Vectors for eliciting immune responses against nondominant epitopes within the hemagglutinin (HA) protein.

Abstract

Methods are provided for producing influenza viruses with altered immunodominant epitopes within their HAs (e.g., with one or more altered residues in one or more of antigenic sites A-E within their HAs), as well as other viral vectors (e.g., influenza VLPs, or non-influenza viruses, or VLPs thereof) that express or have influenza HAs with altered immunogenicity as a result of altering the immunodominant epitopes contained therein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 719,952, filed August 20, 2018, the disclosure of which is incorporated herein by reference.

[0002] Declaration of Government Rights This invention was made with Government support funded by the National Institutes of Health under HHSN272201400008C. The Government has certain rights in this invention. [Background technology]

[0003] Influenza outbreaks in humans are a major public health concern. Annual epidemics (outbreaks of influenza viruses circulating among humans) and sporadic pandemics (outbreaks of new influenza viruses to which humans have no protective immunity) increase morbidity and mortality in human populations and cause significant economic losses. Influenza virus infection or vaccination with current vaccines induces antibodies against highly variable immunodominant epitopes in the head region of the viral surface glycoprotein hemagglutinin (HA). High mutation rates and immune pressures result in the accumulation of mutations in these immunodominant epitopes, which in turn allow the virus to "escape" from circulating antibodies in an individual, who is therefore susceptible to reinfection. As a result of the virus' "escape" from the immune response induced upon infection or vaccination, frequent replacement of vaccine strains is required. Recently, the National Institute of Allergy and Infectious Diseases (NIAID) published a strategic plan to develop a "universal" influenza vaccine that would protect against numerous strains of diverse antigenicity.

[0004] Immunodominance describes the phenomenon that a strong immune response is directed to a subset of antigenic epitopes (i.e., immunodominant antigenic epitopes), while a much weaker immune response is directed to the remaining immunosubdominant epitopes. The immunodominant epitopes of influenza viruses are located in highly variable regions of the HA head (i.e., most antibodies elicited after infection or vaccination are directed to these major antigenic epitopes). The immunosubdominant epitopes are located in conserved regions of the HA head and in the conserved HA stem. Antibodies elicited against the conserved regions of these immunosubdominant epitopes typically react with a broad range of influenza viruses, but because the epitopes are immunosubdominant, the levels of these broadly reactive antibodies are low.

[0005] Several studies have shown that initial antigenic exposure to a novel influenza virus generates high levels of antibodies against major immunodominant epitopes in the highly variable region of the HA head, as well as relatively high levels of antibodies against immunodominant epitopes within the HA stem. Palese and coworkers demonstrated that repeated immunization with multiple chimeric HAs derived from HA subtypes with the same stem region but different head regions increased the levels of stem-reactive antibodies compared to repeated vaccinations with HAs with the same head (Chen et al., 2016; Krammer et al., 2013; Margine et al., 2013; Nachbagauer et al., 2017 and 2015; Krammer et al., 2012 and 2014; Goff et al., 2013).

[0006] Despite this promising data generated with chimeric HA, this approach has drawbacks: (i) the number of HA subtypes available for head region exchange is limited (and not all stem-head combinations are stable); and (ii) current approaches to HA head exchange do not exploit subtype-specific conserved immunodominant epitopes within the head.

[0007] Human influenza virus infections impose a significant burden on individual health, the public health sector, and the global economy. Influenza viruses circulating among humans (i.e., "seasonal" influenza viruses) typically cause annual epidemics that result in 92-356 million illnesses, 140-710,000 hospitalizations, and 12-56,000 deaths annually in the United States alone since 2010 (https: / / www.cdc.gov / flu / about / disease / burden.htm). Epidemics are caused by influenza types A and B. Influenza type A viruses are further divided into subtypes based on the antigenicity of the viral surface glycoproteins HA and neuraminidase (NA). To date, 18 subtypes with HA (H1-H18) and 11 subtypes with NA (N1-N11) have been identified, and HA is classified into two supergroups based on phylogenetic relationships (group 1, H1, H2, H5, H6, H8, H9, H11-H13, H16-18; group 2, H3, H4, H7, H10, H14, H15). However, only viruses of the subtypes H1N1, H2N2, and H3N2 are circulating among humans. Global outbreaks (pandemics) are caused by viruses with HAs that are antigenically different from those previously circulating in humans, and therefore spread rapidly around the world when they encounter immunologically naive populations. There have been four pandemics in the past 100 years. The 1918 pandemic was caused by an H1N1 virus, which was replaced by an H2N2 virus in 1957, causing the "Asian" pandemic. In 1968, H3N2 viruses replaced H2N2 viruses, causing the "Hong Kong" pandemic. H1N1 viruses similar to those circulating in the 1950s re-emerged in 1977 and circulated alongside H3N2 viruses until 2009, when they were replaced by H1N1 viruses with antigenically distinct HAs (the 2009 pandemic).

[0008] Wild waterfowl are a natural reservoir of influenza A viruses and carry most influenza subtypes. Sporadic transmission of avian influenza viruses to humans can cause severe respiratory disease and significant mortality. Highly pathogenic avian influenza of the H5 subtype has infected 840 people, with 454 deaths. Human infection with the H7N9 virus (which emerged in 2013) has caused a 38% mortality rate among 1,625 reported cases (as of May 24, 2018). These avian influenza viruses do not transmit efficiently between humans and have not (yet) caused a pandemic, but H7N9 viruses can transmit between ferrets (an animal model commonly used to study influenza virus transmission) through respiratory droplets (1-4), and H5 viruses with a few mutations can transmit between ferrets through respiratory droplets (5, 6). Any universal vaccine strategy would ideally be applicable / adaptable to these types of viruses. Summary of the Invention

[0009] Disclosed herein is a method for redirecting immune responses in birds or mammals from (frequently mutating) immunodominant epitopes to more conserved nondominant (subdominant) epitopes. Immunization with one or more viruses generated by this method results in the production of more antibodies targeting the conserved immunodominant epitopes, which in turn increases broad spectrum protective immunity. This method significantly outdilutes the immune response to the immunodominant epitopes, resulting in higher levels of antibodies directed to the conserved nondominant epitopes. Influenza vaccines with one or more influenza viruses with modified HA may provide protection against viruses that have undergone drift, as they induce immune responses to nondominant epitopes (i.e., epitopes that are more conserved than the dominant antigenic epitopes on the surface of the hemagglutinin (HA) head of influenza viruses, e.g., human influenza viruses). Thus, vaccination may be required for longer than 1-3 years. Vaccines can contain a mixture of different HA proteins, each with a mutated (e.g., non-naturally occurring) immunodominant epitope to attenuate the immune response to the immunodominant antigenic epitope, thereby increasing the levels of antibodies directed against the immunodominant epitopes.

[0010] As described herein, influenza virus "libraries" (e.g., a mixture of millions of variants) are generated with random mutations at selected positions of highly variable immunodominant antigenic epitopes in the HA head, e.g., from any HA subtype. For example, for H3 HA, influenza viruses with up to 17 mutations in the immunodominant antigenic epitopes of HA have been generated and found to be viable. This virus library is incubated with different sera (e.g., ferret and / or human sera) to filter out variants whose antigenicity is similar to that of the wild type. The individual modified HA sequences (individual ID-EpiMut HAs) can be cloned, sequenced, and tested for reactivity with monoclonal antibodies directed against immunodominant or subdominant epitopes of HA. ID-EpiMut HAs with greater reactivity to antibodies directed against conserved immunodominant epitopes and ID-EpiMut HAs with less reactivity to antibodies directed against immunodominant epitopes are then isolated and, in some cases, pooled into a vaccine formulation.

[0011] In one embodiment, these ID-EpiMut HAs are incorporated into virus-like particles (VLPs) consisting of Ebola virus VP40 matrix protein to study immunization in mice, thus excluding the possibility that other proteins of the influenza virus contribute to immunity. Mouse sera are examined for levels of antibodies directed against immune dominant and immune subdominant epitopes. Vaccination and challenge studies in ferrets are performed with an inactivated influenza vaccine carrying ID-EpiMut HA. Naive or pre-exposed animals are vaccinated, levels of antibodies against immune subdominant epitopes are assessed, and animals are challenged with homologous and heterologous influenza viruses. Vaccines based on ID-EpiMut HA are likely to provide greater cross-protection than vaccines based on wild-type HA.

[0012] Thus, the present disclosure provides a method for eliciting broadly protective antibodies against immune subdominant epitopes within the HA of any subtype. A mixture of influenza viruses, or other vectors (e.g., a mixture of isolated nucleic acids including mRNA and DNA encoding altered HAs, including other viral vectors (e.g., filoviruses, adenoviruses, etc.) or virus-like particles (including Ebola VLPs and influenza VLPs)), or a mixture of polypeptides with altered influenza hemagglutinins (HAs) (altered to have a non-native immune dominant antigenic head as a result of substitutions and / or deletions of residues that form immune dominant epitopes and conserved immune subdominant epitopes) (Figure 2) provides a vaccine that can elicit large amounts of Abs against conserved immune subdominant epitopes within the head and stem, resulting in broader protection than that elicited by current vaccines. Contemplated herein are compositions comprising an HA with one or more altered residues, or an isolated nucleic acid encoding an HA with one or more altered residues, or an individual recombinant virus (e.g., influenza virus, filovirus, adenovirus) or a VLP thereof comprising an isolated HA with one or more altered residues.

[0013] In one embodiment, the recombinant influenza virus produced by this method has a HA with one or more altered residues within one or more immunodominant epitopes of the HA (it is not required that the residues within the epitopes be contiguous or adjacent to each other in the primary amino acid sequence) such that the epitope is altered, e.g., such that it does not bind as efficiently to antibodies specific for said one or more immunodominant epitopes as the (unaltered) immunodominant epitope in the parent HA, and / or when administered, the HA with one or more altered residues within an immunodominant epitope elicits antibodies to conserved immunodominant epitopes within the head and / or stem of the HA. In one embodiment, the recombinant influenza virus produced by this method has 20 or fewer unchanged residues that are part of one or more immunodominant epitopes, e.g., 15, 10, 5, 4, 3, 2, 1, or 0 residues that are unchanged in one or more naturally occurring immunodominant epitopes, e.g., in a particular parent influenza virus. In one embodiment, the recombinant influenza virus produced by this method has 1-10, or 10-20, altered immunodominant residues in two or more immunodominant epitopes. In one embodiment, the recombinant influenza virus produced by this method has 10 or fewer unchanged immunodominant residues, e.g., 5 or 3, in one or two immunodominant epitopes. For example, a recombinant influenza H3 virus produced by this method has five or fewer immunodominant residues at any of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, and 225. For example, a recombinant influenza H5 virus produced by this method has five or fewer immunodominant residues at any of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, and 189.

[0014] In one embodiment, the recombinant influenza virus produced by this method has two or more residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more residues) that result in reduced antibody binding to an antibody that recognizes the immunodominant epitope. Thus, when a residue in the immunodominant epitope is replaced (substituted) with another residue, and these other residues are present in the HA of an influenza virus that infects an animal that has been infected with an HA bearing the immunodominant epitope, the substitution will not result in a memory response (immunological memory), but will instead redirect the immune response to a subdominant epitope in the head or stem of the HA, thereby allowing, for example, a broader immune response to a particular HA subtype, or to a particular clade within an HA subtype. In one embodiment, the recombinant influenza virus produced by this method has 2-5, 5-10, 10-15, 15-20 or more substituted residues in the immunodominant epitope. In one embodiment, the recombinant influenza virus produced by this method has about 10-17 substituted residues in the immunodominant epitope. For example, the recombinant influenza H3 virus produced by this method has 2-5, or 5-10, or 10-15, or 10-17 non-dominant residues at any combination of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, and 225. For example, a recombinant influenza H5 virus produced by this method has 2 to 5, or 5 to 10, or 10 to 15, or 10 to 17 substitutions (to non-dominant residues) at any combination of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, and 189.In one embodiment, the recombinant influenza virus produced by this method has one to two, two to five, or up to ten residues deleted (including, for example, positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, 225 in H3, or positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, 189 in H5), where the deletion in the HA reduces antibody binding to antibodies that recognize immunodominant epitopes.

[0015] In one embodiment, the vaccine comprises a plurality of recombinant influenza viruses with substitutions (or deletions) at positions of the immunodominant epitopes (substitutions to "non-immune dominant residues"). In one embodiment, the vaccine comprises 2-5, or 5-10, or 10-20, or 20-30, or 30-40, or 40-50 different recombinant influenza viruses with substitutions (or deletions) at positions of the immunodominant epitopes. In one embodiment, the vaccine comprises 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 different recombinant influenza viruses with substitutions at positions of the immunodominant epitopes. In one embodiment, the vaccine comprises 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40 different recombinant influenza viruses with substitutions at the positions of immunodominant epitopes.

[0016] In one embodiment, a combination of vectors or viruses encoding the altered HAs of Table 1 or Table 6 are used in a composition administered to a mammal or avian species. [Brief description of the drawings]

[0017] [Figure 1] X-ray structure of the human H3 HA protein, with targeted positions for mutagenesis indicated in red. [Diagram 2] VLPs based on Ebola VP40 with wild-type HA (left) or with HA with randomized amino acid changes at up to 17 amino acid positions (right). [Diagram 3] The three-dimensional structure of H3 HA(4O5N). Shown are the head region (dark grey) and stem region (light grey) (Figure 3A); the five major epitopes A (red), B (blue), C (orange), D (yellow), and E (green) (Figure 3B); the amino acid positions selected for mutagenesis (white), see C.1.2.1 (Figure 3C); and the sequence conservation of over 13,000 novel human H3N2 HAs downloaded from the Influenza Research Database (https: / / www.fludb.org) (Figure 3D). The color scale indicates that the amino acid sequence conservation at each position ranges from 85.5% (gold) to 100% (purple). The highly conserved tyrosine residue at position 98 of the receptor binding pocket is shown in magenta. [Figure 4]Schematic overview of the strategy proposed in this disclosure to increase the amount of broadly reactive Abs against immune subdominant epitopes. (Figure 4A) After infection or vaccination with seasonal influenza virus, a large amount of Abs against highly variable immune dominant epitopes (shown in three consecutive clusters in blue, dark green, and light green) in the stem (black) and head (dark grey) are generated, followed by a smaller amount of Abs against immune subdominant epitopes. (Figure 4B) A new idea is to increase the level of antibodies directed against immune subdominant epitopes by attenuating the immune response against immune dominant epitopes using (repeated) immunization with a mixture of HA proteins (only one mutant HA is shown here) bearing highly mutated non-natural immune dominant antigenic epitopes. [Diagram 5] flowchart. [Figure 6] Representative HA sequences from H3N2 and H5N8 viruses (sequence ID numbers 1 and 6–9). [Figure 7] Representative H5 HA sequence (SEQ ID NO: 2). [Figure 8] Representative H1 HA sequence (SEQ ID NO: 3). [Figure 9] Representative H2 HA sequence (SEQ ID NO: 4). [Figure 10] Representative H7 HA sequence (SEQ ID NO: 5). [Figure 11] H3 variants with substitutions in immunodominant epitope residues. [Figure 12] Clusters and antibody reactivity to wild-type H3 and H3 variants with substitutions in immunodominant epitope residues). [Figure 13] Antibody reactivity to wild-type H3 and H3 variants with substitutions in immunodominant epitopes. [Figure 14] Antibody reactivity to wild-type H3 and selected H3 variants with substitutions in immunodominant epitopes. [Figure 15]A combinatorial set of 10 independent (distinct) H3 variants with substitutions in immune dominant epitopes. [Figure 16] A mixture of H3 variants with substitutions in immunodominant epitopes reacts with antibodies to subdominant epitopes. [Figure 17] Alignment of HA subtypes (see, e.g., Burke et al., PLoS One 9:e112302, (2014)). [Figure 18] Representative antigenic sites within H3 HA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition

[0019] As used herein, the term "isolated" refers to in vitro preparations and / or isolation of nucleic acid molecules (e.g., vectors or plasmids), peptides or polypeptides (proteins), and viruses, such that they are free from association with in vivo materials or are substantially purified from in vitro materials. Isolated virus preparations are generally obtained by in vitro culture and propagation and / or through passaging in eggs, and are therefore substantially free of other infectious materials.

[0020] As used herein, "substantially purified" means that the species of interest is the predominant species, e.g., on a molar basis, more abundant than any other individual species in the composition, and preferably represents at least about 80% of the species present, and in some cases greater than 90%, e.g., 95%, 98%, 99%, or more, of the species present in the composition.

[0021] As used herein, "substantially free" means below the level of detection for a particular infectious agent using standard detection methods for that infectious agent.

[0022] A "recombinant" virus is one that has been manipulated in vitro, for example using recombinant DNA techniques, to introduce an alteration into the viral genome. Reassortant viruses can be produced by recombinant or non-recombinant techniques.

[0023] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or recombinant DNA segment" refers to a nucleic acid (e.g., DNA) that may have been derived from or isolated from a source and subsequently chemically modified in vitro, such that the sequence is not naturally occurring, or the sequence corresponds to a naturally occurring sequence that is not located where it would be in the natural genome. An example of DNA "derived" from a source would be a DNA sequence that is chemically synthesized in substantially pure form after it has been identified as a useful fragment. An example of such DNA that is "isolated" from a source would be a useful DNA sequence that has been excised or removed from the source by chemical means (e.g., the use of a restriction endonuclease). The DNA sequence can be further manipulated (e.g., amplified) by genetic engineering methods for use in the present invention.

[0024] As used herein, a "heterologous" influenza virus gene or a "heterologous" viral segment is derived from an influenza virus source and differs from most other influenza virus genes or most other viral segments in a recombinant (e.g., reassortant) influenza virus.

[0025] The terms "isolated polypeptide," "isolated peptide," and "isolated protein" include polypeptides, peptides, and proteins encoded by cDNA or recombinant RNA, including those of synthetic origin, and any combination thereof.

[0026] The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule. In contrast, "native protein" is used herein to refer to a protein that has been isolated from a natural (i.e., non-recombinant) source. Using molecular biology techniques, recombinant forms of proteins can be produced that have the same properties as the native form.

[0027] Methods for alignment of sequences for comparison are well known in the art, and thus, the percent identity between any two sequences can be determined using a mathematical algorithm.

[0028] These mathematical algorithms can be implemented on a computer to compare sequences and determine sequence identity. Alignments using these programs can be performed using default parameters. Software for performing BLAST analyses is available from the National Center for Biotechnology Information (http: / / www.ncbi.n1m.nih.gov). / ), which are publicly available through the Internet. The algorithm involves first identifying high-scoring sequence pairs (HPSs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequences. T is called the neighborhood word score threshold. These initial neighborhood word hits act as seeds to initiate searches aimed at finding longer HPSs containing these word hits. These word hits are then extended in both directions along each sequence until a cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the accumulation of one or more negatively scoring residue alignments or the end of either sequence causes the cumulative alignment score to fall by an amount X from the maximum achieved, causing the cumulative score to fall below 0.

[0029] In addition to calculating sequence identity, the BLAST algorithm can also perform statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm can be the minimum sum probability (P(N)). This is a measure of the probability that two nucleotide or amino acid sequences will match by chance. For example, a nucleic acid sequence to be examined is considered to be similar to a reference sequence when the minimum sum probability of the nucleic acid sequence to be examined compared to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0030] The BLASTN program (for nucleotide sequences) can use as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and can compare both strands. For amino acid sequences, the BLASTP program can use as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. http: / / www.ncbi.n1m.nih.gov See, e.g., 2003. Alignment can also be performed manually by eye.

[0031] To compare sequences, typically one sequence serves as a reference sequence, and test sequences are compared to this reference sequence.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the sequence identity of test sequences compared to reference sequences based on the designated program parameters.

[0032] Immunodominant epitopes of HA and methods to alter these epitopes

[0033] Both infection and vaccination processes with influenza virus primarily induce antibodies against immune-dominant epitopes in the "head" region of the viral hemagglutinin (HA) protein, the major viral antigen. Mutations in immune-dominant epitopes may allow them to "escape" from circulating antibodies in an individual's body, making individuals who have acquired immunity to previous influenza viruses more susceptible to challenge with mutated "escape variant" viruses. Several strategies are being tested to redirect the recipient's immune response from the (frequently mutated) immune-dominant epitopes in HA to more conserved nondominant epitopes in HA. Targeting conserved nondominant epitopes should increase broad-spectrum protective immunity.

[0034] Infection or vaccination with influenza viruses induces neutralizing antibodies that protect against infection with antigenically closely related viruses. Most neutralizing antibodies are directed against highly variable immunodominant epitopes in the head of the HA. For H3N2 viruses, early studies using antigenic escape mutants against mouse monoclonal antibodies identified five immunodominant epitopes (A–E) in the head of the HA (Wiley et al., 1981 and 1987) (Figure 1). As a result of the large mutation rate of influenza viruses and immune pressure in previously infected and / or vaccinated individuals, mutations accumulate in these immunodominant epitopes, and antibodies circulating in an individual no longer neutralize the virus. This so-called “antigenic drift” is the reason why humans are reinfected with seasonal influenza viruses. Current seasonal influenza vaccine strains are selected based on the antigenic properties of highly variable immunodominant epitopes in the HA head. Therefore, vaccine strains must be replaced with each new cluster or clade of antigenic drift variants. Furthermore, antigenic drift can occur between the time of vaccine selection (February in the Northern Hemisphere) and the start of the influenza season, resulting in a significant loss of vaccine efficacy ("vaccine mismatch").

[0035] Most antibodies (Abs) induced by influenza virus infection or vaccination are directed against highly variable immunodominant antigenic epitopes on the head of HA and therefore react only with closely related viruses. In 1993, Okuno et al. reported monoclonal Abs (mAbs) that neutralize influenza viruses of two different subtypes. This finding was not fully appreciated at the time, and it took another decade before many studies reported mAbs that react with many HAs of the same subtype, or with HAs of different subtypes from the same group, or with HAs from both groups 1 and 2, or with HAs of both influenza A and B. Most of these broadly reactive mAbs bind to the stem of HA, which anchors HA in the membrane and mediates the pH-induced membrane fusion event in late endosomes that releases the viral genome into the cytosol (Figure 1). However, some broadly reactive mAbs react with conserved regions within the head of HA. These mAbs fall into at least two major categories: broadly reactive mAbs that react with conserved immune subdominant epitopes within the central part of the receptor binding site (conserved in most HA subtypes) and broadly reactive mAbs that react with conserved immune subdominant epitopes on the head of HA outside the receptor binding pocket (these epitopes may not be conserved in all subtypes). Conserved immune subdominant epitopes evolve much more slowly than immune dominant epitopes, explaining why antibodies that bind to conserved epitopes react with a greater variety of strains (compared to antibodies that react with immune dominant epitopes). Antibodies directed against conserved immune subdominant epitopes therefore provide protection against viruses of different antigenic clusters within the same subtype or viruses of different subtypes.

[0036] Broadly reactive Abs against conserved regions in HA may pave the way for the development of broadly protective vaccines. However, conserved epitopes are immunosubdominant epitopes, and the detection levels of Abs against these epitopes are much lower than those targeting highly variable major antigenic epitopes in the head of HA, immunodominant epitopes. Therefore, researchers have attempted to shift the focus of the immune response from highly variable major antigenic epitopes in HA, immunodominant epitopes, to conserved immunosubdominant epitopes of HA.

[0037] A study in the 1980s demonstrated that vaccination with HA lacking the head region ("headless HA") elicited stem-reactive Abs, and that these Abs reacted with different subtypes of HA proteins (Graves et al., 1983). Because head removal of HA seemed an attractive strategy to elicit Abs against the conserved stem, various membrane-anchored or secreted versions of headless HA were tested. Although some of these studies led to the generation of broadly protective antibodies upon vaccination, headless HA is less stable and may not fold correctly. In addition, headless HA lacks conserved immunodominant epitopes within the HA head.

[0038] In 2011, Wilson and coworkers reported that the pandemic 2009 H1N1 virus (an antigenically novel influenza virus not circulating in humans prior to 2009) elicited broader cross-reactive antibodies against conserved immunodominant epitopes within the stem of HA than are typically detected after infection or vaccination with seasonal influenza viruses (Wrammert et al., 2011). Other investigators have reported similar results. Furthermore, experimental vaccination against H5 viruses (Ellebedy et al., 2014; Nachbagauer et al., 2014) or H7 viruses (Henry et al., 2016 and 2015; Liu et al., 2017; Krammer et al., 2014; Halliley et al., 2015) induced greater amounts of broadly reactive Abs against conserved immune subdominant epitopes in the stem of the HA than are typically detected after infection or vaccination with seasonal influenza viruses (neither of which are circulating in humans). This effect was strongest upon initial encounter with a novel (previously unencountered) HA; for example, initial vaccination with pandemic 2009 H1N1 vaccine induced high levels of broadly reactive Abs against immune subdominant epitopes in the stem of the HA (Andrews et al., 2015). However, after a second exposure to the pandemic 2009 H1N1 virus, the majority of Abs were directed against immune-dominant epitopes in the head of HA, and the levels of Abs directed against immune-subdominant epitopes in the stem were significantly reduced compared to those measured after the first exposure (Andrews et al., 2015). Taken together, these findings indicate that the first encounter with a novel HA elicits a significant amount of broadly reactive Abs against conserved immune-subdominant epitopes. In contrast, repeated infection or vaccination with seasonal influenza viruses (which differ by only one or two amino acids in one major antigenic epitope and share other major epitopes) primarily stimulates (recalls) Abs against immune-dominant antigenic epitopes.

[0039] Representative methods

[0040] The present disclosure relates to an influenza vaccine based on a different "outdilution" approach to improve antigenic responses to non-dominant epitopes. Customized influenza viruses with strategically antigenically distinct immunodominant epitopes (residues in the immunodominant epitopes are changed to residues that do not react with antibodies that recognize the immunodominant epitopes) in the "head" region of HA are pooled. Thus, the body's response will direct a small amount of antibodies to these antigenically distinct and diverse immunodominant epitopes (also called non-immune dominant epitope residues), while the large amount of conserved non-dominant epitopes shared by the entire pool will generate an immune response that is more likely to prove protective against a group of natural viruses in circulation by enhancing the response to these non-dominant epitopes, as would be expected since these viruses share these conserved non-dominant epitopes.

[0041] In one embodiment, multiple positions in HA (e.g. up to 17 positions in H3) known or suspected to be involved in an epitope are randomly mutated. For H3, 60 live viruses were recovered. The antigenicity of the recovered viruses is evaluated using monoclonal antibodies. Many of the viruses bind to a limited number of mAbs tested, which apparently correspond to non-dominant epitopes. The same approach can be used with a group of subtypes or with only one subtype (e.g. H3N2, H5N1, etc.) that constitute the vaccine cocktail. For the human seasonal H3N2 virus, this method resulted in a virus with a "highly" mutated immunodominant antigenic epitope. The virus was viable, functional, and antigenically distinct from the parent virus.

[0042] In one embodiment, a pan-H3 vaccine is prepared from a virus containing a modified HA that confers protection against multiple antigenic clusters of seasonal human H3N2 viruses. Since a mixture of intrahead immunodominant epitopes has never been detected in nature before, the immune response is focused on conserved immunodominant epitopes. This method allows the generation of millions of HA variants with multiple non-natural mutations in the immunodominant epitopes, resulting in viable viruses with multiple amino acid changes in the immunodominant epitopes that alter their antigenic properties. Unlike other approaches, a vaccine candidate is prepared that (i) has non-natural intrahead immunodominant epitopes, (ii) retains conserved immunodominant epitopes in the HA stem and in the HA head, and (iii) maintains the structural and functional integrity of the HA. As a result, our vaccine candidate should provide greater cross-protection compared to vaccines based on wild-type viruses.

[0043] Influenza vaccine

[0044] Vaccines of the invention include at least one isolated recombinant influenza virus that has desired properties (e.g., a non-native immunodominant epitope in the head and / or one or more conserved immunodominant epitopes in the stem and head of the HA) and maintains the structural and functional integrity of the HA, and optionally includes one or more other isolated viruses (including other isolated viruses with desired properties), one or more immunogenic proteins or glycoproteins (e.g., immunogenic proteins from one or more bacteria, non-influenza viruses, yeast, or fungi) of one or more isolated influenza viruses or one or more other pathogens, and / or isolated nucleic acids (e.g., DNA vaccines) encoding one or more viral proteins (including one or more immunogenic proteins of the isolated influenza viruses of the invention). In one embodiment, the influenza viruses of the invention can be vaccine vectors for influenza viruses or other pathogens.

[0045] Whole virion vaccines can be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. Viruses other than those of the invention (such as those contained in multivalent vaccines) can be inactivated before or after purification, for example with formalin or β-propiolactone.

[0046] Subunit vaccines contain purified glycoproteins. Such vaccines can be prepared by using a suspension of viruses fragmented by treatment with detergents and purifying the surface antigens, for example by ultracentrifugation. Subunit vaccines therefore contain mainly HA proteins but also NA. The detergents used can be, for example, cationic detergents (such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975)), anionic detergents (such as ammonium deoxycholate (Laver and Webster, 1976)) or non-ionic detergents (such as those sold under the name TRITON® X100). Hemagglutinin can also be isolated after treatment of the virions with a protease (such as bromelin) and then purified. Subunit vaccines can be combined with the attenuated viruses of the invention in multivalent vaccines.

[0047] Split vaccines contain virions treated with agents that dissolve lipids. Split vaccines can be prepared as follows: the aqueous suspension of purified virus obtained as described above is treated under stirring with a lipid solvent (ethyl ether, chloroform, etc.) combined with a detergent. The viral particles fragment as the lipids of the viral envelope dissolve. The aqueous phase is recovered, containing the hemagglutinin and neuraminidase, freed from their original lipid environment, and the split vaccine, mainly consisting of the core or its degradation products. The remaining infectious particles are then inactivated if they have not already been inactivated. This split vaccine can be combined with the attenuated virus of the invention in a multivalent vaccine.

[0048] Inactivated vaccinesInactivated influenza virus vaccines are obtained by inactivating replicated viruses using known methods, non-limiting examples of which are formalin treatment, β-propiolactone treatment, etc. Inactivated vaccines of the type that can be used in the present invention can include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain complete inactivated virus, whereas SV vaccines contain purified virus that has been disrupted with detergents that solubilize the lipid-containing viral envelope, followed by chemical inactivation of the remaining virus.

[0049] Additionally, available vaccines include those that contain the isolated surface proteins HA and NA; these are called surface antigen or subunit vaccines.

[0050] Live attenuated virus vaccines A live attenuated influenza virus vaccine (such as a vaccine comprising a recombinant virus of the invention) can be used to prevent or treat influenza virus infection. Attenuation can be achieved in a single step by transferring attenuated genes from an attenuated donor virus to a replicated isolate or reassortant virus according to known methods. Since resistance to influenza A viruses is primarily mediated by the development of an immune response against the glycoproteins HA and / or NA, the genes encoding these surface antigens are derived from the reassortant virus or clinical isolate. The attenuated genes are derived from the attenuated parent. In this approach, the genes that confer attenuation generally do not code for the glycoproteins HA and NA.

[0051] Viruses (donor influenza viruses) that can reproducibly attenuate influenza viruses are available. For example, cold-adapted (ca) donor viruses can be used in the production of attenuated vaccines. Attenuated live reassortant virus vaccines can be generated by combining ca donor viruses with replicated virulent viruses. Reassortant virus progeny are then selected at 25°C (limiting replication of virulent viruses) in the presence of appropriate antisera that inhibit replication of viruses bearing the surface antigens of the attenuated ca donor virus. Useful reassortant viruses are (a) infectious, (b) attenuated in seronegative non-adult mammals and primed adult mammals, (c) immunogenic, and (d) genetically stable. The immunogenicity of ca reassortants parallels their replication levels. Thus, when new wild-type viruses acquired the six transferable genes of the ca donor virus, these viruses were reproducibly attenuated and could be used to vaccinate susceptible mammals, both adult and non-adult.

[0052] Other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses carrying these mutated genes. Attenuating mutations can be introduced into non-coding and coding regions of the genome. Such attenuating mutations can also be introduced into genes other than HA or NA, such as the PB2 polymerase gene. New donor viruses can thus be generated with attenuating mutations introduced by site-directed mutagenesis, and these new donor viruses can be used to generate live attenuated vaccine candidates in a similar manner as described above for the ca donor virus. Similarly, influenza viruses can be used to reassort other suitable known attenuated donor strains to obtain attenuated vaccines suitable for use in vaccinating mammals.

[0053] In one embodiment, such attenuated viruses maintain the viral genes that encode antigenic determinants substantially similar to those of the original clinical isolate, since the goal of an attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking pathogenicity to the extent that the vaccine has minimal chance of inducing a severe disease state in a mammal vaccinated with the vaccine.

[0054] Thus, the viruses in the multivalent vaccine can be formulated as attenuated or inactivated according to known methods and then administered as a vaccine to induce an immune response in animals (e.g., mammals). Methods for determining whether such attenuated or inactivated vaccines maintain similar antigenicity to clinical isolates or high-growth strains derived from clinical isolates are well known in the art. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and nucleic acid screening (e.g., probe hybridization or PCR) to confirm that donor genes encoding antigenic determinants (e.g., HA or NA genes) are not present in the attenuated virus.

[0055] Pharmaceutical Compositions

[0056] A pharmaceutical composition suitable for inoculation (e.g., nasal, parenteral, oral administration) comprises one or more influenza virus isolates (e.g., one or more attenuated or inactivated influenza viruses, and / or subunits thereof, and / or isolated proteins thereof, and / or isolated nucleic acids encoding one or more proteins thereof), and optionally further comprises a sterile aqueous or non-aqueous solution, suspension, emulsion. The composition may further comprise auxiliary agents or excipients known in the art. The compositions of the present invention are generally provided in the form of individual doses (unit doses).

[0057] Conventional vaccines generally contain about 0.1-200 μg (e.g., 30-100 μg) of HA from each of the strains present in the composition. The vaccine-forming primary component of the vaccine composition of the invention can include a single influenza virus or a combination of influenza viruses, such as at least two or three influenza viruses including one or more reassortant viruses.

[0058] Preparations for parenteral administration include sterilized aqueous or non-aqueous solutions, and / or suspensions, and / or emulsions, and may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate). Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration can generally include liposomal solutions containing the liquid dosage forms. Suitable forms of suspended liposomes include emulsions, suspensions, solutions, syrups, elixirs containing inert diluents (purified water) commonly used in the art. Such compositions can also contain adjuvants, wetting agents, emulsifying agents, suspending agents, or any of sweeteners, flavoring agents, and aromatic agents, in addition to inert diluents.

[0059] The compositions of the present invention may further contain salts, buffers, adjuvants, and other substances that are desirable for improving the effectiveness of the compositions when used for administration to an individual. In the context of vaccines, adjuvants, i.e. substances that can enhance a specific immune response, may be used. Typically, the adjuvant and the composition are mixed and then presented to the immune system, or the adjuvant and the composition are presented separately but at the same site in the organism to be immunized.

[0060] Heterogeneity within a vaccine can be created by mixing replicates of influenza viruses for at least two influenza virus strains (e.g., 2-20 strains, or any range or value therein). Vaccines can be provided that provide variation within a single strain of influenza virus using techniques known in the art.

[0061] The pharmaceutical compositions of the present invention may further, or additionally, comprise at least one chemotherapeutic compound, examples of which are immunosuppressants, anti-inflammatory agents, or immune enhancing agents for gene therapy, and chemotherapeutic agents for vaccines, non-limiting examples of which include gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-alpha, interferon-beta, interferon-gamma, tumor necrosis factor-alpha, thiosemicarbazones, methisazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, and ganciclovir.

[0062] The composition may also contain varying but small amounts of endotoxin-free formaldehyde and preservatives that are known to be safe and not contribute to undesirable effects in the organism to which the composition is administered.

[0063] Purpose of the pharmaceutical composition

[0064] Administration of the composition (or the antisera it elicits) can be for "prophylactic" or "therapeutic" purposes. When administered prophylactically, the compositions of the invention, which are vaccines, are provided before any symptoms or clinical signs of pathogen infection appear. Prophylactic administration of the composition serves to prevent or reduce any subsequent infection. When administered prophylactically, the gene therapy compositions of the invention are provided before any symptoms or clinical signs of pathogen infection appear. Prophylactic administration of the composition serves to prevent or reduce one or more symptoms or clinical signs associated with the disease.

[0065] A viral vaccine, when given therapeutically, is given when symptoms or clinical signs of actual infection are detected. Therapeutic administration of the composition serves to alleviate any actual infection. A gene therapy composition, when given therapeutically, is given when symptoms or clinical signs of disease are detected. Therapeutic administration of the compound serves to alleviate the symptoms or clinical signs of the disease.

[0066] Thus, the vaccine compositions of the invention can be given prior to infection (to prevent or reduce anticipated infection) or after actual infection has begun. Similarly, with respect to gene therapy, the compositions can be given prior to any symptoms or clinical signs of a disorder or disease, or after one or more symptoms are detected.

[0067] A composition is said to be "pharmaceutical acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition of the invention is physiologically significant if its presence results in a detectable change in the physiological state of a recipient patient (e.g., enhancing at least one primary or secondary humoral or cellular immune response against at least one strain of infectious influenza virus).

[0068] The "protection" provided need not be absolute, i.e., there need only be a statistically significant improvement compared to a control population or group of mammals, and influenza infection need not be completely prevented or eradicated. Protection may be limited to reducing the severity or rapidity of onset of symptoms or clinical signs of influenza virus infection.

[0069] Administration of pharmaceutical compositions

[0070] Compositions containing one or more influenza viruses with desired properties can confer resistance to one or more pathogens (e.g., one or more influenza virus strains) by passive or active immunization. In active immunization, the live attenuated vaccine composition is administered prophylactically to a host (e.g., a mammal) and the host's immune response to the administration protects against infection and / or disease. For passive immunization, induced antisera are collected and administered to a recipient suspected of suffering from an infection caused by at least one influenza virus strain. The gene therapy compositions of the invention can deliver a desired gene product to prophylactic or therapeutic levels by active immunization.

[0071] In one embodiment, the vaccine is provided to a mammalian female (at or prior to pregnancy or parturition) under conditions and in an amount and for a time sufficient to generate an immune response that serves to protect both the female and her fetus or newborn (through the placenta or through passive incorporation of antibodies in the milk).

[0072] Thus, the invention includes methods of preventing or alleviating a disorder or disease, such as infection by at least one strain of a pathogen. As used herein, a vaccine is said to prevent or alleviate a disease when administration results in full or partial alleviation (i.e., suppression) of the clinical signs or condition of the disease, or when administration results in full or partial immunity of an individual to the disease. As used herein, a gene therapy composition is said to prevent or alleviate a disease when administration results in full or partial alleviation (i.e., suppression) of the clinical signs or condition of the disease, or when administration results in full or partial immunity of an individual to the disease.

[0073] The compositions of the present invention having at least one influenza virus, including an attenuated influenza virus and one or more isolated other viruses, or one or more isolated other viral proteins, or one or more isolated nucleic acid molecules encoding the one or more viral proteins, or a combination thereof, can be administered by any means that achieves the intended purpose.

[0074] For example, administration of such compositions can be by a variety of parenteral routes, including subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral, transdermal, etc. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.

[0075] A typical regimen for preventing, inhibiting, or treating a condition associated with influenza virus involves administering an effective amount of the vaccine composition described herein as a single treatment, or repeatedly administered as increasing or booster doses over a period ranging from one week to up to 24 months, or any range or value therein.

[0076] According to the present invention, an "effective amount" of a composition is an amount sufficient to achieve a desired effect. It is understood that effective administration may depend on the recipient's species, age, sex, health, and weight; type of current treatment (if any); frequency of treatment; and type of desired effect. The effective amount ranges below are not intended to limit the invention, but represent dosage ranges.

[0077] The dose of live, attenuated, or killed virus vaccines for animals (such as adult mammals) is approximately 10 2 ~10 20 Plaque forming units (pfu) / kg, e.g. 10 3 ~10 12 pfu / kg, or 10 2 ~10 10 pfu / kg, or 10 5 ~10 11 pfu / kg, or 10 6 ~10 15 pfu / kg, or 10 2 ~10 10 pfu / kg, or 10 15 ~10 20 pfu / kg, or any range or value therein. For example, a dose of one of the virus isolate vaccines in an inactivated vaccine can range from about 0.1 to 1000 μg of HA protein, such as 0.1 to 10 μg, or 1 to 20 μg, or 30 to 100 μg, or 10 to 50 μg, or 50 to 200 μg, or 150 to 300 μg. However, the dose must be an amount that is safe and effective as determined by conventional methods using existing vaccines as a starting point.

[0078] The dose of immunoactive HA in each dose of replicating viral vaccine can be standardized to contain an appropriate amount (e.g., 30-100 μg, or any range or value therein), or an amount recommended by a government agency or recognized professional organization. The amount of NA can also be standardized, although this glycoprotein can be unstable during purification and storage.

[0079] The dose of immunoactive HA in each dose of the replicating virus vaccine can be standardized to contain an appropriate amount (e.g., 1-50 μg, or any range or value therein), or an amount recommended by the United States Public Health Service (PHS), which is typically 15 μg per component for children over 3 years of age and 7.5 μg per component for children under 3 years of age. The amount of NA can also be standardized, but this glycoprotein can be unstable during purification by processors and during storage (Kendal et al., 1980; Kerr et al., 1975). Each 0.5 ml dose of vaccine can contain approximately 100-500 million virus particles, or 500-2 billion virus particles, or 1-50 billion virus particles, or 1-10 billion virus particles, or 20-40 billion virus particles, or 1-5 billion virus particles, or 40-80 billion virus particles.

[0080] Representative Embodiments

[0081] In one embodiment, a method is provided for generating a plurality of influenza virus nucleic acid molecules encoding hemagglutinins (HA) with a reduced number of immunodominant epitopes. The method includes: introducing random mutations at a plurality of codons in an isolated parent influenza virus nucleic acid molecule encoding a hemagglutinin having an immunodominant epitope, where the mutations are at codons encoding residues within the immunodominant epitopes in the parent hemagglutinin, to provide a library of influenza virus nucleic acid molecules encoding mutant hemagglutinins; introducing the library into cells to provide a library of cells expressing mutant hemagglutinins; introducing the library into cells to provide a library of cells expressing mutant hemagglutinins; and identifying nucleic acid molecules encoding mutant hemagglutinins with substitutions and / or deletions at residue positions that form the immunodominant epitopes, resulting in a reduced number of immunodominant epitopes. In one embodiment, the cells are mammalian cells. In one embodiment, the hemagglutinin (HA) is H1, H2, H3, H5, H6, H7, or H9. In one embodiment, the mutant HA has a non-immune dominant residue at two or more of the following positions within H3: 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225.In one embodiment, within H3, the residue at position 121 is any of Q, R, I, L, V, T, S, F, Y, A; the residue at position 131 is any of R, A, M, K, V, S, Q, C, V, Y, D, E, L; the residue at position 135 is any of Y, G, R, M, K, N, V, W, S, V, P; the residue at position 138 is any of S, W, K, I, R, L; the residue at position 140 is any of L, M, T, S, R, K, M, P; the residue at position 142 is any of N, D, G, Y, Q, E, H, N, L, P; the residue at position 144 is any of T, K, V, G, D, H, L, Q; and the residue at position 145 is any of P, R, W, K. the residue at position 155 is C, F, H, I, R, A, V, S, or N; the residue at position 156 is P, G, S, M, R, T, A, or C; the residue at position 157 is D, P, S, G, I, R, or T; the residue at position 158 is R, V, S, A, K, C, or Q; the residue at position 171 is T, F, L, E, H, C, or R; the residue at position 189 is A, P, T, L, A, S, Y, or R; the residue at position 193 is Q, R, N, T, E, V, or P; the residue at position 212 is V, R, G, S, M, D, or E; and the residue at position 225 is L, P, C, S, Q, G, Y, or F. In one embodiment, within H3, the residue at position 121 is not N, the residue at position 131 is not T, the residue at position 135 is not T, the residue at position 138 is not A, the residue at position 140 is not I, the residue at position 142 is not R, the residue at position 144 is not S, the residue at position 145 is not S, the residue at position 155 is not T, the residue at position 156 is not H, the residue at position 157 is not L, the residue at position 158 is not N, the residue at position 171 is not N, the residue at position 189 is not K, the residue at position 193 is not F, the residue at position 212 is not A and the residue at position 225 is not D.In one embodiment, within H3, the residue at position 121 is N, the residue at position 131 is T, the residue at position 135 is T, the residue at position 138 is A, the residue at position 140 is I, the residue at position 142 is R, the residue at position 144 is S, the residue at position 145 is S, the residue at position 155 is T, the residue at position 156 is H, the residue at position 157 is L, the residue at position 158 is N, the residue at position 171 is N, the residue at position 189 is K, the residue at position 193 is F, the residue at position 212 is A, and the residue at position 225 is D. In one embodiment, the variant HA has a non-immune dominant residue at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, 189 in H5. In one embodiment, within H5, the residue at position 119 is not R, the residue at position 123 is not P, the residue at position 125 is not H, the residue at position 126 is not E, the residue at position 127 is not T, the residue at position 129 is not L, the residue at position 138 is not Q, the residue at position 140 is not A, the residue at position 141 is not S, the residue at position 151 is not I, the residue at position 152 is not K, the residue at position 153 is not K, the residue at position 154 is not N, the residue at position 155 is not D, the residue at position 156 is not A, the residue at position 185 is not A and the residue at position 189 is not N. In one embodiment, within H5, the residue at position 119 is R, the residue at position 123 is P, the residue at position 125 is H, the residue at position 126 is E, the residue at position 127 is T, the residue at position 129 is L, the residue at position 138 is Q, the residue at position 140 is A, the residue at position 141 is S, the residue at position 151 is I, the residue at position 152 is K, the residue at position 153 is K, the residue at position 154 is N, the residue at position 155 is D, the residue at position 156 is A, the residue at position 185 is A, and the residue at position 189 is N.In one embodiment, within H5, residue 119 is L, K, R, S, G, T, E, A, V, F, N; residue 123 is L, Y, I, M, N, S, V, K, G, T, R; residue 125 is L, D, N, G, W, M, I, R, K, F, A, S; and residue 126 is S, R, I, G, N, Q, A, N, R. the residue at position 127 is V, A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; and the residue at position 141 is R. , P, W, K, E, A, M, D, L, Q; the residue at position 151 is T, S, L, Y, K, N, Q; the residue at position 152 is A, P, T, Y, H, E, S, I, F, D; the residue at position 153 is R, Q, T, N, S, F, P, V, K; the residue at position 154 is L, T, D, R, P, S, H; the residue at position 155 is N, G, K, H, T, L, S, I, P, Q; the residue at position 156 is T, F, R, S, D, P, H, G, A, N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, S; and the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, H. In one embodiment, 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 of the codons encoding the residues that form the immunodominant epitope are mutated. In one embodiment, the variant HA has 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 non-immune dominant epitope residues.In one embodiment, the mutant HA has 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17 non-immune dominant epitope residues. In one embodiment, the mutant HA has 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 of the parent's immunodominant epitope residues. In one embodiment, the mutant HA has 1, or 2, or 3, or 4, or 5 of the parent's immunodominant epitope residues. In one embodiment, the nucleic acid molecule is identified using one or more antibodies that recognize the conserved subdominant epitopes. In one embodiment, the cells encoding the nucleic acid molecule are identified as cells that do not bind to one or more antibodies that recognize the immunodominant epitopes. In one embodiment, the nucleic acid molecule encoding the mutant hemagglutinin is sequenced.

[0082] In one embodiment, a method is provided for generating influenza viruses encoding mutant hemagglutinins that have one or more altered residues in one or more immunodominant epitopes compared to the parent influenza virus. The method includes introducing multiple mutations into the HA of the parent influenza virus at residues that form the immunodominant domain, and isolating or generating one or more influenza viruses with mutated HA. In one embodiment, multiple mutations are introduced into antigenic sites A and / or B in the HA, thereby generating a library of influenza viruses with mutant HA. Viruses in the library with different mutations in antigenic sites A and / or B (e.g., viruses that are less reactive with sera and bind to immunodominant epitopes in the parent HA) can be pooled to form a "pan" HA subtype-specific vaccine. In one embodiment, mutations are introduced into a nucleic acid molecule encoding an H3 HA at two or more of residues 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, 225 to encode residues that are not part of an immunodominant epitope in the parent virus, to generate one or more influenza viruses with a mutated H3 HA. In one embodiment, mutations encoding residues that do not form immunodominant epitopes of the parent virus are introduced into two or more of the following residues in a nucleic acid molecule encoding an H5 HA to generate one or more influenza viruses with a mutated H5 HA: 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, and 189.In one embodiment, in the mutated H3 HA, the residue at position 121 is either Q, R, I, L, V, T, S, F, Y, A; the residue at position 131 is either R, A, M, K, V, S, Q, C, V, Y, D, E, L; the residue at position 135 is either Y, G, R, M, K, N, V, W, S, V, P; the residue at position 138 is either S, W, K, I, R, L; the residue at position 140 is either L, M, T, S, R, K, M, P; the residue at position 142 is either N, D, G, Y, Q, E, H, N, L, P; the residue at position 144 is either T, K, V, G, D, H, L, Q; the residue at position 145 is either P, R, W, K; the residue at position 156 is either P, G, S, M, R, T, A, C; the residue at position 157 is either D, P, S, G, I, R, T; the residue at position 158 is either R, V, S, A, K, C, Q; the residue at position 171 is either T, F, L, E, H, C, R; the residue at position 189 is either A, P, T, L, A, S, Y, R; the residue at position 193 is either Q, R, N, T, E, V, P; the residue at position 212 is either V, R, G, S, M, D, E; and the residue at position 225 is either L, P, C, S, Q, G, Y, F. In one embodiment, within H3, the residue at position 121 is not N, the residue at position 131 is not T, the residue at position 135 is not T, the residue at position 138 is not A, the residue at position 140 is not I, the residue at position 142 is not R, the residue at position 144 is not S, the residue at position 145 is not S, the residue at position 155 is not T, the residue at position 156 is not H, the residue at position 157 is not L, the residue at position 158 is not N, the residue at position 171 is not N, the residue at position 189 is not K, the residue at position 193 is not F, the residue at position 212 is not A and the residue at position 225 is not D.In one embodiment, within H3, the residue at position 121 is N, the residue at position 131 is T, the residue at position 135 is T, the residue at position 138 is A, the residue at position 140 is I, the residue at position 142 is R, the residue at position 144 is S, the residue at position 145 is S, the residue at position 155 is T, the residue at position 156 is H, the residue at position 157 is L, the residue at position 158 is N, the residue at position 171 is N, the residue at position 189 is K, the residue at position 193 is F, the residue at position 212 is A, and the residue at position 225 is D. In one embodiment, the residue at position 119 is not R, the residue at position 123 is not P, the residue at position 125 is not H, the residue at position 126 is not E, the residue at position 127 is not T, the residue at position 129 is not L, the residue at position 138 is not Q, the residue at position 140 is not A, the residue at position 141 is not S, the residue at position 151 is not I, the residue at position 152 is not K, the residue at position 153 is not K, the residue at position 154 is not N, the residue at position 155 is not D, the residue at position 156 is not A, the residue at position 185 is not A and the residue at position 189 is not N.In one embodiment, the residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; and the residue at position 126 is S, R, I, G, N, Q, A, N, or R. the residue at position 127 is V, A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; and the residue at position 141 is R, P , W, K, E, A, M, D, L, Q; the residue at position 151 is T, S, L, Y, K, N, Q; the residue at position 152 is A, P, T, Y, H, E, S, I, F, D; the residue at position 153 is R, Q, T, N, S, F, P, V, K; the residue at position 154 is L, T, D, R, P, S, H; the residue at position 55 is N, G, K, H, T, L, S, I, P, Q; the residue at position 156 is T, F, R, S, D, P, H, G, A, N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, S; and the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, H. In one embodiment, the mutant HA has 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17 different residues (non-immune dominant epitope residues) at the parent immune dominant epitope positions.

[0083] In one embodiment, a composition is provided that includes a plurality of different recombinant influenza viruses, each of which encodes a hemagglutinin that includes a non-immune dominant epitope residue at the immune dominant epitope residue site (e.g., antigenic sites A and / or B). The different recombinant influenza viruses thus have different mutations at antigenic sites A and / or B. For example, recombinant influenza viruses that bind immune dominant epitopes in the parent HA with lower reactivity with sera can be pooled to form a "pan" HA subtype specific vaccine. In another embodiment, a composition includes a plurality of different recombinant influenza viruses, each of which encodes a hemagglutinin that includes a non-immune dominant epitope residue at the immune dominant epitope residue site (e.g., antigenic sites A and / or B), and at least two of the plurality of influenza viruses encode different subtypes of hemagglutinin. In one embodiment, one of the plurality of influenza viruses comprises a non-immune dominant epitope at two or more of the following positions within H3: 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, 225. In one embodiment, one of the plurality of influenza viruses comprises a non-immune dominant epitope at two or more of the following positions within H5: 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, 189. In one embodiment, the composition has at least two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more different recombinant influenza viruses.

[0084] Further provided is a method of immunizing an animal, comprising administering to the animal (e.g., human, dog, cat, cow, goat, sheep, horse, pig, bird) an effective amount of a composition comprising multiple viruses as described.

[0085] The invention is illustrated by the following non-limiting examples. EXAMPLES

[0086] Example 1 VLPs can be generated by expressing influenza HA together with a single viral matrix protein, so immune responses to other proteins of the influenza virus (M1, NA, etc.) will not affect the interpretation of the results. VLPs based on Ebola virus VP40 matrix protein are used because humans do not have antibodies to VP40, and therefore VLPs based on Ebola VP40 and expressing HA are efficiently formed.

[0087] We identified 17 amino acid positions in the human H3 HA protein that are known or predicted to affect antigenicity (e.g., 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, and 225; see Figure 1 ) and prepared a gene “library” that encoded all 20 amino acids at each of these 17 positions (resulting in a theoretically possible 20 amino acid sequence) in the genetic background of A / Tokyo / UT-IMS2-1 / 2014 (a clade 3c.2a virus). 17 A library of viruses with "mutated HA" proteins with up to 17 amino acid changes was generated and screened with human sera to identify antigen escape mutants. HA proteins with up to 17 amino acid changes within the antigenic epitope were found to be functional and antigenically distinct from the parent virus.

[0088] "Mutant HA proteins with up to 17 amino acid changes" can induce broadly protective immune responses. Immunization with a mixture of HAs with up to 17 amino acid changes in the positions responsible for immunodominance of the HA head is likely to induce antibodies against HA immunodominant epitopes. Moreover, stronger responses may be elicited against nondominant epitopes, resulting in increased cross-protective immunity compared to current vaccines. Such a vaccine could potentially overcome immune imprinting (biased immune response to the first influenza virus encountered).

[0089] Specifically, a VLP library is prepared with randomized sequences at 17 amino acid positions in the HA protein of the A / California / 7 / 2004 (Cal / 04) virus, a representative example of the "California / 2004" antigen cluster of human H3 viruses. As a control, VLPs containing wild-type HA protein are also generated. All VLPs are treated with sialidase to prevent self-aggregation. The reactivity of monoclonal antibodies against the head and stalk of HA (e.g., more than 20 and more than 10 monoclonal antibodies reacting with the head or stalk of Cal / 04 virus, respectively) is examined with VLPs containing the wild-type protein or the "mutant HA with up to 17 amino acid changes" protein. It is expected that the binding level of antibodies interacting with the stalk of HA will be similar between these two types of VLPs, whereas VLPs containing the "mutant HA with up to 17 amino acid changes" protein will likely have reduced binding to antibodies interacting with the head of HA compared to VLPs containing wild-type HA.

[0090] To test the immunogenicity and protective effect of H3 HA-VLPs against antigenically diverse human H3 viruses, ferrets were immunized twice with Cal / 04 H3 HA-VLPs. Four weeks after the second immunization, antibody titers were determined against VLPs containing wild-type HA protein or "mutated HA with up to 17 amino acid changes" protein. Ferrets were challenged with the homologous Cal / 04 virus or three different human H3 influenza viruses belonging to more recent antigenic clusters (e.g., "Perth / 2009", "Victoria / 2012" and the current 3c.2a1 clade). Virus titers were determined in nasal swabs using plaque assays. Alternatively, or in addition, ferrets will be "pre-immunized" with VLPs containing HA proteins from the "Wuhan / 1995", "Sydney / 1997" and "Fujian / 2002" clusters (these VLPs will be used in sequence to immunize the ferrets; immunizations will be spaced 2 weeks apart). After three sequential "pre-immunizations", the ferrets will be vaccinated with VLPs containing wild-type Cal / 04 HA protein or "mutated Cal / 04 HA with up to 17 amino acid changes" protein and challenged as above to determine whether vaccines eliciting antibodies against non-dominant epitopes can overcome immunogenic imprinting.

[0091] Immunization with wild-type virus protects ferrets against infection with the homologous virus but not with antigenically drifted variants belonging to different antigenic clades. Ferrets immunized with "mutant HA with ~17 amino acid changes" VLPs are better protected against antigenically drifted human H3N2 influenza viruses than ferrets immunized with wild-type virus. This establishes that immunization with "mutant HA with ~17 amino acid changes" proteins elicits broadly protective antibodies, possibly because the immune response is "redirected" toward immunosubdominant epitopes that are more conserved among human H3 viruses than the major immunodominant epitopes.

[0092] Individual mutants and / or libraries with a smaller number of randomized amino acid positions allow the identification of specific mutant HA proteins that elicit cross-protective immune responses against non-dominant epitopes.

[0093] Example 2 Generation and in vitro characterization of HA with multiple mutations in immunodominant epitopes (ID-EpiMut HA)

[0094] Considering that first exposure to a previously unencountered HA elicits high levels of Abs against conserved immune subdominant epitopes, we prepare a mixture of viruses encoding non-native intrahead immune dominant epitopes (i.e., HAs with mutated immune dominant epitopes). Each of these variants is a novel HA to the immune system, thus eliciting increased levels of cross-protective Abs against the conserved immune subdominant epitope regions within the HA. Experimental approach Millions of mutant influenza viruses are generated ("viral libraries") and variants with desired properties (such as non-native immune-dominant epitopes within the head region) are selected.

[0095] Viral library generation and screeningMethods are available for generating influenza virus "libraries" as well as other virus libraries (including Ebola VLP libraries), i.e., mixtures of viruses with random mutations at any or predefined amino acid positions in influenza virus proteins (Li et al., 2016; Ping et al., 2015 and 2016; Taft et al., 2015). Briefly, introduction of mutations at predefined positions in influenza virus cDNA is achieved by PCR or by commercial gene synthesis methods using degenerate oligonucleotides that code for "NNN" at the targeted codons. The resulting PCR or gene mixture is cloned into an RNA polymerase I vector for transcribing influenza virus RNA, resulting in a so-called "plasmid library." Following established protocols for reverse genetics, eukaryotic cells (e.g., 293T human embryonic fibroblast cells) are transfected with the plasmid library, seven RNA polymerase I plasmids to transcribe the seven remaining viral RNA segments (the influenza A virus genome contains eight segments of single-stranded RNA), and four protein expression plasmids to synthesize the viral polymerase proteins (PB2, PB1, PA) and nucleoprotein (NP), all of which are essential for initiating viral replication and transcription. As a result of this approach, a viral library consisting of millions of mutants is generated.

[0096] While classical experimental approaches examine one mutation at a time, our approach allows the simultaneous examination of millions of mutations. Thus, it (i) reproduces many steps of evolution in an experimental setting; (ii) covers a large "sequence space" (including variants not isolated in nature); (iii) allows competition between mutants (a crucial aspect of virus evolution); and (iv) eliminates non-viable mutants during the viral library generation step. The viral library is then screened for various biological characteristics, including antigenicity, receptor binding properties, polymerase activity, and viral titer. The power and versatility of this approach have been demonstrated by modeling the antigenic evolution of seasonal human H1N1 and H3N2 influenza viruses (Li et al., 2016), by isolating polymerase mutants that confer efficient replication capacity to avian influenza viruses in mammalian cells (Taft et al., 2016), and by selecting mutations that increase the titer of influenza A and B vaccine viruses (Ping et al., 2015 and 2016).

[0097] Generation and screening of H5 HA virus librariesTo better understand antigenic differences between pandemic H5N1 influenza viruses, random mutations were introduced at 17 amino acid positions known or suspected to affect the antigenic properties of these viruses (e.g., amino acid positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, and 189; numbers indicate amino acid positions in the mature H5 protein after removal of the signal peptide). These positions are clustered within the highly variable head immunodominant major antigenic epitope of H5N1 viruses. Gene libraries encoding all 20 theoretical amino acids at each of the 17 selected positions were obtained by chemical synthesis. These gene libraries were amplified by PCR, and the PCR products were cloned into an RNA polymerase I vector to generate the H5N1 virus library. In general, the size of the viral library is approximately 10 per ml of supernatant from transfected cells. 4 ~10 7 The viral library therefore contains every possible combination of amino acids at the 17 selected positions (i.e., 20 17 It contains three different combinations of amino acids.

[0098] After generating H5 virus libraries with random mutations at 17 amino acid positions within the HA, these libraries were incubated with ferret sera raised against different H5 viruses. During this selection step, variants with antigenic properties similar to those of currently circulating H5 viruses would be neutralized. The resulting antigenic escape variants were plaque purified in Madin-Darby canine kidney (MDCK) cells (a cell line commonly used to grow influenza viruses), and individual viruses were amplified and sequenced to identify the amino acid changes that caused antigenic escape. After antigenic selection, we isolated over 50 H5 variants that differed from the parent virus by 13–17 amino acids (see Table 1 for examples of selected sequences). This demonstrates the sequence plasticity of the highly variable intrahead immunodominant major antigenic epitope. Importantly, the majority of these variants are antigenically distinct from the parent virus, as demonstrated by hemagglutinin inhibition assays. Similarly, studies with seasonal human H3N2 viruses (see Example 1 and Tables 1B-1C) have yielded variants with multiple amino acid changes in highly variable intrahead immunodominant epitopes that are antigenically distinct from the parental virus. Together, these studies demonstrate that seasonal H3N2 and pandemic H5 viruses with up to 17 amino acid changes in immunodominant epitopes are viable, replicate efficiently, and are antigenically distinct from the parental viruses.

[0099] [Table 1]

[0100] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0101] [Table 3]

[0102] Selection of locations within HA for mutagenesis By generating H3N2 HA variants that harbor non-native immunodominant epitopes within the head, vaccination with a mixture of these viruses may attenuate antibody responses to the immunodominant epitopes and direct the immune response more toward conserved immunodominant epitopes within the head and stem regions of the HA.

[0103] Random mutations at 15 amino acid positionsRandom mutations were introduced at the following 15 amino acid positions in HA that are known or suspected to affect the antigenicity of H3 HA based on published and unpublished data (all numbers representing amino acid positions are relative to the "mature" H3 NA after removal of the signal peptide): 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 158, 159, 189, 193, and 212 (Figure 3). These sites are located primarily in the highly variable immunodominant epitopes A (131, 135, 138, 140, 142, 144, 145), B (155, 156, 158, 159, 189, 193), and C (121, 193) within the head region, and include seven amino acid positions where most seasonal H3N2 cluster changes have occurred (e.g., 145, 155, 156, 158, 159, 189, 193 (84)). Random mutations at these selected positions were introduced into the HA protein of the A / California / 7 / 2004 (CA04) virus, the prototype of the California 2004 (CA04) antigenic cluster of human H3N2 viruses. This older strain was chosen to allow testing of the protective effect of a vaccine based on ID-EpiMut CA04 against recent human H3N2 influenza viruses.

[0104] Briefly, a cDNA library containing random mutations at selected positions in CA04 HA is chemically synthesized, amplified by PCR, and cloned into an RNA polymerase I vector to generate a plasmid library.

[0105] Virus library generation Using this plasmid library with mutant HA, we generate a virus library in the genetic background of the high-yielding A / Puerto Rico / 8 / 34 (PR8) virus (Ping et al., 2015), which gives high virus titers in cultured cells. 6293T cells are transfected with 1 μg of the mutant HA plasmid library, 0.1 μg of each RNA polymerase I plasmid to transcribe the remaining viral RNA (all derived from the high-yield PR8 virus), and 0.1 μg of each polymerase protein expression plasmid for the polymerase and NP proteins. After 48 h, aliquots of supernatant are harvested from the transfected cells and plaque assays are performed in MDCK cells to assess the titer of the viral library. As mentioned above, typically approximately 10 mutated HA plasmids are transfected per ml of cell culture supernatant. 4 ~10 7 pfu mutant viruses are obtained. The library is amplified in AX-4 cells (MDCK cells that overexpress α2,6-linked sialic acid on their cell surface; human influenza viruses bind efficiently to this surface).

[0106] Selection of ID-EpiMut HA variants bearing immunodominant epitopes antigenically distinct from those in the head region of (the majority of) influenza viruses

[0107] To increase the level of antibody responses to immunodominant epitopes within the stem and head of HA, HA variants carrying non-native immunodominant epitopes within the head are generated. To select such variants, the virus library is incubated with a mixture of ferret sera raised against viruses of different antigenic clades and a mixture of human sera from donors of different age groups who had been exposed to a variety of viruses and vaccines during their lifetime.

[0108] Specifically, the virus library is incubated with a mixture of serum at various concentrations, followed by plaque assays in AX-4 cells. Viral plaques are picked from the highest concentration of serum at which plaques are detected. Individual viruses are amplified in AX-4 cells and their HA genes are sequenced. Over 100 individual HA genes are sequenced for each type of serum (human or ferret) and serum concentration.

[0109] Of particular interest are viruses with amino acids not commonly found at each amino acid position (e.g., less than 1% of sequences in the influenza search database). Highest priority is given to HAs with amino acids not frequently detected at each position in any subtype (e.g., less than 1% of sequences in the influenza search database). For all variants, the entire HA gene is sequenced to reveal whether additional mutations have emerged (outside the targeted amino acid positions) that may have compensatory functions, such as a stabilizing effect on HA. The 100 ID-EpiMut HA variants with the least sequence homology to known influenza viruses at the targeted positions are selected for further analysis (Figure 5).

[0110] Reactivity of ID-EpiMut HA variants to H3 HA-specific and stem-specific mAbs

[0111] After isolating ID-EpiMut HA variants with various sequences in the intrahead immune-dominant antigenic epitopes, the reactivity of these HA proteins is examined with a panel of H3 HA-specific Abs. A single population may contain more than 100 different Abs (Yamayoshi et al., 2017; Epstein et al., 2002). We examined the ability of these Abs to neutralize representative human H3N2 influenza viruses of all major antigenic clades (Table 2), and found that most of them neutralized only a portion of the tested viruses. This indicates that these Abs react with the highly variable intrahead immune-dominant antigenic epitopes A–E. However, some of these Abs did neutralize viruses from most major antigenic clusters of human H3N2 viruses (Table 2). Based on competition studies with known stem-reactive mAbs, these Abs were found to react with the head of HA.

[0112] [Table 4]

[0113] In addition, we examined the reactivity of ID-EpiMut HA variants with a panel of stem-reactive mAbs (Yamayoshi et al., 2017 and 2018) or mAbs synthesized based on published sequences (Corti et al., 2011) (Table 3), which recognize either group 2 HA (1417infC10) or group 1 and group 2 HA.

[0114] [Table 5]

[0115] The hemagglutinin inhibition (HI) assay measures the ability of antibodies to inhibit the binding of HA to red blood cells. The HI assay is frequently used to distinguish between antibodies that bind to the head (where the receptor binding pocket that mediates binding to sialic acid on the surface of red blood cells is located) and those that bind to the stem but do not interfere with the hemagglutinin. However, recent H3N2 viruses do not bind to commonly used red blood cells.

[0116] To test the reactivity of the 100 selected ID-EpiMut HA variants, the HA genes of these variants were cloned into protein expression plasmids and transfected into 293T cells. Wild-type CA04 HA protein served as a control. 24 hours after transfection, cells were fixed with 4% paraformaldehyde. HA-expressing cells were incubated with mAb (1 μg / ml) followed by incubation with peroxidase-labeled goat anti-human IgG, Fcγ fragment-specific antibody (Jackson Immuno-Research). TMB (3,3',5,5'-tetramethylbenzidine) solution was added for 5 min at room temperature, and the reaction was stopped by adding H2SO4. Optical density (OD450) was measured at 450 nm using a VersaMax plate reader (Molecular Devices). OD450 values ​​of mock-transfected wells incubated with each mAb were subtracted as background.

[0117] The reactivity of ID-EpiMut HA variants will be compared to that of wild-type HA. Mutants that lose reactivity to cluster-specific Abs (directed against highly variable intrahead immunodominant epitopes) but retain reactivity to broadly reactive H3-specific Abs and stem-specific Abs (directed against conserved immunodominant epitopes) will be identified. Up to 50 ID-EpiMut HA proteins (Figure 5) that fulfill these criteria will be recovered.

[0118] Example 3 Immunogenicity and protective efficacy of the ID-EpiMut vaccineEach variant generated by this method is likely to carry a novel antigen for the immune system. By mixing these variants, the vaccine contains small amounts of each of the non-native intrahead immune-dominant antigenic epitopes, but high amounts of the immune-subdominant epitopes (which are the same in all ID-EpiMut HAs). Such a vaccine will elicit more antibodies directed against the conserved immune-subdominant epitopes compared to vaccines that present only one wild-type HA (current practice for influenza vaccines). Higher levels of antibodies against the conserved immune-subdominant epitopes provide cross-protection. Experimental approach Mouse antisera to individual and mixed ID-EpiMut HA variants will be generated and tested for reactivity. Vaccination and challenge studies will be performed in mice and ferrets to evaluate whether a mixture of non-native immunodominant epitopes can attenuate responses to these epitopes while raising levels of antibodies to immunosubdominant epitopes, resulting in broader protective immunity.

[0119] Generation of virus-like particles harboring individual ID-EpiMut HA variantsIn addition to HA (the major influenza virus antigen), other influenza virus proteins, including NA, NP, matrix (M1) protein, and ion channel (M2) protein, contribute to the antigenicity of the virus. Indeed, immunity against NP and M1 is protective in mouse models. To avoid any confounding effects from these proteins, ID-EpiMut HA variants are presented on the surface of Ebola virus VLPs. By co-expressing Ebola virus VP40 and influenza virus HA, HA-modified VLPs are generated very efficiently. Furthermore, VLPs can be formed very efficiently using a 293T cell line stably expressing VP40. VP40-expressing 293T cells are transfected with protein expression plasmids encoding each of the 50 ID-EpiMut HA variants. After 2–3 days, cells are treated with bacterial neuraminidase, which efficiently releases VLPs from the cells. Cell culture supernatants containing released VLPs were harvested, purified through a sucrose gradient, concentrated by ultracentrifugation, and total protein yield was measured using a BCA assay (Thermo Scientific). The resulting 50 ID-EpiMut-HA / VP40 VLPs, each modified with a single HA mutant (Figure 5), were used to immunize mice.

[0120] Generation of virus-like particles harboring numerous ID-EpiMut HA variantsIn addition to VLPs modified with a single ID-EpiMut HA variant, we will generate eight different ID-EpiMut-HA / VP40 VLPs modified with different ID-EpiMut-HAs (Figure 5). Specifically, we will test five ID-EpiMut-HA / VP40 VLPs modified with each of the 10 ID-EpiMut HA variants, test two ID-EpiMut-HA / VP40 VLPs modified with all 25 ID-EpiMut HA variants, and test one ID-EpiMut-HA / VP40 VLP modified with all 50 ID-EpiMut HA variants (for the first two sets, we will randomly sort the ID-EpiMut HA variants into groups of 10 or 25, respectively). VP40-expressing 293T cells were co-transfected with different protein expression plasmids expressing different ID-EpiMut HAs. Displaying different HA mutants on the surface of the same VLP is likely to result in a smaller population of B cells specific for one particular mutant.

[0121] Generation and characterization of mouse sera directed against ID-EpiMut-HA / VP40 VLPs Mice (BALB / c female mice, Jackson Laboratories; 3 mice per group) were immunized intramuscularly with 10-20 μg of total protein from ID-EpiMut-HA / VP40 VLP, and two weeks later, the same amount of protein from ID-EpiMut-HA / VP40 VLP was injected intramuscularly to boost immunity. Three weeks after the second immunization, blood was collected.

[0122] We examined the reactivity of mouse sera to the following groups of HA proteins: (1) human H3N2 virus HA proteins derived from viruses that had not been amplified in embryonated chicken eggs (thus eliminating the risk of egg-adapted HA mutations affecting antigenicity) and representing all of the major antigenic clades; (2) ID-EpiMut HA variants; and (3) HA proteins representing several other HA subtypes (other subtypes include H1, H5, and H7 (for each of these subtypes, a panel of HA proteins representing the major antigenic clades and subclades was used)).

[0123] ELISA is used with purified HA protein using a protocol that expresses a secreted form of HA stabilized by a trimerization motif ("foldon") (Stevens et al., 2004). Interaction of mouse sera with purified HA protein is detected as described above. To assess the relative contribution of antibodies binding to the highly variable intrahead immunodominant epitopes, the conserved intrastem immunosubdominant epitopes, and the conserved intrahead immunosubdominant epitopes, competition assays are performed with human Abs known to bind these epitopes. Controls include wild-type HA protein and antisera raised against it, as well as the antigenically distant influenza B virus HA protein and antisera raised against it.

[0124] From the 50 ID-EpiMut-HA / VP40 VLPs modified with one EpiMut HA, the top 30 candidates with the highest proportion of antibodies reacting against conserved immunodominant antigen epitopes within the stem and head regions of HA are selected (Figure 5).

[0125] For eight ID-EpiMut-HA / VP40 VLPs modified with multiple EpiMut HAs, candidates were eliminated if they did not elicit increased amounts of antibodies against immune subdominant epitopes (compared to wild-type HA) (Figure 5).

[0126] Immunization of mice with a mixture of ID-EpiMut-HA / VP40 VLPs

[0127] Immunization with a mixture of ID-EpiMut HAs with multiple mutations in immune-dominant epitopes in the head appears to elicit relatively low antibody responses to each of the novel immune-dominant antigens, while enhancing responses to shared epitopes (e.g., conserved immune-subdominant epitopes in the stem and head of HA). To evaluate this, we investigate several different vaccination strategies. In these vaccination strategies, mice (e.g., groups of 5) are primed with a mixture of 10, 15, or 30 ID-EpiMut-HA / VP40 VLPs, each of which is modified with only one ID-EpiMut HA (Table 4a). Mice are either not boosted, boosted with the same ID-EpiMut-HA / VP40 VLPs used in the prime immunization, or boosted with a different set of 10 or 15 ID-EpiMut-HA / VP40 VLPs.

[0128] Similarly, mice were primed with only one ID-EpiMut-HA / VP40 VLP modified with many different mutants (Table 4b), and then either mock boosted, boosted with the same ID-EpiMut-HA / VP40 VLP, or boosted with a different ID-EpiMut-HA / VP40 VLP (this does not apply to mice vaccinated with ID-EpiMut-HA / VP40 VLPs modified with all 50 EpiMut HA mutants), and controls were either primed with HA / VP40 VLPs modified with wild-type HA protein, or primed and boosted with HA / VP40 VLPs modified with wild-type HA protein.

[0129] Serum was collected 28 days after the last immunization and the levels of antibodies against immune subdominant epitopes were examined as described above. Comparison of different vaccination strategies will determine whether two immunizations with the same mixture of ID-EpiMut-HA / VP40 VLPs increase the amount of antibodies against immune subdominant epitopes compared to only one immunization. Comparison of different vaccination strategies will also determine whether a prime / boost regime with different ID-EpiMut-HA / VP40 VLPs increases the amount of antibodies against immune subdominant epitopes compared to a prime / boost regime with the same ID-EpiMut-HA / VP40 VLPs. Furthermore, comparison of different vaccination strategies will determine whether a single VLP decorated with multiple HA mutants (see Table 4b) induces more antibodies against immune subdominant epitopes than multiple VLPs decorated with each single HA mutant (see Table 4a). Comparison of VLPs decorated with 10, 25, or 50 HA variants also provides information regarding the number of different HAs required to attenuate an immune response to an immunodominant epitope within the HA head.

[0130] If a mixture of 10 ID-EpiMut HAs (provided from one or ten VLPs) attenuates the immune response to an immunodominant epitope, similar experiments are performed using mixtures of five or three ID-EpiMut HAs to determine the minimum number of different ID-EpiMut HAs required for this attenuating effect.

[0131] From the different vaccination regimens investigated here, the top 10 are selected (eg, those with the highest levels of antibodies against immune subdominant epitopes) for protection studies in mice (FIG. 5).

[0132] [Table 6]

[0133] [Table 7]

[0134] Challenge studies in mice vaccinated with ID-EpiMut-HA / VP40 VLPs

[0135] Having demonstrated that a mixture of highly mutated intrahead immune-dominant antigenic epitopes attenuates immune responses to these epitopes while enhancing the levels of antibodies to conserved immune-subdominant epitopes, we then determined whether this increased level of antibodies to immune-subdominant epitopes confers broader protection against seasonal human H3N2 viruses compared to the protection induced by vaccines based on wild-type viruses.

[0136] The top 10 vaccination regimens eliciting elevated levels of antibodies against conserved immune subdominant epitopes will be evaluated for protective efficacy of antibodies raised against ID-EpiMut-HA. First, 48 mice will each be vaccinated with the selected vaccination regimen. Recent human H3N2 influenza viruses do not replicate efficiently in mice. Utilizing established strategies for generating mouse-adapted viruses, mouse-adapted variants of CA04 (for homologous challenge) and viruses representing the Perth 2009 (PE09), Victoria 2011 (VI11), and Hong Kong 2014 (HK14) antigenic clusters will be generated for heterologous challenge. To exclude the effect of mouse adaptation mutations on antigenicity, the reactivity of wild-type and mouse-adapted viruses will be compared using sera directed against wild-type HA. In cases where the mouse-adapted variants are antigenically similar to the wild-type virus, the above four mouse-adapted variants will be vaccinated against 10 different strains of the wild-type virus. 6pfu doses were used to challenge each of 12 vaccinated mice. Four mice per challenge group were observed for weight loss, and the remaining eight mice were euthanized 3 and 6 days after challenge (four mice per time point) to assess virus titers in the lungs and nasal turbinates. Vaccination with ID-EpiMut HA elicits Abs that are more broadly protective than those elicited after vaccination with wild-type HA.

[0137] Immunogenicity of ID-EpiMut HA influenza vaccine in ferrets

[0138] The immunogenicity of the top three vaccination regimens in ferrets will be examined (Figure 5). In ferrets, as in humans, the contribution of "internal" influenza viral proteins is less than in mice. Therefore, vaccination and challenge experiments in ferrets will be performed with influenza virus-based vaccines (e.g., viruses with the respective ID-EpiMut HA generated in the genetic background of PR8 virus). Recombinant viruses with ID-EpiMut HA will be inactivated with β-propiolactone (an established procedure for inactivating influenza viruses) and an amount equivalent to 15 μg of HA protein will be used for vaccination.

[0139] Protective effect of ID-EpiMut HA influenza vaccine in ferrets

[0140] We next examined whether vaccination of ferrets with the ID-EpiMut HA influenza vaccine confers broader protection than that induced by a vaccine based on wild-type HA. Ferrets (five per group) were immunized with the top three vaccination schedules (as determined above) (Figure 5). Twenty-eight days after the last immunization, ferrets were intranasally injected with 10 6pfu of the homologous CA04 virus or viruses representing the more recent Perth 2009 (PE09), Victoria 2011 (VI11), or Hong Kong 2014 (HK14) antigenic clusters (heterologous challenge). Starting 1 day after challenge, infected ferrets are weighed daily and nasal wash samples are collected every 2 days to determine virus titers. Vaccination with an inactivated influenza vaccine carrying wild-type HA is expected to protect against challenge with the homologous CA04 virus but provide incomplete protection against heterologous viruses (belonging to different antigenic clusters). The finding that vaccination with ID-EpiMut HA influenza vaccine protects against infection with antigenic drift variants underscores the feasibility of the concept of a universal influenza vaccine.

[0141] [Table 8]

[0142] Five ferrets per group (female, 4-6 months old) are immunized intramuscularly. 28 days after the last immunization, serum is collected and the reactivity of this serum is examined as described above. Control ferrets are immunized with inactivated influenza vaccine carrying wild-type HA. Immunization with ID-EpiMut HA influenza vaccine elicits higher amounts of antibodies directed against conserved immune subdominant epitopes compared to vaccination with vaccine carrying wild-type HA.

[0143] Protective effect of ID-EpiMut HA influenza vaccine in ferrets

[0144] We next examined whether vaccination of ferrets with the ID-EpiMut HA influenza vaccine confers broader protection than that induced by a vaccine based on wild-type HA. Ferrets (five per group) are immunized with the top three vaccination schedules (as determined above) (Figure 5). Twenty-eight days after the last immunization, ferrets are intranasally injected with 106 pfu of the homologous CA04 virus or viruses representing the more recent Perth 2009 (PE09), Victoria 2011 (VI11), or Hong Kong 2014 (HK14) antigenic clusters (heterologous challenge). Starting 1 day after challenge, infected ferrets are weighed daily and nasal wash samples are collected every 2 days to determine virus titers. Vaccination with an inactivated influenza vaccine carrying wild-type HA protects against challenge with the homologous CA04 virus but provides incomplete protection against heterologous viruses (belonging to different antigenic clusters). The finding that vaccination with ID-EpiMut HA influenza vaccine protects against infection with antigenic drift variants underscores the universal influenza vaccine.

[0145] Protective effect of ID-EpiMut HA influenza vaccine in ferrets previously exposed to human influenza virus

[0146] Humans are repeatedly exposed to influenza viruses through natural infection and / or vaccination. To mimic this exposure to multiple influenza viruses, ferrets are sequentially infected with seasonal human H3N2 viruses representing three past antigenic clusters: the Wuhan 1995 (WU95) cluster, the Sydney 1997 (SY97) cluster, and the Fujian 2002 (FU02) cluster (Table 5). Ferrets are then vaccinated with each of the top three vaccination approaches. Twenty-eight days after the last immunization, serum samples are collected from vaccinated ferrets and serum reactivity is examined. Sera from ferrets vaccinated with the ID-EpiMut HA influenza vaccine are more cross-reactive than sera obtained from ferrets immunized with an influenza vaccine carrying a wild-type HA.

[0147] The pre-exposed and vaccinated ferrets were then intranasally injected with 10 6Ferrets were infected with pfu of the homologous CA04 virus or the heterologous PE09, VI11, or HK14 viruses (five ferrets per group, Table 5). Nasal washes were collected every 2 days with daily body weight measurements starting 1 day after challenge as previously described. The ID-EpiMut HA influenza vaccine provides broader protection against viruses with different antigenic clusters than influenza vaccines based on wild-type HA.

[0148] Taken together, the reactivity and neutralization properties of mouse sera raised against the ID-EpiMut HA variants indicate that these sera are more broadly reactive and neutralizing than sera raised against wild-type HA. The vaccination / challenge studies establish that the vaccine approach of the present disclosure provides broader protective immunity than that conferred by current vaccines based on wild-type HA.

[0149] Thus, a mixture of non-native immune-dominant epitopes in the head can attenuate the immune response to these epitopes and redirect the immune response to conserved immune-subdominant epitopes in the HA, thereby increasing the amount of cross-protective antibodies and thereby providing a universal influenza vaccine.

[0150] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0151] References

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[0153] All publications, patents, and patent applications are incorporated herein by reference. Although the present invention has been described herein above with respect to certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present invention is capable of additional embodiments and that several of the details set forth herein may be significantly modified without departing from the underlying principles of the invention. Some aspects of the invention are described below. 1. A method for generating a plurality of influenza virus nucleic acid molecules encoding hemagglutinin (HA) having one or more altered residues within one or more immunodominant epitopes, comprising: providing a library of influenza nucleic acid molecules encoding mutant influenza virus hemagglutinins by introducing random mutations into multiple codons of one or more immune dominant epitopes of an isolated parent influenza nucleic acid molecule encoding an influenza virus hemagglutinin having at least two immune dominant epitopes; introducing the library into cells to provide a library of cells expressing the mutant hemagglutinins; and and identifying mutant hemagglutinins encoded by the library that have one or more substitutions and / or deletions at residues that form the one or more immunodominant epitopes, and thus have a reduced number of immunodominant epitopes compared to the parent hemagglutinin. A method comprising: 2. The method according to item 1, wherein the mutation is introduced into immunodominant epitopes (antigenic sites) A, B, or A and B. 3. The method according to item 1 or 2, wherein the mutation is introduced into immunodominant epitope C, D or E, or any combination thereof. 4. The method according to any one of items 1 to 3, wherein at least 5, 10, 15, or 20 codons, or any integer number between 5 and 20 codons, are mutated. 5. The method according to any one of items 1 to 4, wherein the mutant hemagglutinin is identified using an antibody or other hemagglutinin-binding molecule. 6. The method of claim 5, wherein at least one of the antibodies or other hemagglutinin-binding molecules binds to an immunodominant epitope of the parent hemagglutinin or a different influenza virus of the same HA subtype. 7. The method of claim 5 or 6, further comprising contacting one or more members of the library with at least one antibody or other hemagglutinin-binding molecule that binds to a conserved region within the stem of the hemagglutinin. 8. The method according to any of items 1 to 7, wherein the mutated immunodominant epitope corresponds to residues 121-146 in H3 HA (site A), residues 156-196 in H3 HA (site B), residues 50-57 or residues 275-279 in H3 HA (site C), residues 164, residues 182, or residues 208-217 in H3 HA (site D), or residues 62-83 in H3 HA (site E). 9. The method according to any one of items 1 to 8, wherein the cell is a mammalian cell. 10. The method according to any of items 1 to 9, wherein the hemagglutinin (HA) is H1, H2, H3, H5, H6, H7, or H9. 11. The method according to any one of items 1 to 10, wherein the mutant HA has a substitution at two or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225 in H3, or a deletion at one or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or a combination thereof. 12. In H3, the residue at position 121 is Q, R, I, L, V, T, S, F, Y, or A; the residue at position 131 is R, A, M, K, V, S, Q, C, V, Y, D, E, or L; the residue at position 135 is Y, G, R, M, K, N, V, W, S, V, or P; the residue at position 138 is S, W, K, I, R, or L; the residue at position 140 is L, M, T, S, R, K, M, or P; the residue at position 142 is N, D, G, Y, Q, E, H, N, L, or P; the residue at position 144 is T, K, V, G, D, H, L, or Q; the residue at position 145 is P, R, W, or K; and the residue at position 155 is C, F. , H, I, R, A, V, S, or N; the residue at position 156 is P, G, S, M, R, T, A, or C; the residue at position 157 is D, P, S, G, I, R, or T; the residue at position 158 is R, V, S, A, K, C, or Q; the residue at position 171 is T, F, L, E, H, C, or R; the residue at position 189 is A, P, T, L, A, S, Y, or R; the residue at position 193 is Q, R, N, T, E, V, or P; the residue at position 212 is V, R, G, S, M, D, or E; or the residue at position 225 is L, P, C, S, Q, G, Y, or F. 13. The method according to any of items 1 to 12, wherein the residue at position 121 is not N, the residue at position 131 is not T, the residue at position 135 is not T, the residue at position 138 is not A, the residue at position 140 is not I, the residue at position 142 is not R, the residue at position 144 is not S, the residue at position 145 is not S, the residue at position 155 is not T, the residue at position 156 is not H, the residue at position 157 is not L, the residue at position 158 is not N, the residue at position 171 is not N, the residue at position 189 is not K, the residue at position 193 is not F, the residue at position 212 is not A or the residue at position 225 is not D. 14. The method according to any of items 1 to 13, wherein the non-mutated residue at position 121 is N, the non-mutated residue at position 131 is T, the non-mutated residue at position 135 is T, the non-mutated residue at position 138 is A, the non-mutated residue at position 140 is I, the non-mutated residue at position 142 is R, the non-mutated residue at position 144 is S, the non-mutated residue at position 145 is S, the non-mutated residue at position 155 is T, the non-mutated residue at position 156 is H, the non-mutated residue at position 157 is L, the non-mutated residue at position 158 is N, the non-mutated residue at position 171 is N, the non-mutated residue at position 189 is K, the non-mutated residue at position 193 is F, the non-mutated residue at position 212 is A or the non-mutated residue at position 225 is D. 15. The method according to any one of items 1 to 10, wherein the mutant HA has a substitution at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, 189 in H5, or a deletion at one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, 189, or any combination thereof. 16. The method according to any of items 1 to 10 or 15, wherein the residue at position 119 is not R, the residue at position 123 is not P, the residue at position 125 is not H, the residue at position 126 is not E, the residue at position 127 is not T, the residue at position 129 is not L, the residue at position 138 is not Q, the residue at position 140 is not A, the residue at position 141 is not S, the residue at position 151 is not I, the residue at position 152 is not K, the residue at position 153 is not K, the residue at position 154 is not N, the residue at position 155 is not D, the residue at position 156 is not A, the residue at position 185 is not A or the residue at position 189 is not N. 17. The method according to any one of items 1 to 10 or item 15, wherein the non-mutated residue at position 119 is R, the non-mutated residue at position 123 is P, the non-mutated residue at position 125 is H, the non-mutated residue at position 126 is E, the non-mutated residue at position 127 is T, the non-mutated residue at position 129 is L, the non-mutated residue at position 138 is Q, the non-mutated residue at position 140 is A, the non-mutated residue at position 141 is S, the non-mutated residue at position 151 is I, the non-mutated residue at position 152 is K, the non-mutated residue at position 153 is K, the non-mutated residue at position 154 is N, the non-mutated residue at position 155 is D, the non-mutated residue at position 156 is A, the non-mutated residue at position 185 is A or the non-mutated residue at position 189 is N. 18. The residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; the residue at position 126 is S, R, I, G, N, Q, A, N, or R; and the residue at position 127 is V. , A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; the residue at position 141 is R, P, W, K, E, A, M, D, L, the residue at position 151 is T, S, L, Y, K, N, or Q; the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D; the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, L, S, I, the residue at position 156 is T, F, R, S, D, P, H, G, A, or N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H. 19. The method according to any of items 1 to 18, wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the codons encoding residues forming the immunodominant epitope are mutated. 20. The method according to any of items 1 to 19, wherein the mutant HA has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions. 21. The method according to any of items 1 to 19, wherein the mutant HA has 10, 11, 12, 13, 14, 15, 16, or 17 substitutions. 22. The method according to any of items 1 to 21, wherein the mutant HA has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues without substitution or deletion within the one or more immunodominant epitopes. 23. The method according to any of items 1 to 21, wherein the mutant HA has at least 10, 15, 20, 25, 30, 35, 40, or 45 residues without substitutions or deletions within the immunodominant epitope. 24. The method according to any one of items 1 to 23, further comprising sequencing the nucleic acid molecule encoding the mutant hemagglutinin. 25. A method for producing an influenza virus encoding a mutant hemagglutinin having an altered immunodominant epitope compared to a parent influenza virus, comprising: introducing a mutation in two or more codons for residues at positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225 in a parent H3 HA nucleic acid molecule that encodes a residue that is not a residue of an immunodominant epitope in the parent H3 HA; and Isolating or producing one or more influenza viruses with this mutated H3 HA; The method includes: 26. The method of claim 25, wherein the mutated H3 is recognized by an antibody that binds to a subdominant epitope but is not recognized by an antibody that binds to the immunodominant epitope. 27. In the mutated H3 HA, the residue at position 121 is Q, R, I, L, V, S, F, Y, or A; the residue at position 131 is R, V, S, Q, C, V, Y, D, E, or L; the residue at position 135 is Y, K, N, V, W, G, S, V, or P; the residue at position 138 is W, K, I, F, R, or L; the residue at position 140 is L, M, T, S, R, K, M, Y, or P; the residue at position 142 is N, G, Y, Q, E, H, N, or Q; the residue at position 144 is T, V, G, D, P, H, L, K, or Q; the residue at position 145 is P, D, R, W, or N; and the residue at position 155 is C, I, R, 27. The method of claim 25 or 26, wherein the residue at position 156 is A, V, S, or Q; the residue at position 157 is D, P, S, G, I, Q, R, or T; the residue at position 158 is R, V, S, A, K, C, Q, or G; the residue at position 171 is T, F, L, E, H, V, or R; the residue at position 189 is A, P, T, L, S, Y, or R; the residue at position 193 is Q, R, N, T, E, V, or P; the residue at position 212 is V, R, G, S, M, D, or E; or the residue at position 225 is L, P, C, S, Q, G, Y, or F. 28. The method according to any of items 25 to 27, wherein in H3, the residue at position 121 is not N, the residue at position 131 is not T, the residue at position 135 is not T, the residue at position 138 is not A, the residue at position 140 is not I, the residue at position 142 is not R, the residue at position 144 is not S, the residue at position 145 is not S, the residue at position 155 is not T, the residue at position 156 is not H, the residue at position 157 is not L, the residue at position 158 is not N, the residue at position 171 is not N, the residue at position 189 is not K, the residue at position 193 is not F, the residue at position 212 is not A or the residue at position 225 is not D. 29. The method according to any of items 25 to 28, wherein in H3, the residue at position 121 is N, the residue at position 131 is T, the residue at position 135 is T, the residue at position 138 is A, the residue at position 140 is I, the residue at position 142 is R, the residue at position 144 is S, the residue at position 145 is S, the residue at position 155 is T, the residue at position 156 is H, the residue at position 157 is L, the residue at position 158 is N, the residue at position 171 is N, the residue at position 189 is K, the residue at position 193 is F, the residue at position 212 is A, or the residue at position 225 is D. 30. A method for producing an influenza virus encoding a mutant hemagglutinin having an altered immunodominant epitope compared to a parent influenza virus, comprising: introducing a mutation in two or more codons for residues at positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189 in a parent H5 HA nucleic acid molecule that encodes a residue that is not a residue of an immunodominant epitope in the parent H5 HA; and Isolating or producing an influenza virus with this mutated H5 HA; The method includes: 31. The method of claim 30, wherein the mutated H5 is recognized by an antibody that binds to a subdominant epitope but is not recognized by an antibody that binds to the immunodominant epitope. 32. The method according to item 30 or 31, wherein the residue at position 119 is not R, the residue at position 123 is not P, the residue at position 125 is not H, the residue at position 126 is not E, the residue at position 127 is not T, the residue at position 129 is not L, the residue at position 138 is not Q, the residue at position 140 is not A, the residue at position 141 is not S, the residue at position 151 is not I, the residue at position 152 is not K, the residue at position 153 is not K, the residue at position 154 is not N, the residue at position 155 is not D, the residue at position 156 is not A, the residue at position 185 is not A or the residue at position 189 is not N. 33. The residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; the residue at position 126 is S, R, I, G, N, Q, A, N, or R; and the residue at position 127 is , V, A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; the residue at position 141 is R, P, W, K, E, A, M, D, the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D; the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, the residue at position 156 is T, F, R, S, D, P, H, G, A, or N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H. 34. The method according to any of items 25 to 33, wherein the mutant HA has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 substitutions. 35. A virus produced by the method according to any one of items 25 to 34. 36. A composition comprising a plurality of different recombinant influenza H3 viruses, each encoding a hemagglutinin that contains a plurality of residues that are antigenically different compared to the residues that form an immunodominant epitope in the parent virus, each of the plurality of influenza viruses comprises a substitution at two or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225 in H3, or a deletion at one or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or any combination thereof; composition. 37. The composition of item 36, having at least three, four, or five different viruses with said substitutions. 38. The composition according to item 36, having 5 to 10 different viruses with the substitution. 39. The composition according to item 36, having 10 to 20 different viruses with said substitutions. 40. The composition according to any of items 36 to 39, wherein each of the different viruses has at least 1 to 5 substitutions in antigenic site A or antigenic site B. 41. The composition according to any of items 36 to 39, wherein each of the different viruses has at least 1 to 10 substitutions in antigenic site A and antigenic site B. 42. The composition according to any of items 36 to 41, wherein each different virus has altered binding to an antibody that binds to the corresponding parent hemagglutinin. 43. The composition according to any of items 36 to 42, having one or more viruses in Table 1B. 44. The composition according to any of items 36 to 42, having one or more viruses in Table 6. 45. A composition comprising a plurality of different recombinant influenza H5 viruses, each encoding a hemagglutinin that contains a plurality of residues that are antigenically different compared to the residues that form an immunodominant epitope in the parent virus, each of the plurality of influenza viruses comprises within H5 a substitution at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or a deletion at one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or any combination thereof; composition. 46. ​​The composition according to item 45, having at least three, four, or five different viruses with said substitutions. 47. The composition according to item 45, having 5 to 10 different viruses with said substitutions. 48. The composition according to item 45, having 10 to 20 different viruses with said substitutions. 49. The composition according to any of items 45 to 48, wherein each of the different viruses has at least 1 to 5 substitutions in antigenic site A or antigenic site B. 50. The composition according to any of items 36 to 48, wherein each of the different viruses has at least 1 to 10 substitutions in antigenic site A and antigenic site B. 51. The composition according to any of items 36 to 50, wherein each of the different viruses has altered binding to an antibody that binds to the corresponding parent viral HA. 52. The composition according to item 45, having one or more viruses in Table 1 or Table 6. 53. The composition according to item 36 or 56, having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different recombinant influenza viruses. 54. The composition according to item 36 or 56, having 10 to 20 different recombinant influenza viruses. 55. A method for immunizing an animal, comprising administering to the animal an effective amount of the composition according to any one of items 36 to 54. 56. An H5 HA, wherein in the H5 HA, the residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; the residue at position 126 is S, R, I, G, N, Q, A, N, or R; and the residue at position 127 is V, A, S. , M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; the residue at position 141 is R, P, W, K, E, A, M, D, L, or Q; the residue at position 1 is T, S, L, Y, K, N, or Q; the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D; the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, L, S, I, P, or Q; is T, F, R, S, D, P, H, G, A, or N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H, or the residues at these positions are any combination of these residues.

Claims

1. 1. A method for generating a plurality of influenza virus nucleic acid molecules encoding H3 or H5 hemagglutinins (HA) having one or more altered residues within one or more immunodominant epitopes, comprising: providing a library of influenza virus nucleic acid molecules encoding mutant influenza virus hemagglutinins by introducing random mutations into multiple codons of one or more immune dominant epitopes of an isolated parent influenza virus nucleic acid molecule encoding an influenza virus H3 or H5 hemagglutinin having at least two immune dominant epitopes; introducing the library into cells to provide a library of cells expressing the mutant influenza virus hemagglutinins; and identifying mutant hemagglutinins encoded by the library that have one or more substitutions and / or deletions at residues that form the one or more immunodominant epitopes, resulting in a reduced number of immunodominant epitopes compared to the hemagglutinin of the parent influenza virus, wherein if the mutant hemagglutinin of H3 has substitutions, the mutant hemagglutinin has more than five substitutions compared to the H3 hemagglutinin of the parent influenza virus; A method comprising:

2. In H3HA, residue 121 is Q, R, I, L, V, T, S, F, Y, or A; residue 131 is R, A, M, K, V, S, Q, C, V, Y, D, E, or L; residue 135 is Y, G, R, M, K, N, V, W, S, V, or P; residue 138 is S, W, K, I, R, or L; residue 140 is L, M, T, S, R, K, M, or P; residue 142 is N, D, G, Y, Q, E, H, N, L, or P; residue 144 is T, K, V, G, D, H, L, or Q; the residue at position 145 is P, R, W, or K; the residue at position 155 is C, F, H, I, R, A, V, S, or N; the residue at position 156 is P, G, S, M, R, T, A, or C; the residue at position 157 is D, P, S, G, I, R, or T; the residue at position 158 is R, V, S, A, K, C, or Q; the residue at position 171 is T, F, L, E, H, C, or R; the residue at position 189 is A, P, T, L, A, S, Y, or R; and the residue at position 193 is Q, R, N, T, E, or V or P, or the residue at position 212 is V, R, G, S, M, D, or E, or the residue at position 225 is L, P, C, S, Q, G, Y, or F, or in H5HA, the residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; the residue at position 126 is S, R, I, G, N, Q, A, N, or R; is V, A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; the residue at position 141 is R, P, W, K, E, A, M, D, L, or Q; the residue at position 151 is T, S, L, Y, K, N, or Q; and the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D;2. The method of claim 1, wherein the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, L, S, I, P, or Q; the residue at position 156 is T, F, R, S, D, P, H, G, A, or N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H.

3. The method of claim 1 or 2, wherein the mutant HA has 10 or more substitutions.

4. 1. A method for producing a plurality of influenza viruses comprising a nucleic acid encoding a mutant hemagglutinin having an altered immunodominant epitope relative to a parent influenza virus, comprising: introducing a mutation into two or more codons for residues at positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225 in a parent H3 HA nucleic acid molecule that encodes a residue that is not a residue of an immunodominant epitope in the parent H3 HA; or introducing a mutation into two or more codons for residues at positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189 in a parent H5 HA nucleic acid molecule that encodes a residue that is not a residue of an immunodominant epitope in the parent H5 HA. introducing mutations that encode residues that are not immunodominant epitope residues in HA, where mutations in codons that encode residues that are not immunodominant epitope residues provide a variety of antigenically distinct immunodominant epitopes for immune recognition; and isolating or producing one or more influenza viruses having the mutated H3 or H5 HA, wherein the mutated H3 nucleic acid has more than five codons mutated to encode amino acid substitutions; The method includes:

5. or in said mutated H3 HA, the residue at position 121 is Q, R, I, L, V, S, F, Y, or A; the residue at position 131 is R, V, S, Q, C, V, Y, D, E, or L; the residue at position 135 is Y, K, N, V, W, G, S, V, or P; the residue at position 138 is W, K, I, F, R, or L; the residue at position 140 is L, M, T, S, R, K, M, Y, or P; the residue at position 142 is N, G, Y, Q, E, H, N, or Q; and the residue at position 144 is T, V, G, D, P, H, L, K, or Q. , the residue at position 145 is P, D, R, W, or N; the residue at position 155 is C, I, R, A, V, S, or Q; the residue at position 156 is P, G, S, T, A, or C; the residue at position 157 is D, P, S, G, I, Q, R, or T; the residue at position 158 is R, V, S, A, K, C, Q, or G; the residue at position 171 is T, F, L, E, H, V, or R; the residue at position 189 is A, P, T, L, S, Y, or R; and the residue at position 193 is Q, R, N, T, E, V, or P. or the residue at position 212 is V, R, G, S, M, D, or E, or the residue at position 225 is L, P, C, S, Q, G, Y, or F, or in H5HA, the residue at position 119 is L, K, R, S, G, T, E, A, V, F, or N; the residue at position 123 is L, Y, I, M, N, S, V, K, G, T, or R; the residue at position 125 is L, D, N, G, W, M, I, R, K, F, A, or S; the residue at position 126 is S, R, I, G, N, Q, A, N, or R; and the residue at position 127 is the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, or V; the residue at position 141 is R, P, W, K, E, A, M, D, L, or Q; the residue at position 151 is T, S, L, Y, K, N, or Q; and the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D;5. The method of claim 4, wherein the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, L, S, I, P, or Q; the residue at position 156 is T, F, R, S, D, P, H, G, A, or N; the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S; or the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H.

6. 5. The method of claim 4, wherein in H3HA, the residue at position 121 is N, the residue at position 131 is T, the residue at position 135 is T, the residue at position 138 is A, the residue at position 140 is I, the residue at position 142 is R, the residue at position 144 is S, the residue at position 145 is S, the residue at position 155 is T, the residue at position 156 is H, the residue at position 157 is L, the residue at position 158 is N, the residue at position 171 is N, the residue at position 189 is K, the residue at position 193 is F, the residue at position 212 is A, or the residue at position 225 is D.

7. The method of claim 4, 5 or 6, wherein the mutant HA has at least 6 or at least 10 substitutions.

8. 1. A composition comprising a plurality of different recombinant influenza viruses, each of which comprises a nucleic acid molecule encoding a variant H3 or H5 hemagglutinin (HA) that comprises a plurality of antigenically distinct residues, Each of the plurality of different recombinant influenza viruses of H3 comprises a substitution at two or more of residues at positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or a deletion at one or more of residues at positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or any combination thereof, in the H3 HA, wherein the substitution is a substitution that is identical to that of the parent H3 or each of the plurality of different recombinant influenza viruses of H5 comprises a substitution at two or more of residues at positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or a deletion at one or more of residues at positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or any combination thereof, wherein the substitution is a residue that is not an immunodominant epitope residue of HA. and wherein at least one of the plurality of different H3 recombinant influenza viruses has an H3 hemagglutinin with more than five substitutions compared to the parent H3 HA, and wherein the codon substitutions or deletions provide a variety of antigenically distinct immunodominant epitopes for immune recognition. composition.

9. 9. The composition of claim 8, comprising at least 5 or at least 10 different recombinant influenza viruses having said substitutions.

10. The composition of claim 8, comprising one or more viruses comprising a mutant H3 HA in Table 1B or Table 6 below, wherein the mutations in Table 1B and Table 6 are compared to the influenza A Tokyo 2 (TK / 2) parent virus. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】

11. Each of the plurality of different recombinant influenza viruses comprises an H5 HA, wherein within the H5 HA, residue 119 is L, K, R, S, G, T, E, A, V, F, or N; residue 123 is L, Y, I, M, N, S, V, K, G, T, or R; residue 125 is L, D, N, G, W, M, I, R, K, F, A, or S; residue 126 is S, R, I, G, N, Q, A, N, or R; the residue at position 127 is V, A, S, M, L, K, F, or Y; the residue at position 129 is D, S, G, K, W, R, E, V, Q, A, I, or F; the residue at position 138 is G, D, E, L, A, M, V, F, R, or S; the residue at position 140 is T, G, S, R, D, K, Q, E, C, the residue at position 152 is A, P, T, Y, H, E, S, I, F, or D; the residue at position 153 is R, Q, T, N, S, F, P, V, or K; the residue at position 154 is L, T, D, R, P, S, or H; the residue at position 155 is N, G, K, H, T, L, S, I, P, or Q; the residue at position 156 is T, F, R, S, D, P, H, G, A, or N; and the residue at position 185 is L, D, N, G, E, F, S, L, Q, P, V, M, R, A, or S;the residue at position 189 is Y, S, L, R, K, G, E, F, D, V, E, I, or H, or the residues at these positions are any combination of these residues, or each of the plurality of different recombinant influenza viruses comprises an H3 HA, and the H3 In HA, the residue at position 121 is Q, R, I, L, V, S, F, Y, or A; the residue at position 131 is R, V, S, Q, C, V, Y, D, E, or L; the residue at position 135 is Y, K, N, V, W, G, S, V, or P; the residue at position 138 is W, K, I, F, R, or L; the residue at position 140 is L, M, T, S, R, K, M, Y, or P; the residue at position 142 is N, G, Y, Q, E, H, N, or Q; the residue at position 144 is T, V, G, D, P, H, L, K, or Q; the residue at position 145 is P, D, R, W, or N; and the residue at position 155 is C, I, R, A, V, S, or Q; 11. The composition of claim 8, 9 or 10, wherein the residue at position 6 is P, G, S, T, A, or C, the residue at position 157 is D, P, S, G, I, Q, R, or T, the residue at position 158 is R, V, S, A, K, C, Q, or G, the residue at position 171 is T, F, L, E, H, V, or R, the residue at position 189 is A, P, T, L, S, Y, or R, the residue at position 193 is Q, R, N, T, E, V, or P, the residue at position 212 is V, R, G, S, M, D, or E, or the residue at position 225 is L, P, C, S, Q, G, Y, or F, or the residues at these positions are any combination of these residues.

12. 1. A method for making an immunogenic composition comprising combining at least six different recombinant influenza H3 or H5 viruses, each encoding a hemagglutinin that contains a plurality of residues that are antigenically different compared to the residues that form an immunodominant epitope in the parent virus, Each of the at least six different recombinant influenza H3 viruses comprises a substitution at six or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225 in H3, or a deletion at one or more of positions 121, 131, 135, 138, 140, 142, 144, 145, 155, 156, 157, 158, 171, 189, 193, 212, or 225, or any combination thereof, wherein the substitution is greater than or equal to the H3 position of the parent virus. or each of the at least six different recombinant influenza H5 viruses comprises a substitution at two or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189 in H5, or a deletion at one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189, or any combination thereof, wherein the substitution is a deletion at one or more of positions 119, 123, 125, 126, 127, 129, 138, 140, 141, 151, 152, 153, 154, 155, 156, 185, or 189 in H5, encodes residues that are not immunodominant epitope residues of HA, and wherein codon substitutions or deletions provide a variety of antigenically distinct immunodominant epitopes for immune recognition; method.

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