animal reservoirs may migrate but where human disease has not yet been reported. Filoviruses encode their genomes in the form of single-stranded, negative-sense RNA. Two commonly known members of this family 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 viral family, including closely related siblings (e.g., Ebola Mayinga and Ebola Kikwit), close cousins ​​(Taï Forest and Bundibugyo), distant cousins ​​(Sudan), and distant relatives (Marburg virus). The 2014 Ebola outbreak in West Africa was the largest since viral diseases were first recognized. Arenaviruses are classified into two groups: Old World viruses and New World viruses. The differences between these groups are geographically and genetically distinct. At least eight arenaviruses are known to cause human diseases of varying severity. Aseptic meningitis, a severe human disease causing inflammation involving the brain and spinal cord, can result from lymphocytic choriomeningitis virus (LCMV) infection. Hemorrhagic fever syndromes result from infection with viruses such as Guanarito virus (GTOV), Junín virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV).

[0006] Lassa fever virus (LASV), Ebola (EBOV), and Marburg (MARV) viruses are the most important hemorrhagic fevers in West and Central Africa. Lassa fever is endemic in West Africa, with an estimated infection rate ranging from 300,000 to 1 million, resulting in 5,000 deaths annually. Lassa fever virus (LASV), Ebola (EBOV), and Marburg (MARV) viruses are all containment level 4 pathogens, with high human morbidity and mortality, no established cure, and no currently licensed vaccines for infections caused by these viruses.

[0007] Influenza viruses are members of the Orthomyxoviridae family. There are three types of influenza viruses: 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 tracts. However, highly pathogenic influenza A strains, such as H5N1, can cause systemic infections in livestock, with mortality rates approaching 100%. In 2009, H1N1 influenza was the most common cause of human influenza. A novel strain of H1N1, originating from swine, emerged in 2009 and was declared a pandemic by the World Health Organization. This strain was dubbed "swine flu." H1N1 influenza A viruses were also responsible for the 1918 Spanish flu pandemic, the 1976 Fort Dix pandemic, and the 1977–1978 Russian flu epidemic. Currently, two influenza vaccine approaches are licensed in the United States: inactivated split-virus vaccines and live attenuated virus vaccines. Inactivated vaccines can efficiently induce humoral immune responses but generally induce cell-mediated immune responses poorly. Live virus vaccines cannot be administered to immunocompromised or pregnant patients due to the increased risk of infection.

[0008] Therefore, there is a need to provide effective vaccines that induce broadly neutralizing immune responses to protect against emerging and re-emerging diseases, particularly those caused by viruses such as RNA viruses, including VHF and influenza. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Heeney, Journal of Internal Medicine (2006) 260:399~408 Summary of the Invention [Means for solving the problem]

[0010] In accordance with the present invention, there is provided a method for identifying lead candidate optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against a pathogen, comprising the steps of: i) providing a polypeptide library comprising a plurality of different candidate optimized antigenic pathogen polypeptides, wherein the amino acid sequence of each different candidate is optimized from a plurality of different amino acid sequences of pathogen polypeptides, wherein each different amino acid sequence of the pathogen polypeptide is different from each different amino acid sequence of the pathogen polypeptide and each different amino acid sequence of the pathogen polypeptide comprises an amino acid sequence of a polypeptide of a different isolate, each different isolate being an isolate of a pathogen of the same family as the pathogen against 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 capable of binding to and / or neutralizing pathogens of the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response; and iii) identifying the candidate optimized antigenic pathogen polypeptides bound by one or more of the antigen binding molecules in step (ii) as lead candidate optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against the pathogen. A method is provided that includes:

[0011] Optionally, each different isolate or each of the plurality of different isolates of a pathogen is an isolate of the same subtype or type as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0012] Optionally, each different isolate or each of the plurality of different isolates of a pathogen is an isolate of the same species or genus as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0013] Optionally, the different isolates include isolates of different subtypes or types within the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0014] Optionally, the different isolates include isolates of different species or genus within the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0015] The term "pathogen" as used herein refers to anything that can cause disease, especially an infectious agent that can cause disease, such as a virus, bacterium, fungus, or parasite.

[0016] The term "polypeptide," as used herein, refers to a polymer comprising multiple 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 encompass naturally occurring proteins as well as recombinantly or synthetically produced proteins. Optionally, the polypeptide is a modified polypeptide, e.g., a polypeptide that has been co- or post-translationally modified, such as a glycosylated polypeptide or protein ("glycoprotein"). Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to amino acid side chains. Carbohydrates are attached to proteins by co- or post-translational glycosylation.

[0017] "Pathogen polypeptide" refers to any polypeptide-forming portion of a pathogen. Optionally, the pathogen polypeptide is a structural protein (or portion thereof) of the pathogen. Optionally, the pathogen polypeptide is a surface-exposed structural protein (or portion thereof) 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, the pathogen polypeptide comprises an amino acid sequence of at least 20 amino acid residues. Optionally, the pathogen polypeptide comprises an amino acid sequence of at most 1000, 900, 800, 700, or 600 amino acid residues.

[0018] A fully assembled infectious virus is known as a virion. The simplest virion consists of nucleic acid (single- or double-stranded RNA or DNA) and a capsid protein coat. The capsid is formed as a single or double protein shell and consists of only one or a few structural protein species. Enveloped viruses have an envelope that surrounds their protective protein capsid. The envelope is typically derived from portions of the host membrane (phospholipids and proteins) but contains virally encoded glycoproteins.

[0019] Glycoproteins on the envelope surface serve to identify and bind to receptor sites on the host membrane. The viral envelope then fuses with the host 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 filoviruses, influenza viruses, and human immunodeficiency virus (HIV), enter cells and initiate viral infection. This membrane fusion process is carried out by one or more viral envelope glycoproteins. Fusion can occur on the cell plasma membrane or the endosomal membrane.

[0020] Glycoproteins can help viruses evade the host immune system. Enveloped viruses have great adaptability and can change rapidly to evade the host immune system. Enveloped viruses can cause persistent infections. Examples of enveloped RNA viruses include flaviviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and filoviruses. Retroviruses are enveloped viruses. Enveloped DNA viruses include herpesviruses, poxviruses, and hepadnaviruses.

[0021] Most external viral envelope proteins are glycoproteins, often assembled as dimers or trimers, present as membrane-anchored spikes. Trimeric glycoprotein (GP) spikes on filovirus envelopes mediate all stages of viral entry, including binding, entry, and fusion. Cellular receptor recognition sites are often located in the domain furthest from the viral envelope (distal end), while the proximal domain interacts with the envelope lipid bilayer. Oligosaccharide side chains (glycans) are attached by N-glycosidic or, more rarely, O-glycosidic linkages. Because they are synthesized by cellular glycosyltransferases, the sugar composition of these glycans is similar to that of host cell membrane glycoproteins.

[0022] Filovirus entry on the cell surface has been shown to be mediated by host cell binding factors, including 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), as well as several cell surface proteins, such as integrins, T-cell immunoglobulin and mucin domain-containing (TIM) proteins, and tyrosine protein kinase receptor 3 (TYRO3) family members. After binding to the cell surface, filoviruses are internalized by a macropinocytosis-like process and subsequently transported through early and late endosomes. Next, the viral envelope fuses with the membrane of the late endosome, after which the viral genome enters the cytoplasm. In the cytoplasm, the viral genome replicates and is transcribed, new viral proteins are synthesized, and progeny virions are assembled, which bud from the cell surface.

[0023] The surface glycoprotein GP of Ebola virus (EBOV) is a critical component of many vaccines and a target for neutralizing antibodies. EBOV GP is synthesized as a single polypeptide and then cleaved by a furin-like protease into GP1 and GP2 subunits, which are held together through intersubunit disulfide bonds and noncovalent interactions to form a trimer of GP1-GP2 heterodimers on the viral surface. However, furin cleavage is not sufficient to prime EBOV GP. After cell entry, the virus is ultimately transported to late endosomes, where GP is further primed to remove some "cap" components, thereby triggering the induction of critical membrane fusion events that lead to viral entry. EBOV GP priming is mediated by the cysteine ​​proteases cathepsin B and cathepsin L, which cleave GP1 within the β13-β14 loop. Cathepsin cleavage removes approximately 60% of the amino acids from GP1, including the mucin-like domain, glycan cap, and the outermost β-strand of the proposed receptor-binding region, resulting in a primed form of GP (termed GPcl, 19 kDa GP1 plus GP2). Unlike full-length GP, primed GPcl cannot bind to the endosomal membrane protein Niemann-Pick C1 (NPC1), a host entry factor essential for EBOV infection. The crystal structures of free NPC1-C and its complex with GPcl have been determined (Wang et al., Cell, 2016, 164, 258-268). During Ebola virus infection, the major 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 with GP1.

[0024] Influenza virions contain a segmented, negative-sense RNA genome that encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix protein (M1), proton channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). HA, NA, M1, and M2 are membrane-associated, while NP, PB1, PB2, PA, and NS2 are nucleocapsid-associated proteins. The M1 protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins responsible for virus binding and cell entry and are the source of the major immunodominant epitopes for virus neutralization and protective immunity. Both the HA and NA proteins are considered to be the most important components for prophylactic influenza vaccines.

[0025] With respect to bacteria or fungi, suitable pathogen polypeptides include polypeptides that are essential for the reproduction of the bacteria or fungus, or for the ability of the bacteria or fungus to infect or cause disease in humans. Suitable examples include surface-expressed polypeptides or proteins (e.g., Hu et al., Front. Microbiol. 8:82. doi: 10.3389 / fmicb.2017.00082; Santos and Levitz, Cold Spring Harb Perspect Med. 2014; 4(11): a019711).

[0026] The term "antigenic" as used herein refers to a substance capable of inducing an immune response in a host organism. The immune response can be a humoral and / or cellular immune response. A cellular immune response is the response of cells of the immune system, such as B cells, T cells, macrophages, or polymorphonuclear leukocytes, to a stimulus, such as an antigen or a vaccine. An immune response can include any cell of the body involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. 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 a disease associated with an infection). Methods for measuring immune responses are well known in the art and include, for example, measuring lymphocyte (e.g., B or T cell) proliferation and / or activity, cytokine or chemokine secretion, inflammation, or antibody production.

[0027] If desired, the optimized antigenic pathogen polypeptide can induce antibody production and / or a T cell response in a human or non-human animal to which the polypeptide is administered (either as a polypeptide or expressed, for example, from an administered nucleic acid expression vector).

[0028] The term "antibody," as used herein, refers to an immunoglobulin molecule with a specific amino acid sequence produced by B lymphoid cells. Antibodies are induced in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by specifically reacting with an antigen in some demonstrable manner, and antibodies and antigens are each defined in relation to the other. "Inducing an antibody response" refers to the ability of an antigen or other molecule to induce the production of antibodies.

[0029] "Neutralizing" antibodies or antigen-binding molecules not only bind to pathogens, such as viruses, but also bind in such a way that they inhibit (i.e., reduce) or block infection or the progression of infection. Neutralizing antibodies or antigen-binding molecules can block receptor interaction or bind to viral capsids to inhibit genome uncoating. The term "neutralizing antibody" or "neutralizing antigen-binding molecule" also includes antibodies or antigen-binding molecules that can prevent pathogen, e.g., viral, infection by promoting cytokine responses or promoting uptake and elimination by immune cells. In particular, the term "neutralizing antibody" includes antibodies (or fragments or derivatives thereof) that can inhibit or block pathogen infection (or the progression of infection) through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Only a small subset of the many antibodies that bind to viruses are neutralizing.

[0030] The term "broadly neutralizing antigen binding molecule," as used herein, includes an antigen binding molecule, such as an antibody or a fragment or derivative thereof, that can inhibit (i.e., reduce), neutralize, or prevent infection of at least two different subtypes or species of pathogens, such as at least two different subtypes or species of viruses, at least two different subtypes or species of bacteria, or at least two different subtypes or species of fungi. Optionally, a broadly neutralizing antigen binding molecule can inhibit (i.e., reduce), neutralize, or prevent infection of most or all different subtypes or species of pathogens, such as most or all different subtypes or species of viruses, most or all different subtypes or species of bacteria, or most or all different subtypes or species of fungi. Optionally, a broadly neutralizing antibody can inhibit (i.e., reduce), neutralize, or prevent infection of at least two different types of members of the same pathogen family (e.g., viruses, bacteria, or fungi).

[0031] Optionally, a plurality of different broadly neutralizing antigen-binding molecules are used in step (ii) of the method of the present invention. Optionally, each different broadly neutralizing antigen-binding molecule binds to a different region or epitope of a candidate optimized antigen pathogen polypeptide of the polypeptide library.

[0032] The term "broadly neutralizing immune response," as used herein, refers to an immune response elicited in a subject that is sufficient to inhibit (i.e., reduce), neutralize, or prevent infection and / or progression of at least two different subtypes or species of pathogens, such as at least two different subtypes or species of viruses, at least two different subtypes or species of bacteria, or at least two different subtypes or species of fungi. Optionally, a broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of most or all different subtypes or species of pathogens, such as most or all different subtypes or species of viruses, most or all different subtypes or species of bacteria, or most or all different subtypes or species of fungi. Optionally, a broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of at least two different types of members of the same family of pathogens (e.g., viruses, bacteria, or fungi). Optionally, a broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection of members of at least two different genera of pathogens within the same family (e.g., viruses, bacteria, or fungi).

[0033] Several broadly neutralizing antibodies against pathogens are known. For example, some antibodies have been demonstrated to be capable of neutralizing virus isolates of various subtypes within the Filoviridae family. A systematic analysis of monoclonal antibodies against Ebola virus glycoproteins was described by Saphire et al. (Cell, 2018; 174(4): 938-952). An example of a broadly neutralizing antibody against Ebola virus is the immune-induced macaque antibody CA45 described by Zhao et al., 2017 (Cell 169, 891-904). A broadly neutralizing monoclonal antibody against HIV-1 envelope protein is referenced in Bruun et al. (PLoS ONE 9(10): e109196. doi:10.1371 / journal.pone.0109196). Corti et al. (Curr Opin Virol. 2017 Jun;24:60-69) provide a review of the specificity, antiviral and immunological mechanisms of action, and clinical development of broadly reactive monoclonal antibodies against influenza A and B viruses.

[0034] Optionally, the pathogen is a virus.

[0035] Viruses are classified primarily by phenotypic characteristics, such as morphology, nucleic acid type, mode of replication, host organism, and the type of disease they cause. One scheme for classifying viruses, the Baltimore classification system, places viruses into one of seven groups according to a combination of their nucleic acid (DNA or RNA), stranding (single- or double-stranded), sense, and method of replication: · I: dsDNA viruses (e.g., adenoviruses, herpesviruses, poxviruses); · II: ssDNA viral (+ strand or "sense") DNA (e.g., parvovirus); III: dsRNA viruses (e.g., reoviruses); · IV: (+)ssRNA viral (+strand or sense) RNA (e.g., picornaviruses, togaviruses); · V: (-)ssRNA viral (negative-strand or antisense) RNA (e.g., orthomyxoviruses, filoviruses, arenaviruses, rhabdoviruses); · VI: ssRNA-RT viral (positive strand or sense) RNA with a DNA intermediate in the life cycle (e.g., retroviruses); · VII: dsDNA-RT viral DNA with an RNA intermediate in the life cycle (e.g., hepadnaviruses).

[0036] Optionally, the virus is an RNA virus. RNA viruses include: Group III: viruses have double-stranded RNA genomes; Group IV: Viruses have a positive-sense single-stranded RNA genome. Many well-known viruses are found in this group, including picornaviruses (a family of viruses that includes well-known viruses such as hepatitis A virus, enteroviruses, rhinoviruses, polioviruses, and foot-and-mouth disease virus), SARS virus, hepatitis C virus, yellow fever virus, and rubella virus; Group V: Viruses have negative-sense single-stranded RNA genomes. Ebola and Marburg viruses are well-known members of this group, along with influenza virus, Lassa virus, measles, mumps, and rabies.

[0037] The classification of different RNA virus families according to the Baltimore classification is given in the table below: [Table 2]

[0038] Optionally, the virus is an emerging or re-emerging RNA virus. Examples of emerging or re-emerging RNA viruses include Ebola virus, Marburg virus, Lassa virus, influenza virus, MERS coronavirus, Hendra virus, and Nipah virus.

[0039] Optionally, the virus is a filovirus or arenavirus. Optionally, the virus is an Ebola virus or a Marburg virus. Optionally, the virus is a Lassa virus. Optionally, the virus is an influenza virus.

[0040] Optionally, the pathogen is a DNA virus. Optionally, the pathogen is a member of the Poxviridae family, such as monkeypox virus.

[0041] DNA viruses include: Group I: Viruses have double-stranded DNA. The viruses that cause chickenpox and herpes are found in this group. Group II: The virus has single-stranded DNA.

[0042] The Baltimore classification of different DNA virus families is given in the table below: [Table 3]

[0043] Optionally, the pathogen is a reverse transcribing virus. Reverse transcribing viruses include: Group VI: Viruses have single-stranded RNA viruses that replicate through a DNA intermediate. Retroviruses are included in this group, of which HIV is a member. Group VII: Viruses have a double-stranded DNA genome and replicate using reverse transcriptase. Hepatitis B virus can be found in this group.

[0044] The term "subtype," as used herein, refers to a genetic variant or strain of a pathogen (e.g., a virus, bacterium, or fungus). For example, the genus Ebolavirus is a virological taxon within the family Filoviridae. Members of this genus are called Ebola viruses. Six known Ebola virus subtypes are named for the regions where they were first identified: Bundibugyo, Reston, Sudan, Tai Forest, Zaire, and Bombali. Influenza A viruses are classified into subtypes based on 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, "H7N2 virus" designates an influenza A virus subtype that has an HA7 protein and an NA2 protein. Similarly, an "H5N1" virus has an HA5 protein and an NA1 protein.

[0045] The naming of naturally occurring variant viruses in the Filoviridae family 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 that differ from the reference virus to which it is compared, such as unique antigenic properties, host range, or disease manifestations it causes. "Virus variants with simple differences in genomic sequence are not given individual strain status due to the absence of a recognizably distinct viral phenotype." Thus, a strain is a genetically stable virus variant that differs from the natural reference virus (the typical variant) in that it causes significantly different observable phenotypes of infection (such as different types of disease, infecting different types of hosts, or being transmitted by different means). "Genetically stable" means that the genomic changes associated with the phenotypic changes are largely conserved over time through natural selection. The degree of genomic sequence variation is irrelevant to the classification of a variant as a strain, since distinct phenotypes sometimes result from several mutations. "Observable phenotype" means, for example, that in comparable animal experiments, researchers can distinguish between animals infected with a reference control virus and those infected with a suspected novel strain, even without knowing which virus the animals were administered and without any information regarding the differences between the two viruses. Designating a viral variant as a strain is the responsibility of an international expert group. To date, no natural filovirus strains conforming to this definition have been reported. For example, all genetic variants described for EBOV cause similar hemorrhagic fevers in humans and experimental animals and are transmitted in the same way. None of the known EBOV genetic variants can be distinguished from other variants solely from a clinical standpoint. Indeed, its diversity appears to be limited to subtle differences in growth kinetics and in vitro plaque formation, or slight changes in disease duration in experimental animals, ultimately resulting from limited but stable genome sequence differences.This is also true for the different genetic variants of MARV, RAVV, BDBV, RESTV, and SUDV (currently there is only one isolate of TAFV and no isolates of LLOV).

[0046] According to Kuhn et al., a naturally occurring genetic filovirus variant is a naturally occurring filovirus whose genomic consensus sequence differs by ≤10% from the sequence of a reference filovirus (the prototypic virus of a particular filovirus species), but is not identical to the reference filovirus, and does not cause an observable different phenotype of disease. (Filovirus strains are genetic filovirus variants, but most genetic filovirus variants are not filovirus strains, as defined by strain definition.)

[0047] Another scheme for classifying viruses is that of the International Committee on Taxonomy of Viruses (ICTV). The system shares many features with the classification systems for cellular organisms, such as the taxon structure. However, this naming system differs in several ways from other taxonomic codes. Virus classification begins at the order level and proceeds as follows, with taxon suffixes in italics: eyes (-virales) Family (-viridae) Subfamily (-virinae) Genus(-virus) seed

[0048] Species names often take the form of [disease] viruses, especially those related to higher plants and animals.

[0049] The establishment of an order is based on the presumption that the virus families it contains most likely evolved from a common ancestor. The majority of virus families remain uncharacterized. As of 2017, nine orders, 131 families, 46 subfamilies, 803 genera, and 4,853 species of viruses have been defined by the ICTV. The orders are: Caudovirales, Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Ortervirales, Picornavirales, Bunyavirales, and Tymovirales. These orders encompass viruses with diverse host ranges. · Caudovirales are tailed dsDNA (group I) bacteriophages. · Herpesvirales contains large eukaryotic dsDNA viruses. · Ligamenvirales contains linear dsDNA (group I) Archaean viruses. · Mononegavirales includes non-segmented (negative) stranded ssRNA (group V) plant and animal viruses. · Nidovirales consists of (+)strand ssRNA (group IV) viruses that have vertebrate hosts. · Ortervirales contains single-stranded RNA and DNA viruses that replicate through a DNA intermediate (groups VI and VII). · Picornavirales contains small (+)stranded ssRNA viruses that infect a variety of plant, insect, and animal hosts. · Tymovirales contains monopartite (+)ssRNA viruses that infect plants. · Bunyavirales contains tripartite (-) ssRNA viruses (group V).

[0050] According to the ICTV, a viral species is "a monophyletic group of viruses whose properties can be distinguished from those of other species by several criteria."

[0051] The term "isolate" as used herein refers to a pure pathogen sample obtained from an infected individual. Virus-infected cells already contain a population of genomes after just one round of replication, and the virions derived from these genomes are slightly different from each other. Similarly, a sample taken from an infected individual contains a large number of virions, many of which are very slightly different. Consequently, an "isolate" refers to a population, and the "sequence" of an "isolate" is the consensus sequence of the population of genomes present in the analyzed sample. A virus isolate can be defined as an "example of a particular virus." A natural filovirus isolate is an example of a specific natural filovirus or an example of a specific genetic variant. Isolates can be identical in consensus sequence or individual sequence, or slightly different from each other.

[0052] Optionally, the one or more broadly neutralizing antigen-binding molecules comprise antibodies obtained from, or derived from, a subject exposed to a pathogen of the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0053] Optionally, the one or more broadly neutralizing antigen-binding molecules comprise antibodies obtained from, or derived from, a subject exposed to a pathogen of the same subtype or type as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0054] Optionally, the one or more broadly neutralizing antigen-binding molecules comprise antibodies obtained from, or derived from, a subject exposed to a pathogen of the same species or genus as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0055] Optionally, the one or more broadly neutralizing antigen-binding molecules comprise a non-antibody antigen-binding protein. For example, the one or more broadly neutralizing antigen-binding molecules may comprise a designed ankyrin repeat protein (DARPin), an aptamer, an anticalin, or a T cell receptor molecule.

[0056] DARPins are genetically engineered antibody-mimetic proteins that typically exhibit highly specific and high-affinity target protein binding. They are derived from natural ankyrin proteins and contain repeated structural units that form stable protein domains with large potential target interaction surfaces. Typically, DARPins contain four or five repeats, of which the first (N-capping repeat) and last (C-capping repeat) serve to provide a hydrophilic surface. DARPins correspond to the average size of natural ankyrin repeat protein domains. Proteins with fewer than three repeats (i.e., the capping repeat and one internal repeat) do not form sufficiently stable tertiary structures. The molecular weight of a DARPin depends on the total number of repeats: [Table 4]

[0057] 10 12 It is possible to generate libraries of nucleic acids encoding DARPins with random potential target-interacting residues, with a diversity of more than 100 variants. From these libraries, DARPins that bind to desired selected targets with picomolar affinity and specificity can be selected using ribosome display or phage display, which uses a signal sequence that allows cotranslational secretion. In this way, by screening a library of DARPins, it is possible to identify one or more DARPins that bind to and / or neutralize more than one subtype of pathogen. Library-based screening for identifying DARPins is described, for example, in Hartmann et al. (Molecular Therapy: Methods and Clinical Development 2018 Vol. 10: 128-143).

[0058] Optionally, the one or more antigen-binding molecules described in step (ii) of the method of the present invention comprise a broadly neutralizing antibody (or a fragment or derivative thereof that retains broadly neutralizing activity), such as a broadly neutralizing monoclonal antibody (BNmAb) (or a fragment or derivative thereof that retains broadly neutralizing activity).

[0059] Optionally, the one or more antigen-binding molecules described in step (ii) of the method of the present invention comprise antibodies obtained from, or derived from, subjects who have survived an outbreak of a pathogen of the same subtype, type, or family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0060] Optionally, the one or more antigen-binding molecules described in step (ii) of the method of the present invention comprise antibodies obtained from, or derived from, subjects who have survived an outbreak of a pathogen of the same species, genus, or family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0061] The term "outbreak," as used herein, refers to the occurrence of a disease in a defined facility (e.g., a hospital or medical center), community, geographic region, or period of time with more cases than would normally be expected. An outbreak can occur in a limited geographic area or can spread across several countries. It can last for days, weeks, or years. The number of cases indicating the existence of an outbreak varies depending on the pathogen, the size and type of the exposed population, previous experience or lack of exposure to the disease, and the duration and location of the outbreak. Thus, outbreak status correlates with the normal frequency of the disease in the same region, in the same community, and during the same season of the year. The existence of an outbreak can be established by comparing current information with historical incidence rates in a population or community during the same time of year to determine whether the number of observed cases exceeds the expected number.

[0062] Where appropriate, a pathogen outbreak can refer to the presence of more cases of disease caused by the pathogen than would normally be expected in a region (e.g., continental region) or country, or in a population or community over one or more seasons or years.

[0063] Optionally, an outbreak of a pathogen (eg, a virus) is the occurrence of more cases of disease caused by the pathogen than would normally be expected in a region (eg, a continental region) over a season.

[0064] Optionally, an outbreak of a pathogen (eg, a virus) is the occurrence of more cases of disease caused by the pathogen than would normally be expected in a population over a season.

[0065] Examples of continental regions include the African region: North Africa: Algeria; Canary Islands; Ceuta; Egypt; Libya; Madeira; Melilla; Morocco; Sudan; Tunisia; Western Sahara; East 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; West Africa: Benin; Burkina Faso; Cape Verde; Côte d'Ivoire; Gambia; Ghana; Guinea; Guinea-Bissau; Liberia; Mali; Mauritania; Niger; Nigeria; Saint Helena; Senegal; Sierra Leone; Togo; · Southern Africa: Botswana; Lesotho; Namibia; South Africa; Swaziland.

[0066] Optionally, the subject from which the antibody is obtained or derived is a human or non-human mammalian subject.

[0067] Candidate optimized antigenic pathogen polypeptides of the polypeptide library can be expressed using any suitable expression system, suitable examples include mammalian cells, or yeast, or insect, or bacterial cells.

[0068] Optionally, the candidate optimized antigenic pathogen polypeptides of the polypeptide library are expressed on the cell surface of an expression system, where cell surface expression increases the likelihood that the candidate optimized antigenic pathogen polypeptides will be correctly folded.

[0069] Optionally, the candidate optimized antigenic pathogen polypeptides are screened for binding by one or more antigen-binding molecules by flow cytometry. For example, cells expressing the candidate optimized antigenic pathogen polypeptides can be used in a flow cytometry assay.

[0070] Optionally, the candidate optimized antigenic pathogen polypeptides are screened for binding by one or more broadly neutralizing antigen-binding molecules using a first assay (e.g., flow cytometry) and for binding by one or more broadly neutralizing antigen-binding molecules using a second assay (e.g., a neutralization assay).

[0071] Optionally, the pathogen is a virus, the candidate optimized antigenic pathogen polypeptide is a candidate optimized antigenic viral polypeptide, and the pathogen peptide is a viral polypeptide.

[0072] Optionally, the polypeptide library is a viral pseudotype library comprising a plurality of different viral pseudotypes, each different viral pseudotype comprising a different candidate optimized antigenic pathogen polypeptide, e.g., a different candidate optimized antigenic viral polypeptide (e.g., a viral glycoprotein).

[0073] Optionally, in step (ii), the candidate optimized antigenic viral polypeptides are screened for binding by one or more broadly neutralizing antigen binding molecules by screening the viral pseudotypes for binding and / or neutralization by one or more antigen binding molecules.

[0074] Pseudotyping is the process of producing a virus or viral vector by combining it with foreign viral envelope proteins. The result is a pseudotyped viral particle. Pseudotyped particles lack the genetic material to produce additional viral envelope proteins and therefore cannot transmit phenotypic changes to progeny viral particles. A "pseudotype" can be defined as a hybrid viral particle containing a protein nucleocapsid ("core") that encapsulates a nucleic acid (RNA or DNA) genome, enclosed in a lipid "envelope" membrane derived from the host cell. This envelope is acquired during budding as the core exits the cell and contains 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 be derived from the virus under study. Many pseudotypes also have a foreign gene, called a "transgene," engineered into their genome. In the presence of susceptible cells, the envelope protein binds to a cellular receptor that allows entry into the cell, ultimately resulting in transgene expression. Rhabdoviruses (e.g., varicella-zoster virus, VSV) and retroviruses (e.g., lentiviruses) are widely used as cores for pseudotyping. A key feature of retroviruses is their ability to reverse transcribe their dimeric single-stranded RNA into double-stranded deoxyribonucleic acid (dsDNA) copies, which are then integrated into the cellular genome through the use of viral and cellular enzymes. In retroviral pseudotypes, this usually results in the expression of a transduction / reporter gene, the latter of which can be easily quantified. Reporter gene expression is directly correlated with the efficiency of viral envelope / receptor interaction and inversely correlated with whether individual antibody responses or antiviral agents can interfere with the natural viral entry and replication process.

[0075] Binding of viral pseudotypes to broadly neutralizing antigen-binding molecules can be measured using any suitable technique known to those skilled in the art, such as hemagglutinin inhibition (HI) assays or enzyme-linked immunosorbent assays (ELISAs). ELISA analysis of antibody binding to glycoprotein (GP) is described in Saphire et al., 2018 (Cell 174(4): 938-952) in connection with the analysis of monoclonal antibodies against Ebola virus GP.

[0076] The production of retroviral pseudotypes and their use in pseudotype neutralization 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).

[0077] Representatives of all seven genera of retroviruses have been used in pseudotyping studies, but to date, only gammaretrovirus or lentivirus pseudotypes have been widely used. Lentiviruses are a genus of the Retroviridae family and, unlike gammaretroviruses, can infect nonproliferating cells, making them amenable for gene therapy applications involving highly differentiated or quiescent cells (e.g., G0 cell cycle phase), including muscle or neurons. The most common lentiviral vector used for pseudotyping is HIV-1, although simian immunodeficiency virus (SIV) has also been used.

[0078] Generation of retroviral pseudotypes is achieved through the simultaneous introduction of a foreign envelope protein gene, a core retroviral gene, and a cloned version of a transgene (e.g., a reporter or therapeutic gene) into producer cells, usually a highly transfectable cell line such as human embryonic kidney (HEK) 293 clone 17T cells (American Type Culture Collection #CRL-11268) (Pear et al., 1993, PNAS USA 90: 8392-8396).

[0079] 1. Envelope Plasmid. The envelope gene of the study virus is cloned into an appropriate expression plasmid. The gene is 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 utilize different, usually strong, constitutive gene promoters (e.g., human cytomegalovirus (CMV) immediate-early gene), which may affect the efficiency of pseudotype generation.

[0080] 2. Retroviral gag-pol plasmid. The gag and pol genes encode a polyprotein that is then cleaved to release the structural proteins found within the core (including matrix, capsid, and nucleocapsid), and proteins involved in viral replication (protease, reverse transcriptase, and integrase) that are responsible for processing the structural proteins, converting the ssRNA viral genome to dsDNA, and ensuring integration (of the transgene) into the host cell genome. Additionally, in lentiviral gag-pol constructs, the rev gene is included. The Rev protein is involved in the transport of viral mRNA from the nucleus to the cytosol for translation.

[0081] 3. Transfer / reporter plasmids: These are genes that are stably integrated into the host cell DNA, from which they are expressed via various cis-acting transcriptional elements. The transfer plasmid contains a packaging signal upstream of the gene to ensure incorporation of the viral RNA containing the gene into the viral core during pseudotype generation.

[0082] After the cellular machinery transcribes and translates the transfected gene, the RNA dimer of the transgene (the region between the long terminal repeats; LTRs) is incorporated into the pseudotype via the packaging signal. Because the transfer plasmid is the only plasmid engineered to contain the packaging signal, no other nucleic acids are incorporated into the mature pseudotype particle. A domain at the N-terminus of Gag targets the nucleocapsid to the cell plasma membrane, into which the envelope protein is inserted. Pseudotype particles budded from the cell are enclosed in the cell membrane to form the viral envelope.

[0083] Pseudotyped viruses are released into the culture medium of producer cells. This supernatant can be titrated on target cells to measure the concentration of functional particles. They bind to cells via envelope protein-receptor interactions, followed by membrane fusion and internalization. The pseudotyped genome carrying the transgene / reporter gene is integrated into the host cell DNA and expressed there. The reporter gene expression level correlates with the level of transduction by viable particles. Because only the transgene is present in the pseudotype, viral proteins are not produced in the target cells, preventing further pseudotype production and propagation. This provides safety when working with pseudotypes compared to working with wild-type virus. Green fluorescent protein (GFP)-based pseudotypes are easily titrated using fluorescence microscopy or flow cytometry, luciferase pseudotypes by luminometry, and β-galactosidase (β-gal) pseudotypes by color reaction.

[0084] Many standard serological assays measure only antibody binding, not inhibition of virus infectivity (hemagglutinin inhibition (HI) and ELISA). Neutralization assays allow for sensitive detection of functional antibody responses. However, for highly contained viruses (e.g., Ebola), these assays are not widely applicable due to the need for high biosecurity laboratories and specially trained personnel. One way to circumvent this problem is to use retroviral and lentiviral particles pseudotyped with pathogen envelopes, such as "surrogate viruses," for use in neutralization assays. When using a pseudotyping strategy, only the viral envelope protein is required, eliminating the possibility of escape of recombinant or native viruses. These pseudotypes are replication-deficient and cannot generate replication-competent progeny.

[0085] Pseudotypes are excellent serum reagents for virus neutralization assays because the virions can contain reporter genes and have heterologous viral envelope proteins on their surface. The introduction of these reporter genes into target cells depends on the function of the viral envelope proteins, and therefore the titer of neutralizing antibodies against the envelope can be measured by reporter gene introduction and reduction of expression. PV neutralization assays have now been developed for a wide range of RNA viruses from multiple virus families (see Temperton et al., Table 1, above).

[0086] Pseudotype-based influenza neutralization assays have been shown to be highly efficient for measuring broadly neutralizing antibodies, making them ideal serological tools for studying cross-reactive responses to multiple subtypes with pandemic potential (Corti et al., 2011, Science 333 (6044): 850-856).

[0087] The production of lentiviral vectors pseudotyped with filovirus glycoproteins is described in Sinn et al., 2017 (Methods Mol Biol. 2017;1628:65-78).

[0088] An example of a suitable general method for producing viral pseudotypes is as follows:

[0089] For transfection, 5 × 10 6 24 hours after seeding HEK-293T cells, a complex containing plasmid DNA and PEI, which facilitates DNA delivery into the cells, is added. Retroviral gag-pol and reporter plasmids are co-transfected with the required envelope plasmids.

[0090] An example of a suitable neutralization assay is as follows:

[0091] 1 x 10 in a 96-well plate 5 Approximately 100 × TCID50 of pseudotyped virus, resulting in a relative light unit (RLU) output of 1 × 10, was incubated with serum dilutions for 1 hour at 37% (5% CO2) followed by 1 × 10 4 Target cells were added. These were incubated for an additional 48 hours, after which the medium was removed and replaced with a 50:50 mixture of fresh medium and luciferase reagent. Luciferase activity was detected after 2.5 minutes by reading the plate on a luminometer. For all results, background RLU (virus alone or DEnv) was estimated prior to analysis.

[0092] Saphire et al. (supra) describe three independent assays for the evaluation of mAb neutralization in connection with the analysis of monoclonal antibodies against Ebola virus GP: i) biologically contained EBOV (ΔVP30) (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 iii) Replication-competent vesicular stomatitis virus (rVSV) carrying the EBOV GP.

[0093] Neutralization of Ebola ΔVP30-RenLuc virus An Ebola virus in which the reporter gene Renilla luciferase replaces the viral transcription factor VP30 (Ebola ΔVP30-RenLuc virus) was used to complement a Vero cell line stably expressing VP30 in trans (Vero VP30), thus allowing analysis at BSL-3 (Halfmann et al., 2008). A total of 5 × 10 Ebola ΔVP30-RenLuc virus diluted in minimal essential medium containing 2% fetal bovine serum was used to infect the cells. 3 The focus-forming units are incubated with 50 μg / ml of monoclonal antibody for 3 hours at 37° C. The virus / antibody mixture is inoculated onto 9×10 cells in a 96-well plate at a multiplicity of infection (MOI) of 0.001. 3 The cells were added to Vero VP30 cells seeded the day before at 1000 cells / well and incubated at 37°C and 5% CO for 3 days. When used, guinea pig complement (Cedarlane) was added to minimal essential medium at a final concentration of 10%. The live cell luciferase substrate, EnduRen (Promega), was then incubated with the cells for 3 hours, after which luciferase levels were measured as relative light units (RLU) using a Tecan M1000 plate reader (Tecan). Assays were performed in duplicate, with known neutralizing (GP 133 / 3.16) and non-neutralizing monoclonals (VP35 5 / 69.3.2) serving as positive and negative controls, respectively. Antibodies that neutralize the luciferase signal by ≥95% are defined as strong neutralizers, 50%-94% inhibition of the luciferase signal is considered to be moderate neutralizers, and neutralizers with 49% or lower inhibition are classified as weak / non-neutralizers.

[0094] Neutralization of authentic EBOV Assays to assess neutralization of authentic EBOV are performed according to the method described by Holtsberg et al. (Holtsberg et al., 2015, J Virol. 2015; 90:266-278). Vero E6 cells are seeded at 2.5 × 10 cells per well in the inner 60 wells of a black 96-well plate. -4 Virus infection was performed 24 hours after seeding at 1000 cells / well. Antibodies were serially diluted twice in Vero growth medium (Eagle's minimum essential medium containing Earle's salts and L-glutamine, 5% fetal bovine serum (FBS), and 1% penicillin-streptomycin) to the desired final concentration (50 μg / ml), mixed with an equal amount of live EBOV, and incubated at 37°C for 1 hour with mixing every 15 minutes. The antibody / virus mixture was then added to Vero cells at an MOI of 0.2, incubated at 37°C for 1 hour, washed with PBS, and growth medium alone was added to all wells. The plates were then incubated at 37°C for an additional 48 hours. Cells were then fixed with 10% neutral buffered formalin, and the percentage of infected cells was determined by indirect immunofluorescence assay using the EBOV-specific human mAb KZ52 and goat anti-human IgG conjugated to Alexa Fluor 488 (Molecular Probes) as the secondary antibody. Images are acquired with 20 fields per well using a 20x objective on an Operetta high-content imaging system (Perkin-Elmer). Operetta images are analyzed by a custom algorithm built from the image analysis functions available in Harmony software (Perkin-Elmer). The percentage of inhibition for each antibody is determined relative to control cells incubated with medium alone. Antibodies that reduced the percentage of infected cells by >80% are classified as strong neutralizers, while antibodies that reduced infection by 50%-79% and <50% are considered moderate and weak / non-neutralizers, respectively.

[0095] Neutralization of rVSV-EBOV GP Recombinant vesicular stomatitis virus (VSV) expressing both eGFP and a recombinant surface GP in place of VSV G (rVSV-EBOV) has been previously described (Wec et al. (e.g., Wong et al., 2010, Virol. 84:163-175; Wong et al., 2016, Science; 354:350-354; Wong et al., 2010, Virol. 84:163-175). For the neutralization assay, 6.0 × 10 Vero cells were used. 4 Cells were seeded at 100 cells / well and cultured overnight at 37°C and 5% CO2 in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU / ml penicillin, and 100 μg / ml streptomycin. The next day, the virus was incubated with serial 3-fold dilutions of antibody starting at 330 nM (approximately 50 μg / ml) in serum-free EMEM for 1 hour at room temperature before infecting Vero cell layers in 96-well plates. The amount of virus used for infection was determined based on titration of virus stock to achieve a final infection of 35-50% (MOI of approximately 0.1 infectious units / cell) in control wells without antibody. Virus was incubated with cells in 50% v / v EMEM supplemented with 2% FBS, 100 IU / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO2 for 14–16 hours, after which cells were fixed and nuclei were stained with Hoescht's staining. rVSV infectivity was measured by counting EGFP-positive cells relative to the total number of cells indicated by nuclear staining using a Cellinsight CX5 automated microscope and accompanying software (Thermo Scientific). Infection levels in control wells lacking antibody were set to 100%, and infections were normalized to that value for each antibody dilution tested in triplicate. Mean values ​​were determined and the full 9-point dilution curve was used to calculate the half-maximal inhibitor concentration, IC 50 is determined using GraphPad Prism version 6. IC 50 Antibodies with a β-glucosidase activity of 5 nM or less are considered to be strong neutralizers, and antibodies with a β-glucosidase activity of 5 nM or less are considered to be strong neutralizers. <IC 50Antibodies with <50 nM and ≤50 nM are considered to be moderate and weak / non-neutralizers, respectively. The non-neutralizing fraction, an indicator of antibody potency, is also determined by using the antibody at the highest concentration tested, 330 nM, and measuring the GFP signal compared to that of untreated control cells. Fractions that reduce the signal by ≥98%, 50-98%, and less than 50% are considered to be strong, moderate, and weak / non-neutralizers, respectively.

[0096] Methods for screening polypeptide libraries are described in Bruun et al. (PLoS ONE 9(10): e109196).

[0097] Optionally, the method of the present invention further comprises the step of generating a polypeptide library.

[0098] Optionally, the polypeptide library is generated by expressing 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 that encodes a different candidate optimized antigenic pathogen polypeptide of the polypeptide library.

[0099] Optionally, different candidate optimized pathogen polypeptides are expressed in or on the surface of mammalian cells. Suitable methods are well known to those skilled in the art.

[0100] Optionally, the nucleotide sequence of each different nucleic acid of the nucleic acid library is optimized for expression of the encoded polypeptide in mammalian cells.

[0101] Optionally, 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.

[0102] Optionally, the pathogen is a virus, the candidate optimized antigenic pathogen polypeptide is a candidate optimized antigenic viral polypeptide, and the pathogen peptide is a viral polypeptide.

[0103] Optionally, the nucleic acid library is a viral pseudotype vector library, wherein each different nucleic acid in the library is part of an expression vector for producing a viral pseudotype comprising an encoded viral 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 viral polypeptide encoded by a different nucleic acid sequence in the viral pseudotype vector library.

[0104] Optionally, the viral pseudotype vector library may be prepared in a volume of at least 2, 3, 5, 10, 20, 30, 40, 50, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 Contains different members.

[0105] Optionally, the expression vector is also a vaccine vector.

[0106] Examples of vaccine vectors include viral vaccine vectors, bacterial vaccine vectors, RNA vaccine vectors, or DNA vaccine vectors.

[0107] Viral vaccine vectors use live viruses to deliver nucleic acids (e.g., DNA or RNA) into human or non-human animal cells. The nucleic acid contained in the virus encodes one or more antigens that, when expressed in 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 properties of nucleic acid vaccines with those of live attenuated vaccines. Like nucleic acid vaccines, viral vaccine vectors deliver nucleic acids to the host for the production of antigenic proteins that can be tailored to stimulate a broad range of immune responses, including antibodies. Helper T cells (CD4 + T cells), and cytotoxic T lymphocytes (CTL, CD8 + T cells) mediate immunity. Unlike nucleic acid vaccines, viral vaccine vectors also have the potential to actively invade and replicate in host cells, further activating the immune system like adjuvants, much like live attenuated vaccines. Therefore, viral vaccine vectors generally contain live attenuated viruses genetically engineered to carry nucleic acids (e.g., DNA or RNA) encoding protein antigens from unrelated organisms. Viral vaccine vectors can generally generate stronger immune responses than nucleic acid vaccines, but for some diseases, viral vectors are used in combination with other vaccine technologies in a strategy called heterologous prime-boost. In this system, one vaccine is administered as a priming step, followed by vaccination with an alternative vaccine as a booster. The heterologous prime-boost strategy aims to provide a stronger overall immune response. Viral vaccine vectors can be used as both a prime and boost vaccine as part of this strategy. Viral vaccine vectors are reviewed in Ura et al., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013;2:97-105).

[0108] Optionally, the viral vaccine vector is based on a viral delivery vector, such as a poxvirus (e.g., 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), a bacterial delivery vector (e.g., Salmonella, E. coli), an RNA expression vector, or a DNA expression vector.

[0109] Optionally, the vector is a pEVAC-based expression vector. The pEVAC expression vector is described in more detail in Example 7 below.

[0110] In other embodiments, different candidate optimized antigenic pathogen polypeptides are expressed in or on the surface of bacterial, yeast, or insect cells.

[0111] Optionally, the method of the present invention further comprises generating a nucleic acid library by synthesizing a plurality of different nucleic acids, each different nucleic acid comprising a different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide.

[0112] Optionally, the method of the present invention further comprises the steps of: i) obtaining amino acid sequences of pathogen polypeptides and / or nucleotide sequences encoding the pathogen polypeptides of 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 or encoded amino acid sequence of the pathogen polypeptide and is different from each of the obtained or encoded amino acid sequences.

[0113] Optionally, generating a plurality of different nucleotide sequences in step (ii) above includes the steps of: performing a multiple sequence alignment of the amino acid or nucleotide sequences obtained in step (i) above; identifying from the multiple sequence alignment an amino acid sequence or encoded amino acid sequence that is highly conserved among polypeptides of 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 a sequence that encodes the highly conserved amino acid sequence or encoded amino acid sequence identified from the multiple sequence alignment.

[0114] An amino acid sequence that is highly conserved or encoded among polypeptides of different pathogen isolates can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, or 800 amino acid residues in length.

[0115] Optionally, the number of amino acid sequences of pathogen polypeptides, or the number of nucleotide sequences encoding pathogen polypeptides, of different pathogen isolates may be at least 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10 6 , 10 9 , or 10 12 Typically, the more sequences used for the multiple sequence alignment, the better.

[0116] Optionally, the method of the present invention further comprises the steps of identifying an amino acid sequence or encoded amino acid sequence that is an ancestral amino acid sequence from the multiple sequence alignment; and including the sequence encoding the ancestral amino acid sequence identified from the multiple sequence alignment in one or more of the different generated nucleotide sequences.

[0117] The inclusion of one or more nucleotide sequences encoding an ancestral amino acid sequence can be advantageous because the ancestral amino acid sequence, which is highly conserved with respect to the extant amino acid sequence, is expected to be structurally and / or functionally important for the survival and / or reproduction of the pathogen. Similarly, pathogen isolates may be highly diverse (especially isolates of emerging or re-emerging pathogens, such as emerging or re-emerging RNA viruses), and the evolutionary distance between these two pathogen populations may be large, so a vaccine designed to work against a pathogen population in one patient will not work in a different patient. However, the most recent common ancestor is more closely related to each of the two pathogen populations than to each other. Thus, a vaccine designed based on a common ancestor will be more likely to be effective against a larger population of circulating strains.

[0118] Ancestral sequence reconstruction (ASR) is discussed in Randall et al. (Nat. Commun. 7:12847 doi: 10.1038 / ncomms 12847 (2016)). The authors define ASR as "the process of analyzing modern sequences in an evolutionary / phylogenetic tree context to infer ancestral sequences at specific nodes in the tree." Ancestral sequence reconstruction (ASR) is used in the study of molecular evolution. Unlike traditional evolutionary approaches to studying proteins, ASR vertically probes for statistically inferred ancestral proteins within nodes of the tree by horizontally comparing related protein homologs from the ends of different branches of the phylogenetic tree (see Figure 1). A phylogenetic tree is a branching diagram that shows the evolutionary relationships among various biological species or other entities based on 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 homologs of a protein of interest are selected, aligned in a multiple sequence alignment (MSA), and a phylogenetic tree is constructed with statistically estimated sequences at the branch nodes. These sequences are called "ancestors." The process of synthesizing the corresponding DNA, transforming it into cells, and producing the protein is called "reconstruction."

[0119] Ancestral sequences are typically calculated using maximum likelihood (ML) methods, although Bayesian methods have also been implemented. Because ancestry is inferred from phylogenies, the morphology and composition of the phylogeny play a major role in the output ASR sequence. ASR does not require the actual sequence of the ancestral protein / DNA to be recreated, but rather sequences that are likely to be similar to the sequence at the node. Maximum likelihood (ML) methods work by generating sequences where the residue at each position is predicted to be the most likely to occupy that position by the inference method used. Typically, this is a score matrix (similar to those used in BLAST or MSA) calculated from extant sequences. Alternative methods include maximum parsimony (MP), which constructs sequences based on a sequence evolution model, typically with the idea that sequences with the fewest nucleotide sequence changes represent the most efficient and most likely pathway for evolution to occur. MP is often considered the least reliable reconstruction method, perhaps because it oversimplifies evolution to an extent that is not applicable on billion-year timescales. Other methods include Bayesian methods, which include consideration of residue uncertainty. Such methods are sometimes used to complement ML, but typically result in more ambiguous sequences (i.e., sequences containing residue positions where no clear substitutions can be predicted). In such instances, several ASR sequences often result that encompass most of the ambiguity relative to each other.

[0120] The ASR method and algorithm are described in Joy et al., 2016, PLOS The following is a more detailed description based on the description in Computational Biology 12(7): DOI:10.1371 / journal.pcbi.1004763.

[0121] If necessary, the ASR should be at least 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10 6 , 10 9 , or 10 12In some cases, the more sequences used, the better.

[0122] Optionally, each of the sequences used for the multiple sequence alignment is the full-length sequence of a pathogen polypeptide of a pathogen isolate.

[0123] Any attempt at ancestry reconstruction begins with a phylogeny. Generally, a phylogeny is a tree-based hypothesis about the order in which populations (called taxa) are related by descent from a common ancestor. Observed taxa are represented by the terminal or terminal nodes of the tree, which are progressively connected by branches to their common ancestor, typically represented by a branching point in the tree called an ancestral node or junction. Ultimately, all lineages converge to the most recent common ancestor of the entire sample of taxa. In the context of ancestry reconstruction, phylogenies are often treated as if they were known quantities (Bayesian approaches being an important exception). Because there can be a vast number of phylogenies that are roughly equally valid for 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 hypothesized evolutionary model to a given phylogeny. When a model contains one or more free parameters, the overall goal is to estimate these parameters based on measured features in observed taxa (sequences) that descend from a common ancestor. Parsimony methods are an important exception to this paradigm. They are based on the empirical rule that trait-state variation is rare, without any attempt to quantify that rarity.

[0124] maximum savings method Parsimony refers to the principle of selecting the simplest of competing hypotheses. In the context of ancestry reconstruction, parsimony strives to find a distribution of ancestral states within a given tree that minimizes the total number of trait-state changes required to explain the states observed at the tip of the tree. This maximum parsimony method is one of the earliest formulated 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 trait states parsimoniously through two traversals of a rooted binary tree. The first stage is a post-order traversal that proceeds from the tip of the tree toward the root by visiting descendant (child) nodes before their parents. First, a set of possible trait states S for the i-th ancestor is determined based on the observed trait states of its descendants. iEach assignment is the intersection of the trait states of the ancestors and their descendants. If the intersection is the empty set, it is a union. The latter case means that a trait state change occurred between the ancestor and one of its two direct descendants. Each such event contributes to a cost function in an algorithm that can be used to distinguish between alternative trees based on maximum parsimony. Next, a preorder traversal of the tree is performed, proceeding from the root toward the tips. A trait state is then assigned to each descendant based on the trait state it shares with its parent. Because the root has no parent nodes, trait states may need to be chosen arbitrarily, especially if more than one possible state is reconstructed at the root. Parsimony is intuitively preferable and highly efficient, so in some cases it is still used to seed ML optimization algorithms with initial phylogenies (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 problems: 1. Variation in evolutionary rates. Fitch's method assumes that all trait-state transitions are equally likely; therefore, any transition incurs the same cost for a given tree. This assumption is often unrealistic and can limit the accuracy of such methods. For example, in nucleic acid evolution, transitions tend to occur more frequently than transversions. This assumption can be relaxed by assigning different costs to specific trait-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 "minimum evolution" heuristic underlying such methods results in the assumption that change is rare and is therefore inappropriate when change is the rule 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 between lineages. Parsimony methods implicitly assume that the same amount of evolutionary time has passed on all branches of the tree. Because of this, they do not account for variation in branch lengths within a tree, which is often used to quantify the passage of evolutionary or chronological time. This limitation makes the technique prone to estimating, for example, that a single change occurred on a very short branch, rather than multiple changes on a very long branch. This shortcoming is addressed by model-based methods (both ML and Bayesian) that estimate the stochastic process of evolution as it unfolds along each branch of the 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: Even without a statistical model underlying the method, the estimates have no clear uncertainty.

[0125] Maximum likelihood method (ML) ML methods of ancestral state reconstruction treat trait states at internal tree nodes as parameters and attempt to find parameter values ​​that maximize the probability of the data (observed trait states) given a hypothesis (a model of evolution and the phylogeny associated with the observed sequences or taxa). Some of the earliest ML approaches to ancestry reconstruction were developed in the context of gene 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 have been 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).

[0126] These approaches use the same probabilistic framework used to estimate phylogenetic trees (Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of molecular evolution. 1981;17(6):368-76). Briefly, the evolution of gene sequences is modeled by a time-reversible continuous-time Markov process. In the simplest of these, all traits undergo independent state transitions (e.g., nucleotide substitutions) at a constant rate over time. This basic model is often extended to allow for different rates at each branch of the tree. Indeed, mutation rates can also change over time (e.g., due to environmental changes); this can be modeled by allowing a rate parameter to evolve along the tree rather than increasing the number of parameters. The model defines the transition probability from state i to j along a branch of length t (in units of evolutionary time). The likelihood of a phylogeny is calculated from a nested sum of transition probabilities corresponding to the hierarchical structure of the proposed tree. At each node, the likelihood of its descendants is summed over all possible ancestral trait states at that node:

number

[0127] The problem of ancestral reconstruction is not to calculate the overall likelihood of alternative trees, but to find the combination of trait states at each ancestral node with the best marginal likelihood. Generally, there are two approaches to this problem. First, one can work upward from the descendants of the tree, progressively assigning to each ancestor the most likely trait state considering only its direct descendants. This approach is called marginal reconstruction. It is similar to a greedy algorithm, which makes locally optimal choices at each stage of an optimization problem. While this can be very efficient, it is not guaranteed to reach a global optimum for the problem. Instead, one can try to find a joint combination of ancestral trait states across the entire tree that jointly maximizes the likelihood of the data. Thus, this approach is called joint reconstruction. Because it is not as fast as marginal reconstruction, it is less likely to be trapped by a local optimum in the non-convex objective functions that modern optimization methods and heuristics are designed to avoid. In the context of ancestry reconstruction, this means that marginal reconstructions may assign trait states to direct descendants that deviate from the joint distribution of ancestral trait states that is a local optimum but far from the global optimum. Joint reconstructions are more computationally complex than marginal reconstructions. Nevertheless, efficient algorithms for joint reconstructions have been developed with time complexity that is roughly linear with the number of observed taxa or sequences.

[0128] ML-based ancestry reconstruction methods tend to provide greater accuracy than MP methods when there is variation in evolutionary rates between traits (or between sites within a genome). However, these methods are still unable to accommodate variation in evolutionary rates over time, also known as heterotachy. If the evolutionary rate of a particular trait accelerates along a phylogenetic branch, the amount of evolution that has occurred along that branch will be underestimated for a given length of the branch, assuming a constant evolutionary rate for that trait. Additionally, heterotachy can be difficult to distinguish from variation in evolutionary rates between traits.

[0129] Because ML (unlike maximum parsimony) requires researchers to specify a model of evolution, its accuracy can be affected by the use of a grossly inaccurate model (model misspecification). Furthermore, ML can only provide a single reconstruction of a trait state (often called a "point estimate"); if the likelihood surface is highly non-convex and contains multiple peaks (local optima), a single point estimate may not provide an adequate representation, and a Bayesian approach may be more appropriate.

[0130] Bayesian inference Bayesian inference uses the likelihood of observed data to update a researcher's thinking, or a prior distribution to generate a posterior distribution. In the context of ancestry reconstruction, the goal is to estimate the posterior probability of the ancestral trait state at each internal node of a given tree. Furthermore, these probabilities can be integrated over the parameters of the evolutionary model and the posterior distribution over the space of all possible trees. This is an application of Bayes' theorem:

number

[0131] One of the first implementations of a Bayesian approach to ancestral sequence reconstruction was developed by Yang and colleagues, who defined prior distributions using ML estimates of evolutionary models and trees, respectively. Their approach is thus an example of an empirical Bayes method for calculating posterior probabilities of ancestral trait states, a method 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). Regarding the formulation of Bayes' rule above, empirical Bayes methods anchor the empirical estimates of models and trees obtained from the data, effectively removing them from the posterior likelihood and the prior term in the equation. Furthermore, 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, in the denominator, the empirical distribution of site patterns in that alignment of observed nucleotide sequences (i.e., the assignment of nucleotides to tree tips) rather than exhaustively calculating P(D) for all possible values ​​of S given θ. Computationally, empirical Bayes methods are similar to ML reconstructions of ancestral states, except that rather than searching for ML assignments of states based on the respective probability distributions at each internal node, the probability distributions themselves are reported directly.

[0132] Empirical Bayesian methods for ancestry reconstruction require researchers to assume that evolutionary model parameters and trees are known without error. When the size or complexity of the data makes this an unrealistic assumption, it may be more prudent to adopt a fully hierarchical Bayesian approach and estimate a joint posterior distribution for ancestral trait states, models, and trees (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 Bayesian method for ancestry reconstruction by sampling ancestral sequences from this joint posterior distribution using Markov chain Monte Carlo (MCMC) methods. A similar approach has also been used to reconstruct the evolution of fungal species' symbiosis with algae (lichenification) (Lutzoni F, Pagel M, Reeb V. Major fungal lineages are derived from lichen symbiotic ancestors. Nature. 2001;411(6840):937-40). For example, the MCMC Metropolis-Hastings algorithm searches for a joint posterior distribution by accepting or rejecting parameter assignments based on the ratio of posterior probabilities.

[0133] Thus, the empirical Bayesian approach calculates the probability of various ancestral states for a particular tree and evolutionary model. By expressing the reconstruction of ancestral states as a set of probabilities, one can directly quantify the uncertainty in assigning any particular state to an ancestor. On the other hand, the hierarchical Bayesian approach averages these probabilities over all possible trees and evolutionary models in proportion to how similar these trees and models are given the observed data.

[0134] A fully Bayesian approach is limited to the analysis of a relatively small number of sequences or taxa because the space of all possible trees quickly becomes so vast that it becomes computationally infeasible for linkage samples to converge in a reasonable amount of time.

[0135] Pathogens, particularly emerging or re-emerging pathogens, such as emerging or re-emerging RNA viruses, evolve at extremely rapid rates, often at orders of magnitude more rapid than mammals or birds. For these organisms, ancestry reconstruction can be applied over much shorter timescales, e.g., to reconstruct global or local precursors of epidemics spanning decades rather than millions of years. It has been proposed that such reconstructed strains be compared with sequences isolated from current-day patients and used as targets for vaccine design efforts (Gaschen et al., Science. 2002;296(5577):2354-60).

[0136] According to method embodiments of the present invention, any suitable method of ARS can be used to identify an amino acid sequence that is an ancestral amino acid sequence or an encoded amino acid sequence from a multiple sequence alignment.

[0137] Optionally, identifying the ancestral amino acid sequence from the multiple sequence alignment includes performing maximum parsimony ancestral sequence reconstruction (MP-ASR).

[0138] Optionally, identifying the ancestral amino acid sequence from the multiple sequence alignment includes performing maximum likelihood ancestral sequence reconstruction (ML-ASR).

[0139] Optionally, identifying the ancestral amino acid sequence from the multiple sequence alignment includes performing Bayesian Ancestral Sequence Reconstruction (BI-ASR).

[0140] There are many software packages available that perform ancestral sequence reconstruction. See the table below (Joy et al., 2016, PLOS Computational Biology 12(7): DOI:10.1371 / journal.pcbi.1004763) provides a representative sample of the wide variety of packages that implement methods of ancestry reconstruction with different strengths and specialties. [Table 5]

[0141] Most of these software packages are designed to analyze 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 phylogenetic analysis of DNA and protein sequence alignments by ML. Ancestry reconstruction can be performed using the codeml program. HyPhy, Mesquite, and MEGA are also software packages for 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. 125-81) implements a simultaneous ML method for ancestral sequence reconstruction that can be easily adapted to reconstruct more generalized ranges of discrete ancestral trait states, such as geographic locations, by specifying customized models in its batch language (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). 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 traits using both maximum parsimony and ML methods, as well as several visualization tools for interpreting the results of ancestry 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 also a modular system, but it places more emphasis on ease of use than on analytical customization. As of version 5, MEGA allows users to reconstruct ancestral states using maximum parsimony, ML, and empirical Bayes methods.

[0142] Bayesian analysis of gene sequences can confer greater robustness against model misspecification. MrBayes (Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17(8):754-5) allows estimation of ancestral states at ancestral nodes using a fully 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 probabilistically maps phylogenetic variation (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) analyzes discrete or continuous traits in a Bayesian framework, evaluates evolutionary models, reconstructs ancestral states, and detects evolutionary correlations between pairs of traits.

[0143] Other software packages are more geared towards the analysis of qualitative and quantitative traits (phenotypes). For example, the ape package in the statistical computing environment R (Paradis E. Analysis of phylogenetics and evolution with R. New York: Springer; 2006) provides ancestral state reconstruction methods for both discrete and continuous traits through the ace function, which includes ML. Note that ace performs reconstruction by calculating scaled conditional likelihoods instead of marginal or joint likelihoods used by other ML-based ancestry reconstruction methods, which may negatively affect the accuracy of reconstruction at nodes other than the root. Phyrex implements a maximum parsimony-based algorithm for reconstructing ancestral gene expression profiles (by wrapping around the baseml function in PAML) (Rossnes R, Eidhammer I, Liberles DA. Phylogenetic reconstruction of ancestral character states for gene expression and mRNA splicing data. BMC bioinformatics. 2005;6(1):127).

[0144] Several software packages also reconstruct phylogeography. BEAST (Bayesian Evolutionary Analysis by Sampling Trees) (Bouckaert R, Heled J, Kuehnert 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 The analyses of diversification in R. Methods in Ecology and Evolution. 2012;3(6):1084-92 were performed using Mk2 (a continuous-time Markov model of binary trait 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 is an R package that provides methods for ancestral state reconstruction under the BiSSE model. Lagrange performs analyses on the evolutionary reconstruction of geographic ranges of 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 past patterns of gene flow and ancestral geographic location. 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) estimates ancestral states 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) estimates past biogeography by examining isolated geographic distributions.

[0145] Genome rearrangements provide valuable information for interspecies comparative genomics. 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 genetic marker ancestry reconstruction. 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 examine the history of gene rearrangements. Count (Csuos M. Count: evolutionary analysis of phylogenetic profiles with parsimony and likelihood. Bioinformatics. 2010;26(15):1910-2) reconstructs the evolution of gene family size. 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) analyzes the gain and loss of genetic features coded by binary traits. PARANA(Patro R, Sefer E, Malin J, Marcais G, Navlakha S, Kingsford C. Parsimonious reconstruction of network evolution. Algorithms for Molecular Biology. 2012;7(1):1) performs parsimony-based estimation of ancestral biological networks that represent gene losses and duplications.

[0146] There are also several web server-based applications that allow researchers to use ML methods for ancestral reconstruction of different trait types without the need 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 reconstructing ancestral genomes by identifying and locating 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 ML-based probabilistic reconstruction of ancestral sequences using gap trait models to reconstruct 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.

[0147] The candidate optimized antigenic pathogen polypeptides of the polypeptide library can include one or more regions of the amino acid sequence identified through the ARS. Optionally, the ancestral amino acid sequence, or each region thereof, of the candidate optimized antigenic pathogen polypeptide is at least 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues in length. Optionally, the ancestral amino acid sequence, or each region thereof, of the candidate optimized antigenic pathogen polypeptide is at most 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, or 800 amino acid residues in length.

[0148] Optionally, the candidate optimized antigenic pathogen polypeptides of the polypeptide library comprise an amino acid sequence that has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity along its entire length to the amino acid sequence of one or more pathogen polypeptides of the different isolates from which the candidate optimized antigenic pathogen polypeptides are optimized.

[0149] Optionally, the method of the present invention includes optimizing the codons of the different generated nucleotide sequences for optimal expression of the encoded candidate optimized antigenic pathogen polypeptide in an expression system. Codon optimization takes advantage of the degeneracy of the genetic code but does not change the amino acid sequence of the encoded polypeptide. Due to degeneracy, one protein can be encoded by many alternative nucleic acid sequences. Codon preference (codon usage bias) varies in each organism, which can pose challenges when expressing recombinant proteins in heterologous expression systems and can result in low and unreliable expression.

[0150] Any suitable expression system may be used. Some suitable examples are well known to those skilled in the art, including expression in mammalian, yeast, insect, or bacterial cells. Optionally, the expression system comprises mammalian cells. Optionally, the expression system comprises yeast, insect, or bacterial cells.

[0151] Codon optimization methods are well known to those skilled in the art. Codon optimization algorithms can be used to design codon-optimized nucleotide sequences that encode amino acids. Such algorithms aim to provide codon-optimized sequences that maximize the expression of a polypeptide or protein in a desired expression system. Examples of suitable codon optimization algorithms include the GeneOptimizer™ algorithm (ThermoFisher), the OptimumGene™ algorithm (GenScript), and GeneGPS® (ATUM).

[0152] If necessary, the method of the present invention also includes other sequence optimizations to maximize protein expression in desired expression systems.Such gene optimization takes into account codon usage bias and other sequence-related parameters related to gene expression, such as transcription, splicing, translation and mRNA degradation.Examples of such sequence-related parameters are shown below (parameters are categorized below as affecting transcription efficiency, translation efficiency or protein refolding, but some parameters may affect more than one of these steps): [Table 6]

[0153] Gene optimization algorithms, such as GeneOptimizer™ and OptimumGene™, take some of these parameters into account.

[0154] Genetic optimization of human protein expression in E. coli is discussed by Maertens et al. (Protein Science 2010 Vol. 19:1312-1326).

[0155] Optionally, the methods of the invention include optimizing the different nucleotide sequences for antigenicity of the encoded candidate optimized antigenic pathogen polypeptide.

[0156] Antigenicity optimization may include any of the following: (a) deletion or modification of a nucleic acid sequence encoding an amino acid sequence that is thought to inhibit the production and / or function of anti-pathogen polypeptide antibodies (e.g., deletion or modification of a mucin-like domain—see, e.g., Reynard et al., Journal of Virology, 2009, 9596-9601); (b) region swapping to recover one or more potentially lost encoded epitopes; (c) Site-specific mutations, for example, of N-linked glycosylation sites. Typically, site-specific mutations are designed to delete N-linked glycosylation sites, but there may be situations in which it is desirable to introduce additional sites, for example, to mask epitopes that elicit non-neutralizing antibodies. The ability of glycosylation to sterically block antibody binding to HA and thus provide protection against the host immune response has been demonstrated for influenza viruses. Sun et al. (Journal of Virology, 2013, 87(15):8756-8766) demonstrated that antibodies induced by viruses with multiple glycosylation sites have broader neutralizing activity than antibodies induced by viruses with fewer glycosylation sites; (d) changes to enhance stability (e.g., disulfide bond formation, reduced degradation of the encoded polypeptide by serine proteases); (e) glycan removal (improving B cell access); (f) Insertion of nucleic acid sequences, for example to insert a nucleic acid sequence encoding a desired epitope.

[0157] Antigenic optimization of the ectodomain of HIV-1 gp120 has been described by Joyce et al. (J Virol. 2013 Feb;87(4):2294-306).

[0158] Optionally, the different pathogen isolates include different pathogen isolates from outbreaks of pathogens of the same subtype as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0159] Optionally, the different pathogen isolates include different pathogen isolates from outbreaks of pathogens of different subtypes but of the same type as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0160] Optionally, the different pathogen isolates include different pathogen isolates from outbreaks of pathogens of a different type but the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0161] Optionally, the different pathogen isolates include different previous pathogen isolates of the same subtype, type, or family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0162] Optionally, the different pathogen isolates include different previous pathogen isolates of pathogens of the same species, genus, or family as the pathogen against which it is desired to induce a broadly neutralizing immune response.

[0163] Optionally, the methods of the present invention for identifying lead candidate optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against a pathogen are in vitro methods.

[0164] In accordance with the present invention, there is provided a method for identifying nucleic acid sequences encoding optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against a pathogen, comprising the steps of: i) immunizing a human or 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 the invention; ii) determining whether a broadly neutralizing immune response is induced in a human or non-human animal after immunization in step (i); and iii) if it is determined from step (ii) that a broadly neutralizing immune response is induced in a human or non-human animal, identifying the nucleic acid sequence as a nucleic acid sequence encoding an optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response against the pathogen. Also provided is a method comprising:

[0165] Optionally, whether a broadly neutralizing immune response is induced in a human or non-human animal is determined by determining whether antibodies in serum obtained from the human or non-human animal bind to more than one pathogen subtype within the same family as the pathogen to which a broadly neutralizing immune response is desired.

[0166] Optionally, whether a broadly neutralizing immune response is induced in a human or non-human animal is determined by determining whether antibodies in serum obtained from the human or non-human animal bind to more than one pathogen type within the same family as the pathogen to which a broadly neutralizing immune response is desired.

[0167] Any suitable non-human animal may be used. Optionally, the non-human animal is a mammal. Optionally, the mammal is a guinea pig or a mouse. Optionally, the non-human animal is a bird.

[0168] According to the present invention, i) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or is identical to SEQ ID NO: 1; ii) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or to SEQ ID NO:2; iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:4; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:5; v) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:7; or vi) a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:8 Also provided is an isolated nucleic acid molecule comprising the sequence or its complement.

[0169] According to the present invention, i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, or identical to SEQ ID NO: 10; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 12; or iii) a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 14. Also provided is an isolated nucleic acid molecule comprising the sequence or its complement.

[0170] According to the present invention, i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, or identical to SEQ ID NO: 19; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:21; iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:23; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:25; v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:27; vi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:29; or vii) a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:31. Also provided is an isolated nucleic acid molecule comprising the sequence or its complement.

[0171] According to the present invention, i) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:1, or is identical to the amino acid sequence encoded by SEQ ID NO:1; ii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:2, or identical to the amino acid sequence encoded by SEQ ID NO:2; iii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:4, or identical to the amino acid sequence encoded by SEQ ID NO:4; iv) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:5, or identical to the amino acid sequence encoded by SEQ ID NO:5; v) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:7, or is identical to the amino acid sequence encoded by SEQ ID NO:7; vi) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:8, or identical to the amino acid sequence encoded by SEQ ID NO:8; vii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:10, or identical to the amino acid sequence encoded by SEQ ID NO:10; viii) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:12, or is identical to the amino acid sequence encoded by SEQ ID NO:12; or ix) Further provided is an isolated polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:14, or is identical to the amino acid sequence encoded by SEQ ID NO:14.

[0172] According to the present invention, i) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, or identical to SEQ ID NO:3; ii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:6; iii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:9; iv) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11, or identical to SEQ ID NO:11; v) is at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 13; or vi) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, or identical to SEQ ID NO: 15 An isolated polypeptide comprising the amino acid sequence is also provided.

[0173] According to the present invention, i) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, or identical to SEQ ID NO: 18; ii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:20; iii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:22; iv) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:24; v) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:26; vi) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:28; or vii) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30, or identical to SEQ ID NO: 30 An isolated polypeptide comprising the amino acid sequence is also provided.

[0174] The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between sequences, or otherwise referred to as sequence identity.Sequence identity is often measured in terms of percentage identity (or similarity or homology), and the higher the percentage, the more similar the two sequences are.The homolog or variant of a given gene or protein has a relatively high degree of sequence identity when aligned using standard methods.Methods for aligning 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. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids' Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from the National Center for Biotechnology Information (NCBI, Bethesda, MD) and from several sources on the Internet, and is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0175] The sequence identity between nucleic acid sequences or amino acid sequences can be determined by comparing the alignment of sequences.If the equivalent position in the compared sequences is occupied by the same nucleotide or amino acid, the molecules are identical at that position.The score of the alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at the position shared by the compared sequences.When comparing sequences, optimal alignment may require the introduction of gaps into one or more of the sequences to take into account possible insertions and deletions in the sequence.The sequence comparison method may also use gap penalties, so that for the same number of identical molecules in the compared sequences, a sequence alignment with as few gaps as possible will achieve a higher score than an alignment with many gaps, since it reflects a higher relationship between the two compared sequences.The calculation of maximum percent identity involves producing an optimal alignment, taking gap penalties into account.

[0176] Suitable computer programs for performing sequence comparisons are widely available in the commercial and public sectors. 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; programs available at http: / / www.ebi.ac.uk / fasta), Clustal W2.0 and X2.0 (Larkin et al. al., 2007, Bioinformatics 23: 2947-2948; program available at http: / / www.ebi.ac.uk / tools / clustalw2), and the EMBOSS pairwise alignment algorithm (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 at http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using the default parameters.

[0177] For example, sequence comparison may be performed using the "needle" method of the EMBOSS pairwise alignment algorithm, which determines the optimal alignment of two sequences (including gaps) when considered over their entire length and provides a percentage identity score. The default parameters for amino acid sequence comparison ("Protein molecule" option) are: gap extension penalty: 0.5, gap opening penalty: 10.0, matrix: Blosum62.

[0178] Sequence comparison may be performed over the entire length of the reference sequence.

[0179] Also provided in accordance with the present invention are isolated nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0180] Also provided in accordance with the present invention are isolated nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:13 and a polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0181] The present invention also provides a composition comprising a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, and a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0182] The present invention also provides a composition comprising a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:13, and a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0183] The present invention also provides a combined preparation comprising (i) a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, and (ii) a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0184] The present invention also provides a combined preparation comprising (i) a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, and (ii) a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

[0185] The present invention also provides a composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0186] The present invention also provides a composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0187] The present invention also provides a fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0188] The present invention also provides a fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0189] The present invention also provides a combined preparation comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:6, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0190] The present invention also provides a combined preparation comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:13, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0191] The term "combination preparation," as used herein, refers to a "kit of parts" in the sense that the combination components (i) and (ii) defined above can be administered independently or by using different fixed combinations of distinct amounts of the combination components (i) and (ii). The components can be administered simultaneously or sequentially. When the components are administered sequentially, the interval between administrations is preferably selected so that the therapeutic effect of the combined use of the components is greater than the effect obtained by using only one of the combination components (i) and (ii).

[0192] The components of the combined preparation may be present in one combined unit dosage form, or may be present as a first unit dosage form of component (i) and a second unit dosage form of separate component (ii). The ratio of the total amount of combined component (i) to combined component (ii) administered in the combined preparation may be varied to address, for example, the needs of a patient subpopulation being treated, or the needs of a single patient, which may be due, for example, to the patient's particular disease, age, sex, or weight.

[0193] Preferably, there is at least one beneficial effect, such as an enhancement of the effect of component (i), or component (ii), or a mutual enhancement of the effects of the combination components (i) and (ii), such as a more than additive effect, additional beneficial effect, fewer side effects, less toxicity, or a combined therapeutic effect compared to the effective dosage of one or both of the combination components (i) and (ii), and very preferably a synergistic effect of the combination components (i) and (ii).

[0194] The combined preparation of the present invention can be provided as a pharmaceutical combined preparation for administration to a mammal, preferably a human. Component (i) can be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if necessary, and / or component (ii) can be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if necessary.

[0195] Further provided in accordance with the present invention is an isolated nucleic acid molecule that encodes an amino acid sequence encoded by a nucleic acid of the present invention.

[0196] The present invention further provides isolated nucleic acid molecules encoding amino acid sequences encoded by the nucleic acids of the present invention that have been codon-optimized for expression in mammalian cells.

[0197] The present invention further provides isolated nucleic acid molecules encoding the amino acid sequences encoded by the nucleic acids of the present invention that have been gene-optimized for expression in mammalian cells.

[0198] The present invention also provides an isolated nucleic acid encoding a polypeptide of the present invention.

[0199] The present invention also provides an isolated nucleic acid molecule encoding a polypeptide of the present invention, wherein the nucleic acid is codon-optimized for expression in a mammalian cell.

[0200] Also provided in accordance with the present invention is an isolated nucleic acid molecule encoding a polypeptide of the present invention, wherein the nucleic acid is gene-optimized for expression in a mammalian cell.

[0201] The present invention also provides a vector comprising the nucleic acid of the present invention.

[0202] Optionally, the vector further comprises a promoter operably linked to the nucleic acid.

[0203] Optionally, the promoter is for expression of a polypeptide encoded by the nucleic acid in a mammal.

[0204] Optionally, the promoter is for expression of the polypeptide encoded by the nucleic acid in yeast, bacterial, or insect cells.

[0205] 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 (e.g., an RNA vaccine vector or a DNA vaccine vector).

[0206] The nucleic acid molecule of the present invention can comprise a DNA or RNA molecule. In the embodiment where the nucleic acid molecule comprises an RNA molecule, it is recognized that the molecule can comprise an RNA sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to any of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 12, 14, 19, 21, 23, 25, 27, 29, or 31, or a complement thereof, in which each "T" nucleotide is replaced with "U".

[0207] For example, where an RNA vaccine vector comprising a nucleic acid of the invention is provided, it is recognized that the nucleic acid sequence of the nucleic acid of the invention is an RNA sequence and therefore can comprise, for example, an RNA nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, any of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 12, 14, 19, 21, 23, 25, 27, 29, or 31, or a complement thereof, in which each "T" nucleotide is replaced with a "U".

[0208] Also provided in accordance with the present invention is an isolated cell comprising or transfected with a vector of the present invention.

[0209] The present invention also provides viral pseudotyped particles comprising the polypeptides of the present invention.

[0210] The present invention also provides a method for producing viral pseudotyped particles, comprising transfecting a host cell with a vector comprising a nucleic acid of the present invention.

[0211] The present invention also provides a fusion protein comprising a polypeptide of the present invention.

[0212] The present invention further provides a pharmaceutical composition comprising a nucleic acid of the invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0213] The present invention also provides a pharmaceutical composition comprising a vector of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0214] The present invention also provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0215] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant to enhance the immune response in the subject to the polypeptide of the composition or the polypeptide encoded by the nucleic acid.

[0216] The present invention also provides a method for inducing an immune response against a pathogen in a subject, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0217] Optionally, the pathogen is a virus. Optionally, the virus is a member of the families Filoviridae, Arenaviridae, or Orthomyxoviridae.

[0218] The present invention also provides a method for inducing an immune response to a virus of the Filoviridae or Arenaviridae family in a subject, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0219] The present invention also provides a method for immunizing a subject against a pathogen, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0220] Optionally, the pathogen is a virus. Optionally, the virus is a member of the families Filoviridae, Arenaviridae, or Orthomyxoviridae.

[0221] The present invention further provides a method for immunizing a subject against a virus of the Filoviridae family, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0222] The present invention also provides a method for inducing an immune response to a virus of the Filoviridae family in a subject, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0223] 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 to, or identical to, 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 to, or identical to any of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, 12, or 14.

[0224] 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 to, or identical to, the 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 to, or identical to, any of SEQ ID NOs: 3, 6, 9, 11, 13, or 15.

[0225] The present invention further provides a method for immunizing a subject against a virus of the Arenaviridae family, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0226] The present invention also provides a method for inducing an immune response to a virus of the Arenaviridae family in a subject, the method comprising the step of administering to the subject a nucleic acid of the present invention, a polypeptide of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0227] 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 to, or identical to, 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 to, or identical to any of SEQ ID NOs: 19, 21, 23, 25, 27, 29, or 31.

[0228] Optionally, a 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 to, or identical to, the 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 to, or identical to, any of SEQ ID NOs: 18, 20, 22, 24, 26, 28, or 30.

[0229] Any suitable route of administration may be used. Administration methods 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, in solid forms suitable for dissolution or suspension in liquid before injection, or as emulsions. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets of the types previously described. Administration can be systemic or local.

[0230] Compositions can be administered in any suitable manner, for example, with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Preparations for parenteral administration include sterile aqueous or non-aqueous 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 (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present.

[0231] Some of the compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts 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 inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines, and substituted ethanol amines.

[0232] Administration can be achieved by single dose or multiple doses.In the context of the present disclosure, the dose administered to a subject must be sufficient to induce beneficial therapeutic response in the subject over time, or to inhibit or prevent infection.The required dose varies from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the infection to be treated, the specific composition used and its administration method.Appropriate dose can be determined by those skilled in the art by routine experimentation alone.

[0233] 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 is compatible with 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 contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any common pharmaceutical carrier, such as sterile saline solution or sesame oil, can be used. The vehicle can also contain conventional pharmaceutical auxiliary materials, such as pharmaceutically acceptable salts to adjust osmotic pressure, buffers, preservatives, etc. Other vehicles that can be used with the compositions and methods provided herein are saline and sesame oil.

[0234] In some embodiments, the composition comprises a pharmaceutically acceptable carrier and / or an adjuvant. For example, the adjuvant can be alum, Freund's complete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide).

[0235] Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. See Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15 th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compositions, such as one or more influenza vaccines, and additional agents.

[0236] Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, buffered salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0237] Optionally, the compositions of the invention are administered intramuscularly.

[0238] Optionally, the composition is administered intramuscularly, intradermally, subcutaneously by needle, or by gene gun or electroporation.

[0239] The present invention also provides a nucleic acid expression vector that includes a multiple cloning site containing KpnI and NotI endonuclease sites.

[0240] Optionally, the multiple cloning site comprises the nucleic acid sequence of SEQ ID NO:16.

[0241] Optionally, the nucleic acid expression vector is a nucleic acid expression vector and a viral pseudotype vector.

[0242] Optionally, the nucleic acid expression vector is a vaccine vector.

[0243] Optionally, the nucleic acid expression vector comprises, from 5' to 3' direction: a promoter; a splice donor site (SD); a splice acceptor site (SA); and a terminator signal, with a multiple cloning site located between the splice acceptor site and the terminator signal.

[0244] Optionally, the promoter comprises the CMV immediate early 1 enhancer / promoter (CMV-IE-E / P) and / or the terminator signal comprises the terminator signal of the bovine growth hormone gene (Tbgh) lacking a KpnI restriction endonuclease site.

[0245] Optionally, the nucleic acid expression vector further comprises an origin of replication and a 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.

[0246] Optionally, the nucleic acid expression vector comprises the nucleic acid sequence of SEQ ID NO: 17 (pEVAC).

[0247] The polypeptide of the present invention can comprise one or more conservative amino acid substitutions.Conservative amino acid substitutions are those substitutions that, when made, will least interfere with the properties of the original protein, that is, will preserve the structure and particularly function of the protein, and will not be significantly changed by such substitutions.Examples of conservative substitutions are shown below: [Table 7]

[0248] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains.

[0249] In general, the substitutions expected to produce the greatest changes in protein properties will be non-conservative changes, such as (a) a hydrophilic residue, e.g., seryl or threonyl, substituting (or being substituted by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline substituting (or being substituted by) any other residue; (c) a residue having an electronegative side chain, e.g., lysyl, arginyl, or histidyl, substituting (or being substituted by) a residue having an electronegative side chain, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, substituting (or being substituted by) a residue having no side chain, e.g., glycine.

[0250] In certain embodiments of the present invention, sequence alignment and ancestral sequence reconstruction (ASR) can be used to identify highly conserved immune targets that a pathogen cannot change and that will inevitably be present in future pandemics of that virus family, even the most variable RNA viruses. Synthetic gene technology can be used to produce computer-generated viral genes that are highly expressed and can be easily cloned into expression vectors, such as the pEVAC vector (which has proven to be a highly versatile expression vector for generating viral pseudotypes and for direct DNA vaccination of animals and / or humans).

[0251] The pEVAC vector can be used to generate large gene panels for rapid generation of viral pseudotypes. This allows for probing of libraries of viral pseudotypes, each with its own unique viral protein, with a large panel of monoclonal antibodies. This process ensures that inserts generate conformationally correct viral surface proteins to present the most accessible targets for the virus's Achilles' heel. Narrowing the candidate selection by pseudotyping and mAb binding provides a shortlist of top candidates for testing by vaccination. This can be done in guinea pigs, where a streamlined process of pEVAC-vaccine insert delivery is performed. If necessary, this allows for shuttle of vaccine inserts from the DNA pEVAC vector to various viral vectors based on advanced, designed, convenient cloning sites. Because chimpanzee adenovectors (ChAds) were widely used in Phase I to evaluate most Ebola virus vaccine candidates for the West African outbreak, we chose to use the same vector for direct comparison in humans. For screening in guinea pigs, we used DNA priming with the pEVAC-vaccine insert followed by the ChAd-vaccine insert.

[0252] In a particular embodiment of the present invention:

[0253] 1) High-throughput "deep" sequencing technologies provide viral variation data from current and past pandemics. Analysis of this data can identify structurally highly conserved regions that preserve known B- and T-cell epitopes, which can be used as a basis for designing optimal vaccine inserts.

[0254] 2) Human monoclonal antibody (mAb) technology allows the generation of antiviral mAbs against vaccine targets, e.g., viral envelope proteins, and identifies epitope-rich regions targeted by broadly neutralizing monoclonal antibodies (BNmAbs).

[0255] 3) Optimal gene design and synthesis incorporates digitally modeled conserved scaffolds of the genes identified in (1) to include the broadest NmAb epitopes in these scaffolds (BN epitopes may not be optimally presented on naturally conserved GPs).

[0256] 4) Downstream information of convenient cloning sites matching the requirements of vaccine and pseudotype vectors can be taken into consideration during the design and synthesis of RNA and codon-optimized synthetic genes as vaccine inserts, allowing rapid and highly efficient cloning and shuttling into different screening (i.e., lentiviral pseudotype; PV) and vaccine (i.e., MVA, ChAd, VSV, DNA, etc.) vectors.

[0257] 5) Viral pseudotypes (lentiviruses) generated from digitally designed inserts will be screened for functionality in vitro via transduction and infection studies. In addition, neutralization assays will be performed using a panel of BNmAbs and patient sera to ensure that known epitopes are preserved.

[0258] 6) By narrowing the selection to the best class of vaccine inserts, several synthetic vaccine inserts are validated for immunogenicity in guinea pigs using rapid DNA priming (and, if necessary, adenoviral boosting), a method that produces highly reproducible titers. In vivo screening will identify which are most immunogenic and produce the greatest breadth of neutralization.

[0259] The central role of viral glycoproteins in cell attachment, fusion, and uncoating makes them an important antigenic target for viral vaccines and monoclonal antibody therapy, pioneered during the West African Ebola outbreak. Analysis of EBOV GP sequences across species demonstrated high levels of diversity at the nucleotide and amino acid levels (only approximately 60–65% nt identity). Current conventional filovirus vaccine approaches require polyvalent GP-targeted vaccines, encoding species-specific GPs that are more conserved (approximately 97–98% identity in the GP nucleotide sequence). While vaccines using older strains of EBOV (rVSV.ZEBOV = Kikwit) have been shown to provide cross-protection (Henao-Restrepo AM, Lancet 2015), there are concerns that this may limit their efficacy against future outbreaks of other diverse, highly pathogenic filoviruses.

[0260] The inventors can achieve dramatic improvements in vaccine efficacy against novel viral variants based on sequence data (including, optionally, e.g., pandemic sequence data) and generate synthetic, optimized vaccine inserts to provide the broadest possible vaccine protection against future pandemics of variable RNA viruses. In certain embodiments, the inventors' novel vaccine technology: (1) Pandemic pathogen sequence (2) Broadly antiviral neutralizing monoclonal antibodies (BNmAbs) derived from pandemic survivors; and (3) computational modeling methodology. (4) Synthetic gene technology and antigen display technology (5) High-throughput virus binding and neutralization screening (6) In vivo immune selection and vaccine efficacy readout Combine the two.

[0261] The final product is a novel immunogen used to elicit the broadest spectrum of protective immune responses. We provide proof of concept that a next-generation single vaccine insert indeed induces a broad neutralization profile against the Ebolavirus genus (Zaire, Sudan, and Bundibugyo) and further targets the more distantly related filovirus, Marburg virus.

[0262] Embodiments of the present invention will now be described, by way of example only, in the following examples and with reference to the accompanying drawings, in which: The present invention provides, for example, the following items. (Item 1) 1. A method for identifying lead candidate optimized antigenic pathogen polypeptides capable of inducing a broadly neutralizing immune response against a pathogen, comprising: i) providing a polypeptide library comprising a plurality of different candidate optimized antigenic pathogen polypeptides, wherein the amino acid sequence of each different candidate is optimized from a plurality of different amino acid sequences of pathogen polypeptides, wherein each different amino acid sequence of the pathogen polypeptides is different from each different amino acid sequence of the pathogen polypeptides, and each different amino acid sequence of the pathogen polypeptides comprises the amino acid sequence of a polypeptide of a different isolate, each different isolate being an isolate of a pathogen of the same family as a pathogen against 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 capable of binding to and / or neutralizing pathogens of the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response; and iii) identifying the candidate optimized antigenic pathogen polypeptide bound by one or more of the antigen binding molecules in step (ii) as a lead candidate optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response against said pathogen. A method comprising: (Item 2) Item 1. The method of item 1, wherein the one or more broadly neutralizing antigen-binding molecules comprise antibodies obtained from, or derived from, a subject exposed to a pathogen of the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response. (Item 3) 3. The method of item 1 or 2, wherein the one or more broadly neutralizing antigen-binding molecules comprise a non-antibody antigen-binding protein. (Item 4) 4. The method of claim 3, wherein the one or more broadly neutralizing antigen-binding molecules comprise a designed ankyrin repeat protein (DARPin), an anticalin, an aptamer, or a T cell receptor molecule. (Item 5) 10. The method of any of the preceding items, wherein the candidate optimized antigenic pathogen polypeptides of the polypeptide library are expressed in or on the surface of mammalian cells. (Item 6) 5. The method of any of items 1 to 4, wherein the candidate optimized antigenic pathogen polypeptides of the polypeptide library are expressed in or on the surface of bacterial, yeast, or insect cells. (Item 7) Item 11. The method of any preceding item, wherein the pathogen is a virus, the candidate optimized antigenic pathogen polypeptide is a candidate optimized antigenic viral polypeptide, and the pathogen peptide is a viral polypeptide. (Item 8) 8. The method of 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 viral polypeptide. (Item 9) In step (ii), the candidate optimized antigenic viral 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. The method of claim 8. (Item 10) 8. The method of any of items 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 cytometry assay. (Item 11) 10. The method of any preceding item, further comprising generating said polypeptide library. (Item 12) 12. The method of 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. (Item 13) 13. The method of claim 12, wherein the different candidate optimized pathogen polypeptides are expressed in or on the surface of mammalian cells. (Item 14) 14. The method of claim 12 or 13, wherein the nucleotide sequence of each different nucleic acid of the nucleic acid library is codon-optimized and optionally gene-optimized for expression of the encoded polypeptide in a mammalian cell. (Item 15) 15. The method of any of items 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. (Item 16) 16. The method of any of items 12 to 15, wherein the pathogen is a virus, the candidate optimized antigenic pathogen polypeptide is a candidate optimized antigenic viral polypeptide, and the pathogen peptide is a viral polypeptide. (Item 17) 17. The method of claim 16, wherein the nucleic acid library is a viral pseudotype vector library, each different nucleic acid in the library is part of an expression vector for producing a viral pseudotype comprising the encoded viral 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, the viral pseudotype library comprising a plurality of different viral pseudotypes, each different viral pseudotype comprising a different candidate optimized viral polypeptide encoded by a different nucleic acid sequence in the viral pseudotype vector library. (Item 18) 18. The method of any of items 15 to 17, wherein the expression vector is also a vaccine vector. (Item 19) 19. The method of claim 18, wherein the vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector. (Item 20) 20. The method of item 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. (Item 21) 21. The method of any of items 15 to 20, wherein the vector is a pEVAC-based expression vector. (Item 22) 13. The method of claim 12, wherein the different candidate optimized antigenic pathogen polypeptides are expressed in or on the surface of bacterial, yeast, or insect cells. (Item 23) 23. The method of any of items 12 to 22, further comprising generating the nucleic acid library by synthesizing a plurality of different nucleic acids, each different nucleic acid comprising a different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide. (Item 24) i) obtaining the amino acid sequences of said pathogen polypeptides and / or the nucleotide sequences encoding said pathogen polypeptides of said different pathogen isolates; and ii) generating a plurality of different nucleotide sequences, each different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide, the encoded amino acid sequence of each different candidate optimized antigenic pathogen polypeptide being optimized from the obtained or encoded amino acid sequence of the pathogen polypeptide and different from each of the obtained or encoded amino acid sequences. 24. The method of claim 23, further comprising: (Item 25) The generation of the plurality of different nucleotide sequences in step (ii) of Item 24 is performing multiple sequence alignment of the amino acid or nucleotide sequences obtained in step (i) of item 24; identifying highly conserved or encoded amino acid sequences among the polypeptides of the different pathogen isolates from the multiple sequence alignment; and 25. The method of claim 24, comprising generating a plurality of different nucleotide sequences, each different nucleotide sequence encoding a different candidate optimized antigenic pathogen polypeptide, and one or more of the different nucleotide sequences comprising a sequence encoding a highly conserved amino acid sequence or encoded amino acid sequence identified from the multiple sequence alignment. (Item 26) Identifying an amino acid sequence or encoded amino acid sequence that is an ancestral amino acid sequence from the multiple sequence alignment; and including a sequence encoding the ancestral amino acid sequence identified from said multiple sequence alignment in one or more of said different generated nucleotide sequences. 26. The method of claim 25, further comprising: (Item 27) 27. The method of any of items 24 to 26, comprising codon-optimizing, optionally codon-optimizing, the different generated nucleotide sequences for optimal expression of the encoded candidate optimized antigenic pathogen polypeptide in an expression system. (Item 28) 28. The method of item 27, wherein the expression system comprises mammalian cells. (Item 29) 28. The method of claim 27, wherein the expression system comprises yeast, bacterial, or insect cells. (Item 30) 30. The method of any of items 24 to 29, comprising optimizing the different nucleotide sequences for antigenicity of the encoded candidate optimized antigenic pathogen polypeptide. (Item 31) The antigenicity optimization comprises: deletion or modification of a nucleic acid sequence encoding an amino acid sequence that inhibits the production and / or function of an anti-pathogen polypeptide antibody (e.g., deletion or modification of a mucin-like domain); region swapping to recover one or more potentially lost encoded epitopes; For example, site-directed mutagenesis of N-linked glycosylation sites; changes to enhance stability (e.g., disulfide bond formation, reducing degradation of the encoded polypeptide by serine proteases); Glycan removal; Insertion of nucleic acid sequences, e.g., to insert nucleic acid sequences encoding desired epitopes. Item 31. The method according to Item 30, comprising any one of the following: (Item 32) The method of any of the preceding items, wherein the one or more broadly neutralizing antigen-binding molecules described in step (ii) of item 1 comprise a broadly neutralizing antibody, preferably a broadly neutralizing monoclonal antibody (BNmAb). (Item 33) The method according to any of the preceding items, wherein the one or more antigen-binding molecules described in step (ii) of item 1 comprise antibodies obtained from, or antibodies derived from, subjects who survived an outbreak of a pathogen of the same family, and optionally the same subtype or type, as the pathogen against which it is desired to induce a broadly neutralizing immune response. (Item 34) 34. The method of claim 33, wherein the subject from which the antibody is obtained or derived is a human or non-human mammalian subject. (Item 35) 35. The method of item 33 or 34, wherein the one or more antigen-binding molecules comprise a broadly neutralizing monoclonal antibody (BNmAb). (Item 36) 10. The method of any of the preceding items, wherein the different pathogen isolates comprise different pathogen isolates from outbreaks of a pathogen of the same subtype as the pathogen against which it is desired to induce a broadly neutralizing immune response. (Item 37) 10. The method of any of the preceding items, wherein the different pathogen isolates comprise different pathogen isolates from an outbreak of a pathogen of a different subtype but of the same type as the pathogen to which it is desired to induce a broadly neutralizing immune response. (Item 38) 10. The method of any of the preceding items, wherein the different pathogen isolates comprise different pathogen isolates from outbreaks of pathogens of a different group but the same family as the pathogen against which it is desired to induce a broadly neutralizing immune response. (Item 39) The method of any of the preceding items, wherein the different pathogen isolates comprise different previous pathogen isolates of the same subtype, type, or family as the pathogen against which it is desired to induce a broadly neutralizing immune response. (Item 40) The method of any of the preceding items, wherein each candidate optimized antigenic pathogen polypeptide comprises at least 20 amino acid residues. (Item 41) Item 11. The method of any preceding item, wherein the pathogen is a virus. (Item 42) 42. The method of claim 41, wherein the virus is an RNA virus. (Item 43) 43. The method of item 41 or 42, wherein the virus is an emerging or re-emerging RNA virus. (Item 44) 44. The method of any of items 41 to 43, wherein the virus is a filovirus, arenavirus, or orthomyxovirus. (Item 45) 44. The method of any of items 41 to 43, wherein the virus is Ebola virus or Marburg virus. (Item 46) 44. The method of any of items 41 to 43, wherein the virus is Lassa virus. (Item 47) Item 11. The method of any preceding item, wherein the pathogen polypeptide is a viral glycoprotein. (Item 48) 10. The method according to any of the preceding items, which is an in vitro method. (Item 49) 1. A method for identifying a nucleic acid sequence encoding an optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response against a pathogen, comprising: i) immunizing a human or non-human animal with a nucleic acid comprising a nucleic acid sequence encoding a lead candidate optimized antigenic pathogen polypeptide identified by the method described in any of the preceding items; ii) determining whether a broadly neutralizing immune response is induced in said human or said non-human animal after immunization in step (i); and iii) if it is determined from step (ii) that a broadly neutralizing immune response is induced in said human or said non-human animal, identifying said nucleic acid sequence as a nucleic acid sequence encoding an optimized antigenic pathogen polypeptide capable of inducing a broadly neutralizing immune response against said pathogen. A method comprising: (Item 50) 50. The method of claim 49, comprising determining whether a broadly neutralizing immune response is induced in the human or non-human animal by determining whether antibodies in serum obtained from the human or non-human animal bind to and / or neutralize more than one pathogen subtype. (Item 51) 51. The method of item 49 or 50, wherein the non-human animal is a mammal. (Item 52) 52. The method of claim 51, wherein the mammal is a guinea pig or a mouse. (Item 53) 51. The method of item 49 or 50, wherein the non-human animal is a bird. (Item 54) i) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or is identical to SEQ ID NO: 1; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:2; iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:4; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:5; v) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:7; or vi) is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8, or is identical to SEQ ID NO:8 An isolated nucleic acid molecule comprising a nucleic acid sequence, or its complement. (Item 55) i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, or identical to SEQ ID NO: 10; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 12; or iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 14. An isolated nucleic acid molecule comprising a nucleic acid sequence, or its complement. (Item 56) i) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, or identical to SEQ ID NO: 19; ii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:21; iii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:23; iv) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:25; v) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:27; vi) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:29; or vii) at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or identical to SEQ ID NO:31. An isolated nucleic acid molecule comprising a nucleic acid sequence, or its complement. (Item 57) i) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:1, or is identical to the amino acid sequence encoded by SEQ ID NO:1; ii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:2, or identical to the amino acid sequence encoded by SEQ ID NO:2; iii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:4, or identical to the amino acid sequence encoded by SEQ ID NO:4; iv) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:5, or identical to the amino acid sequence encoded by SEQ ID NO:5; v) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:7, or is identical to the amino acid sequence encoded by SEQ ID NO:7; vi) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:8, or identical to the amino acid sequence encoded by SEQ ID NO:8; vii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:10, or identical to the amino acid sequence encoded by SEQ ID NO:10; viii) at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO:12, or identical to the amino acid sequence encoded by SEQ ID NO:12; ix) is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by SEQ ID NO: 14, or is identical to the amino acid sequence encoded by SEQ ID NO: 14 An isolated polypeptide comprising an amino acid sequence. (Item 58) i) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, or identical to SEQ ID NO:3; ii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:6; iii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:9; iv) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11, or identical to SEQ ID NO:11; v) is at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO: 13; or vi) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, or identical to SEQ ID NO: 15 An isolated polypeptide comprising an amino acid sequence. (Item 59) i) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, or identical to SEQ ID NO: 18; ii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:20; iii) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:22; iv) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:24; v) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:26; vi) at least 95%, 96%, 97%, 98%, or 99% identical to, or identical to, SEQ ID NO:28; or vii) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30, or identical to SEQ ID NO: 30 An isolated polypeptide comprising an amino acid sequence. (Item 60) 57. An isolated nucleic acid encoding the amino acid sequence encoded by the nucleic acid of items 54, 55, or 56, which has been codon-optimized, and optionally gene-optimized, for expression in mammalian cells. (Item 61) 62. An isolated nucleic acid encoding a polypeptide according to claim 57, 58, or 59, which has been codon-optimized, and optionally gene-optimized, for expression in mammalian cells. 62. A vector comprising the nucleic acid of items 54, 55, 56, 60, or 61. (Item 63) 63. The vector of item 62, further comprising a promoter operably linked to the nucleic acid. (Item 64) 64. The vector of item 63, wherein the promoter is for expression of the polypeptide encoded by the nucleic acid in mammalian cells. (Item 65) 64. The vector of item 63, wherein the promoter is for expression of the polypeptide encoded by the nucleic acid in yeast or insect cells. (Item 66) 66. The vector according to any of items 62 to 65, which is a vaccine vector. (Item 67) 67. The vector of item 66, which is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector. (Item 68) 66. An isolated cell comprising the vector of any of items 62 to 65. (Item 69) 60. A pseudotyped viral particle comprising the polypeptide of items 57, 58, or 59. (Item 70) 70. A method for producing the pseudotyped virus particle of item 69, comprising transfecting a host cell with the vector of any one of items 62 to 64. (Item 71) 60. A fusion protein comprising the polypeptide of items 57, 58, or 59. (Item 72) 62. A pharmaceutical composition comprising the nucleic acid of item 54, 55, 56, 60, or 61, and a pharmaceutically acceptable carrier, excipient, or diluent. (Item 73) 68. A pharmaceutical composition comprising a vector according to any of items 62 to 64, 66 or 67, and a pharmaceutically acceptable carrier, excipient or diluent. (Item 74) 60. A pharmaceutical composition comprising the polypeptide of item 57, 58, or 59 and a pharmaceutically acceptable carrier, excipient, or diluent. (Item 75) 75. The pharmaceutical composition of any of items 72 to 74, further comprising an adjuvant for enhancing the immune response in a subject to the polypeptide of the composition or the polypeptide encoded by the nucleic acid. (Item 76) 72. A method for inducing an immune response against a virus of the Filoviridae family in a subject, the method comprising administering to the subject the nucleic acid of any of items 54, 55, 60, or 61, the polypeptide of item 57 or 58, the vector of any of items 62 to 64, 66, or 67, or the pharmaceutical composition of any of items 72 to 75. (Item 77) 75. A method of immunizing a subject against a virus of the Filoviridae family, comprising administering to the subject a nucleic acid according to any of items 54, 55, 60, or 61, a polypeptide according to item 57 or 58, a vector according to any of items 62 to 64, 66, or 67, or a pharmaceutical composition according to any of items 72 to 75. (Item 78) 76. A method for inducing an immune response against a virus of the Arenaviridae family in a subject, the method comprising administering to the subject the nucleic acid of any of items 56, 60, or 61, the polypeptide of item 59, the vector of any of items 62 to 64, 66, or 67, or the pharmaceutical composition of any of items 72 to 75. (Item 79) 76. A method for immunizing a subject against a virus of the Arenaviridae family, comprising administering to the subject the nucleic acid of any of items 56, 60, or 61, the polypeptide of item 59, the vector of any of items 62 to 64, 66, or 67, or the pharmaceutical composition of any of items 72 to 75. (Item 80) 80. The method of any of items 76 to 79, wherein the composition is administered intramuscularly. (Item 81) A nucleic acid expression vector containing a multiple cloning site containing KpnI and NotI endonuclease sites. (Item 82) 82. The vector of Item 81, wherein the multiple cloning site comprises the nucleic acid sequence of SEQ ID NO: 16. (Item 83) 83. The vector according to item 81 or 82, which is an expression vector and a viral pseudotype vector. (Item 84) 84. The vector according to any of items 81 to 83, which is a vaccine vector. (Item 85) 85. The vector according to any of items 81 to 84, comprising, in 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. (Item 86) 86. The vector of item 85, wherein the promoter comprises a CMV immediate-early 1 enhancer / promoter and / or the terminator signal comprises the terminator signal of the bovine growth hormone gene lacking a KpnI restriction endonuclease site. (Item 87) 87. The vector of any of items 81 to 86, further comprising an origin of replication and a nucleic acid encoding resistance to an antibiotic. (Item 88) 88. The vector of item 87, wherein the origin of replication comprises a pUC-plasmid origin of replication and / or the nucleic acid encodes resistance to kanamycin. (Item 89) 89. The vector according to any of items 81 to 88, comprising the nucleic acid sequence of SEQ ID NO: 17. (Item 90) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 91) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:13 and a polypeptide comprising the amino acid sequence of SEQ ID NO:15. (Item 92) A composition comprising a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, and a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 93) A composition comprising a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, and a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15. (Item 94) A combined preparation comprising (i) a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6, and (ii) a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 95) A combined preparation comprising: (i) a first nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13; and (ii) a second nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15. (Item 96) A composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 97) A composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:13, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:15. (Item 98) A fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:6 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 99) A fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:13 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:15. (Item 100) A combined preparation comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:6, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO:9. (Item 101) A combined preparation comprising (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 15. (Item 102) 76. A nucleic acid according to any of items 54, 55, 60 or 61, a polypeptide according to item 57 or 58, a vector according to any of items 62 to 64, 66 or 67, or a pharmaceutical composition according to any of items 72 to 75 for use as a medicament. (Item 103) 76. The nucleic acid according to any of items 54, 55, 60 or 61, the polypeptide according to item 57 or 58, the vector according to any of items 62 to 64, 66 or 67, or the pharmaceutical composition according to any of items 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 family Filoviridae. (Item 104) Use of a nucleic acid according to any of items 54, 55, 60 or 61, a polypeptide according to item 57 or 58, a vector according to any of items 62 to 64, 66 or 67, or a pharmaceutical composition according to any of items 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 family Filoviridae. (Item 105) 76. A nucleic acid according to any one of items 56, 60, or 61, a polypeptide according to item 59, a vector according to any one of items 62 to 64, 66, or 67, or a pharmaceutical composition according to any one of items 72 to 75, for use as a medicament. (Item 106) 76. The nucleic acid according to any of items 56, 60, or 61, the polypeptide according to item 59, the vector according to any of items 62 to 64, 66, or 67, or the pharmaceutical composition according to any of items 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. (Item 107) 75. Use of a nucleic acid according to any of items 56, 60, or 61, a polypeptide according to item 59, a vector according to any of items 62 to 64, 66, or 67, or a pharmaceutical composition according to any of items 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. (Item 108) 97. The nucleic acid according to item 90 or 91, the composition according to item 92, 93, 96 or 97, the combined preparation according to item 94, 95, 100 or 101, or the fusion protein according to item 98 or 99 for use as a medicament. (Item 109) 97. The nucleic acid according to item 90 or 91, the composition according to item 92, 93, 96 or 97, the combined preparation according to item 94, 95, 100 or 101, or the fusion protein according to item 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 family Filoviridae. (Item 110) Use of a nucleic acid according to item 90 or 91, a composition according to item 92, 93, 96 or 97, a combined preparation according to item 94, 95, 100 or 101, or a fusion protein according to item 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. [Brief explanation of the drawings]

[0263] [Figure 1] FIG. 1 shows an illustration of the phylogenetic tree and its relationship to the ancestral sequence reconstruction.

[0264] [Figure 2] Figure 2 shows a phylogenetic tree comparing Ebola virus and Marburg virus. Numbers indicate the percent confidence of the branches.

[0265] [Figure 3] FIG. 3 shows the plasmid map of pEVAC.

[0266] [Figure 4] Figure 4 shows the results of the challenge study in the Ebola challenge model, which was lethal to non-vaccinated guinea pigs (group 1, bottom line), while all vaccinated guinea pigs (group 2, top line) were protected (left) and continued to gain weight (right).

[0267] [Figure 5] Figure 5 shows the results of a pseudotyped virus neutralization assay demonstrating the strength of neutralizing antibody responses to target antigens expressed on the surface of pseudotyped viruses representing all Ebola virus species and Marburg virus. The strength of neutralization is shown as a heat map, with red (darkest shading) indicating very strong neutralization, decreasing as we move from orange to yellow (lighter shading), and white indicating no neutralization / equivalent to the negative control value. T2-4 and T2-6 are nucleic acid vaccines encoding lead candidate optimized antigenic Ebola polypeptides in combination with the T2-11 Marburg candidate, currently undergoing preclinical testing with serum samples collected from immunized guinea pigs.

[0268] [Figure 6] Figure 6 shows the results of a study to determine the efficacy of nucleic acid vaccines encoding different lead candidate optimized antigenic pathogenic polypeptides identified using an embodiment of the method of the present invention. Antibody binding was measured by incubating two cell populations bearing two different group 1 influenza A glycoproteins (H1 pandemic and seasonal) on their surface with pooled mouse serum. Any bound antibodies were then detected with a secondary antibody, and results were recorded using a flow cytometer. Binding was significantly increased before and after vaccination with all constructs, but not after vaccination with PBS (control). Overall, the vaccine candidates outperformed those from COBRA in both cases (*).

[0269] [Figure 7] Figure 7 shows the results of a study determining the binding of cells expressing two different group 1 influenza A glycoproteins (seasonal H1N1, and pandemic-origin H1N1) on their cell surface by mouse sera from animals immunized with either COBRA or DIOS HA gene antigens.

[0270] [Figure 8] Figure 8 shows the results of cross-HA group binding (left panel) and pseudotype neutralization (right) of H7N9 (A / Shanghai 2 / 2013) by sera from DIOS or COBRA DNA-immunized mice. In the right panel, the top curve is for CR9114, the two curves descending from the two lowest starting points on the left of the graph are for H1N1, and the remaining two curves are for H1N1pdm. DETAILED DESCRIPTION OF THE INVENTION

[0271] Examples of non-optimized Ebola and Marburg virus ancestral nucleic acid sequences (i.e., sequences that have not been codon-optimized or gene-optimized) are provided below, along with gene-optimized nucleic acid sequences that encode candidate antigenic pathogen polypeptides.

[0272] methodology For a given viral species, candidate primary sequences are downloaded, e.g., from GenBank (and any other available sources, e.g., outbreak data), and filtered to exclude identical sequences, sequences outside the range of the protein of interest, and sequences with many ambiguous nucleotides. A multiple sequence alignment of the filtered sequences is generated (typically using MAFFT) and manually checked to ensure that the 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: outgroup sequences, midpoint rooting, center-of-the-tree, or a tree that maximizes the association between root-to-tip distance and sampling time. Ancestral sequences are generated using HyPhy assuming MG94 with an F3x4 model of codon substitution to ensure that known epitopes are preserved. A phylogenetic tree with both primary and ancestral sequences is generated using IQTREE to check the placement of ancestral strains. The ancestral sequence is then modified in several ways: by region deletion (e.g., removal of the mucin-like domain); region swapping (to restore epitopes that may be lost); and mutation of specific sites (e.g., in the fusion domain of filoviruses), including editing of N-linked glycosylation sites and introducing mutations to enhance stability. [Example]

[0273] Example 1 Ebola Sudan Ancestors (T2-4) Non-optimized (SEQ ID NO: 1) [ka] [ka] Gene optimization (SEQ ID NO: 2) [ka] Amino acid sequence encoded by the non-optimized and gene-optimized sequence (SEQ ID NO: 3): [ka]

[0274] Example 2 Comprehensive ancestor of Ebola virus (T2-6) Non-optimized (SEQ ID NO: 4) [ka] Gene optimization (SEQ ID NO: 5) [ka] Amino acid sequence encoded by the non-optimized and gene-optimized sequence (SEQ ID NO: 6): [ka]

[0275] Example 3 Marburg virus ancestor (T2-11) Non-optimized (SEQ ID NO: 7) [ka] Gene optimization (SEQ ID NO: 8) [ka] Amino acid sequence encoded by the non-optimized and gene-optimized sequence (SEQ ID NO:9): [ka]

[0276] Example 4 Layer 2-4 (SUDV_anc_-MLD) Sudan Ebola virus ancestral sequence lacking the mucin-like domain (minus, "-") Nucleotide sequence (SEQ ID NO: 10): [ka] Amino acid sequence (SEQ ID NO: 11): [ka]

[0277] Example 5 Layer 2-6 (SUDV_EBOV-TAFV-BDBV_anc_-MLD) Ancestral sequences for four species of Sudan, Zaire, Tai Forest, and Bundibugyo Ebola viruses lacking the mucin-like domain Nucleotide sequence (SEQ ID NO: 12): [ka] [ka] Amino acid sequence (SEQ ID NO: 13): [ka]

[0278] Example 6 Layer 2-11 (RAVV_MARV_anc) Ancestral sequences for strains Marburg virus and Raven virus Nucleotide sequence (SEQ ID NO: 14) [ka] [ka] Amino acid sequence (SEQ ID NO: 15): [ka]

[0279] Example 7 pEVAC expression vector Figure 3 shows a map of the pEVAC expression vector. The sequence of the vector's multiple cloning site is shown below, followed by its complete nucleotide sequence. Sequence of pEVAC multiple cloning site (MCS) (SEQ ID NO: 16): [ka] The full sequence of pEVAC (SEQ ID NO: 17): CMV-IE-E / P:248-989 CMV immediate early 1 enhancer / promoter KanR:3445-4098 Kanamycin resistance SD:990-1220 Splice Donor SA:1221-1343 splice acceptor Tbgh:1392-1942 Terminator signal from bovine growth hormone pUC-ori:2096-2769 pUC plasmid replication origin [ka] [ka] [ka]

[0280] Example 8 Lead candidate optimized antigenic Ebola polypeptides capable of inducing broadly neutralizing antibody responses Following the 2014 West African outbreak, there was significant interest in developing a vaccine against Ebola. Programs currently in clinical development have so far taken the "classical approach" of vaccine development, using Ebola and / or Marburg virus surface glycoproteins (GPs) from one to three strains expressed in a viral vector backbone. Antigenic specificity is derived exclusively from the EBOV strain involved; for example, Merck uses the Kikwit GP; GSK uses the Mayinga EBOV and Gulu SUDV strains; Crucell and Profectus Biosiences both use Marburg virus along with Zaire and Sudan Ebola strains; and Novavax's approach is unique, using the 2014 Makona EBOV strain.

[0281] Table 1 below shows flow cytometry assay results illustrating the strength of antibody binding to target antigens representing all Ebola virus species (subtypes) and Marburg virus. Binding strength is depicted by a heatmap, with red (darkest shading in grayscale) representing very strong binding, orange to yellow (lighter shading in grayscale) representing decreasing binding, and white representing no binding / negative control values. Serum samples 1-22 were collected from individuals immunized with other Ebola virus vaccine candidates. T2-4 and T2-6 are nucleic acid vaccines encoding lead candidate optimized antigenic Ebola polypeptides in combination with the T2-11 Marburg candidate, currently undergoing preclinical testing with serum samples collected from immunized guinea pigs. Table 1 [Table 1]

[0282] Example 9 Protection achieved by trivalent Lassa, Ebola, and Marburg virus vaccine (Tri-LEMvac) in an Ebola challenge model The inventors have developed a trivalent vaccine (Tri-LEMvac) that will generate combined vaccine efficacy against future outbreaks of variants of the hemorrhagic fever Lassa, Ebola, and Marburg viruses.

[0283] We bioinformatically designed synthetic glycoprotein sequences from the GPC open reading frames of LASV (L), EBOV (E), and MARV (M) from all available arenavirus and filovirus databases. These conserved sequences comprise neutralizing antibody and T cell-enriched epitopes for each of these viruses. To ensure that these synthetically engineered LASV, EBOV, and MARV envelopes were functional and antigenic, they were pseudotyped and quality-controlled for both binding and neutralization against a panel of broadly neutralizing antibodies. Herein, we selected the vaccine-derived vector Modified Vaccinia Ankara (MVA) for the construction of a trimeric LEM vaccine.

[0284] The modified vaccinia Ankara (MVA) vaccine platform is a non-replicating strain (i.e., does not replicate in human cells) and is one of the third-generation smallpox vaccines and most advanced recombinant poxvirus 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 strong promoters and stable insertion sites (Orubu et al., Pone, 2012,7(6)e0040167). MVA was selected for the following reasons: 1) it can stably express multiple independent ORFs via compatible expression cassettes with strong and time-regulated promoters, allowing for cost-effective trivalent LEM vaccination in a single vaccine lot; 2) it can induce robust B and T cell immune responses in animals and humans, especially when primed or boosted with DNA or RNA vectors; and 3) it can heat-stabilize vaccine lots for storage and transportation in developing countries lacking a cold chain (Frey et al., Vaccine, 2015, 33(39):5225-34). Proof of principle for the trivalent vaccine candidate has been demonstrated by i) validation of cassettes for independent L, E, and M GPC expression and epitope presentation; and ii) preclinical efficacy with filovirus challenge. Challenge study results are shown in Figure 4. The Ebola challenge model was fatal to non-vaccinated guinea pigs (group 1, bottom line), but all vaccinated guinea pigs (group 2, top line) were protected (left) and continued to gain weight (right).

[0285] Example 10 Pseudotyped virus neutralization assay Figure 5 shows the results of a pseudotyped virus neutralization assay illustrating the strength of neutralizing antibody responses to target antigens expressed on the surface of pseudotyped viruses representing all Ebola virus species and Marburg virus. The strength of neutralization is shown by a heat map, with red (darkest shading when viewed on a grayscale) representing very strong neutralization, orange to yellow (lighter shading when viewed on a grayscale) representing decreasing neutralization, and white representing no neutralization / equal to the negative control value.

[0286] T2-4 and T2-6 are nucleic acid vaccines each encoding a lead candidate optimized antigenic Ebola polypeptide in combination with the T2-11 Marburg candidate, currently in preclinical testing with serum samples collected from immunized guinea pigs.

[0287] The results indicate that administering the combination of T2-6 and T2-11 vaccine inserts resulted in a synergistic increase in the breadth of the immune response.

[0288] Example 11 Antibody binding assay Figure 6 shows the results of the antibody binding assay. Antibody binding was measured by incubating two cell populations bearing two different group 1 influenza A glycoproteins (H1 pandemic and seasonal) on their surface with pooled mouse serum. Any bound antibodies were then detected with a secondary antibody, and the results were recorded using a flow cytometer. Binding was significantly increased before and after vaccination with all constructs, but not after vaccination with PBS (control). Overall, the DIOS vaccine candidate outperformed the COBRA vaccine in both cases ( * ).

[0289] Example 12 Comparison of immune responses induced by two different computational approaches Four groups of six mice were immunized five times at two-week intervals with 25 μg of four separate pEVAC plasmids encoding HA gene antigens designed either by a method according to an embodiment of the present invention (DIOS) or by conventional methods (COBRA).

[0290] Antibody-based FACS was performed on cells expressing two different group 1 influenza A glycoproteins (seasonal H1N1 and pandemic-origin H1N1) on their surface. These were used to test mouse sera from animals immunized with either the COBRA or DIOS HA gene antigens. The results are shown in Figure 7.

[0291] Overall, the DIOS HA gene antigen matched or significantly outperformed the COBRA HA gene antigen ( ** p<0.01, *** p<0.001).

[0292] Example 13 Cross-HA group binding and pseudotype neutralization of H7N9 (A / Shanghai 2 / 2013) We tested whether the DIOS-H1N1pdm vaccine of Example 12 (which produces higher levels of antibody binding to pandemic H1 HA antigens than H1N1-COBRA) can elicit antibodies that recognize and bind to different group 2 viral HAs, such as the viral HA from the potentially pandemic H7N9 strain A / Shanghai / 2 / 2013.

[0293] Figure 8 shows the cross-HA group binding (left panel) and pseudotype neutralization (right) results of H7N9 (A / Shanghai 2 / 2013) by sera from DIOS- or COBRA DNA-immunized mice. The H7 binding data (left), confirmed by the pseudotype neutralization data (right), show that H1N1pdm-vaccinated mice exhibited the highest neutralization compared to the other groups. The DIOS-H1N1pdm vaccine induced significantly more binding than the other groups tested and was comparable to the positive control broadly neutralizing monoclonal antibodies F16 (Corti et al., 2011, Supra) and CR9114 (Dreyfus et al., Science, 2012; 337(6100): 1343-1348).

[0294] These results support the conclusion that the DIOS-H1N1pdm immunogen cross-neutralizes H7 and that cross-HA group immune protection is possible for vaccines produced by the methods of the present invention.

[0295] Example 14 Lassa virus glycoprotein This example describes a Lassa virus glycoprotein ancestral sequence produced using methods according to an embodiment of the invention, and modifications of the ancestral sequence to improve its immunogenicity by stabilizing its structure.

[0296] Lassa fever is a hemorrhagic disease caused by an Old World arenavirus known as Lassa virus (LASV). The virus was first isolated in Nigeria in 1969 and is now endemic in West Africa. Due to the high morbidity and mortality associated with Lassa hemorrhagic fever, LASV is classified as a Category A pathogen.

[0297] Lassa virus is an enveloped, ambisense RNA virus with a bipartite genome. The virus particle is enveloped by a mature glycoprotein (GP) trimeric spike that mediates viral entry. Similar to other class 1 viral fusion proteins, the envelope glycoprotein precursor (GPC) is translated as a single polypeptide and proteolytically cleaved into three subunits. Processing initially occurs in the endoplasmic reticulum (ER) by cellular signal peptidases. The GPC is then transported to the cis-Golgi apparatus and processed by the cellular proprotein convertase subtilisin kexin isozyme-1 / site-1 protease (SKI-1 / S1P) to produce a noncovalently linked, stable signal peptide (SSP) / GP1 / GP2 heterotrimer. Unlike other class 1 fusion proteins, the relatively long signal peptide of the GPC is not cleaved, which serves a chaperone-like function necessary for the correct transport and processing of the GP. The SSP interacts with the cytoplasmic 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.

[0298] The Lassa virus glycoprotein ancestral sequence (construct 1) for lineages III and IV (L-10) was generated using methods according to embodiments of the present invention. The parent ancestral sequence (construct 1) was then independently modified to provide (A) SOSEP (construct 2); and (B) FLEP (construct 4), as well as a glycan knockout called NtoK to stabilize other flexible heterotrimers and prevent dissociation of the glycoprotein ectodomain from the noncovalently linked transmembrane domain (to provide constructs 3 and 5).

[0299] (A) Two cysteine ​​residues were introduced at positions 207 and 360 to form a disulfide bridge (SOS) between the extracellular and transmembrane domains of GP. To facilitate complete cleavage of these two domains, the furin cleavage site was altered from RRLL to RRRR at positions 256–259. A glutamic acid to proline mutation (EP) at position 329 prevents structural rearrangements and makes the protein less flexible.

[0300] (B) The furin cleavage site (256-RRLL-259) between the C-terminus of the ectodomain and the N-terminus of the transmembrane domain was replaced with a flexible linker with the sequence 256-GGGGSGGGGS-265. In addition, the EP mutation in (A) was introduced at position 335.

[0301] Variants of both designs were generated that further contained an asparagine-to-lysine mutation at positions 272 or 278 to inactivate the glycosylation motif for SOSEP-NtoK or FLEP-NtoK, respectively, which may block access of some neutralizing antibodies, such as 37.7H. Construct 1: Lassa virus glycoprotein ancestral sequences for lineages III and IV (L-10=LASV_III_IV_anc) Amino acid sequence (SEQ ID NO: 18): [ka] DNA sequence (SEQ ID NO: 19): [ka] [ka] Construct 2: SOSEP-variant of construct 1 (L-10-SOSEP) Amino acid sequence (SEQ ID NO: 20): [ka] DNA sequence (SEQ ID NO:21): [ka] [ka] Construct 3: SOSEP variant of construct 1 with an N to K mutation (L-10-SOSEP-NtoK) Amino acid sequence (SEQ ID NO: 22): [ka] DNA sequence (SEQ ID NO: 23): [ka] Construct 4: FLEP variant of construct 1 (L-10-FLEP) Amino acid sequence (SEQ ID NO: 24): [ka] DNA sequence (SEQ ID NO: 25): [ka] Construct 5: FLEP variant of construct 1 with an N to K mutation (L-10-FLEP-NtoK) Amino acid sequence (SEQ ID NO:26): [ka] DNA sequence (SEQ ID NO: 27): [ka] Example 15 Lassa virus nucleoprotein

[0302] This example describes a Lassa virus nucleoprotein ancestral sequence produced using methods according to an embodiment of the invention. Construct 6: Ancestral Lassa virus nucleoprotein sequence of Nigerian Lassa isolates (L-NP-1=L-NP-CovAnc-1_N) Amino acid sequence (SEQ ID NO:28): [ka] DNA sequence (SEQ ID NO: 29): [ka] [ka] Example 16 Lassa virus nucleoprotein

[0303] This example describes a Lassa virus nucleoprotein ancestral sequence produced using methods according to an embodiment of the invention. Construct 7: Ancestral sequence of Lassa virus nucleoprotein from Sierra Leone isolates (L-NP-1=L-NP-CovAnc-2_SL) Amino acid sequence (SEQ ID NO: 30): [ka] DNA sequence (SEQ ID NO: 31): [ka] [ka]

Claims

[Claim 1] The invention described in the specification.

Images