WO2013093514A2 - Vaccines - peptides - Google Patents

Abstract

The present invention provides influenza peptide antigens which may be used to induce T cell responses, and not only to the particular influenza strain from which peptide antigen was derived, but also to peptides derived from other influenza strains. These peptides, and vaccines comprising these peptides, are useful in inducing T cell immunity to influenza and advantageously the T cell immunity is to more than one type of influenza.

Description

VACCINES - PEPTIDES

Field of the inventio

The present invention relates to peptides useful for inducing ceil mediated immunity to influenza, In particular the present invention relates to peptides that are useful for provoking a memory T cell response, such as an. enhanced CD4⁺ T-cell response. The peptides of the present invention can be used to induce T cell immunity to influenza and used in the treatment or prophylaxis of an influenza infection, especially i patients who are immunologically naive to an influenza virus. It is particularly preferred that the peptides of the present invention cause cross-reactive T cell responses, meaning the peptides can generate a cellular immune response that will abrogate the consequences of infection with various different types and subtypes of influenza. Therefore the peptides of the invention are useful for inducing cell mediated immunity to more than one strain of influenza, such as several influenza strains or preferably substantially all influenza strains. The invention further comprehends peptide- based vaccine compositions comprising such peptides and the use of such peptides and vaccine compositions in the treatment or prevention of influenza.

Background to the invention

Despite widespread vaccination initiatives, influenza remains a major cause of mortality and morbidity. Each year between 250 000 and 500 000 deaths are attributed to seasonal influenza with associated annual healthcare costs of $14billion in the US alone. Vaccination programmes aim to minimise the burden of seasonal influenza, with the majority of vaccines available at the time of writing designed to generate protective antibody-mediated immunity.

This serological protection is highly strain specific, especially if generated using killed virus preparations. The success of seasonal vaccination programmes is dependent upon both the reliable predictive modelling of strain circulation and the lack of viral coat protein mutation enabling immune evasion during a flu season.

Furthermore, influenza can extend beyond its usual seasonal impact by shifting its antigenic profiie significantly enough to escape from protective immunity on a global scale. If such pandemic strains cany traits of high virulence and pathogenicity then associated mortality can be devastating, as seen in the 1918 outbreak. influenza viruses can evade established protective immune responses by two distinct mechanisms: The gradual antigenic drift of viral surface epitopes results from low fidelity viral replication and adoption of mutations which eventually allows escape from established serological immunity. Less common, but with significant impacts on global health, is the emergence of entirely new viral strains arising from the reassortment of influenza virus RNA from different strains in a common host. The emerging novel pathogen can result in a pandemic where the new flu strain can spread rapidly through communities which lack protective immunity to novel viral proteins. In the context of these events where there are no pre-existing protective antibodies, T cells may mediate protection or limit the severity of influenza associated illness (Kreijtz JH et al., Vaccine 25 612-620 2007). Pre-existing T cell responses have been shown to modulate influenza severity in the context of existing antibodies (McMichael et al, N Engl J Med 309, 13-17, 1983) but the role of protective cell mediated immunity (CM.) in seronegative individuals naive to a particular flu strain is not understood.

Lee et al. (J Clin Invest 118, 3478-3490, 2008) showed that memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. However, the experiments were carried out ex vivo and do not necessarily accurately reflect the clinical picture.

Despite of these reports, at the time of writing the main focus of research remains the search for a 'super-antibody' that is capable of targeting all known subtypes of the influenza A virus. in My 2011, Corti et al. (Science 28 July 2011: 1205669) reported on the isolation of a neutralising monoclonal antibody that recognised the hemagglutinin (HA) glycoprotein of all 16 known subtypes of influenza A and neutralised both group 1 and group 2 influenza A viruses using a single-cell culture method for screening large numbers of human plasma cells. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 HI and the group 2 H3 subtypes analysed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. Based on these results, it was reported that the antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency. Announcing these findings, Dr. A. Lanzavecehia, who led the study, also opined that approaches to developing a universal vaccine that did not rely on antibodies were unlikely to work (report in The Independent, 29 July 201 1).

Nevertheless, there remains a need in the art for new therapeutic agents for use in the prevention and treatment of influenza viral infections, including influenza A, especially in patients who do not have pre-existing protective antibodies for the strain of virus that is the cause of the infection.

Summary of the invention

The present inventors have identified a role for CD4^† T cells, thai recognise particular peptide antigens i limiting disease severity in influenza infections. The inventors then identified influenza peptide antigens which may be used to induce T cell responses, and not only to the particular influenza strain from which the peptide antigen was derived, hut also to peptides derived from other influenza strains. These peptides, and vaccines comprising these peptides, are useful in inducing T cell immunity to influenza and advantageously the T cell immunity is to more than one strain of influenza.

The peptides of the present invention have been experimentally identified and validated as being cross-protective peptides. The peptides of the present invention induce cross-reactive T cell responses. Cross reactivity means that T cells recognise and respond to a corresponding antigen from a different influenza strain even it is not identical in sequence to the antigen initially inducing that T cell line.

The present invention provides a peptide comprising a sequence having at least 85% identity to a peptide selected from the group consisting of;

SEQ ID NO: 1 (MSLLTEVETYVLSIV),

SEQ ID NO: 2 (EVETYVLSIVPSGPLKA),

SEQ ID NO: 3 (EALMEWLKTRPILSPLTK),

SEQ ID NO: 4 (TRPILSPLTKGILGFVF),

SEQ ID NO: 5 (LTKGILGFVFTLTVPSER),

SEQ ID NO: 13 (LYDKEEXRRIWRQANNGEDA), SEQ ID NO: 16 (ELIRMVKRGINDRNFWR),

SEQ ID NO: 17 (RMCNILKGKFQTAAQRA ),

SEQ ID NO: 21 (KGILGFVFTLTVPSERGL),

SEQ ID NO: 22 (DPNNMDRAVKLYRKLKRE),

SEQ ID NO: 24 (REITFHGAKEIALSYSAG),

SEQ ID NO: 25 (ARQMVQA RAIGTHPSSS),

SEQ ID NO: 27 (VRELVLYDKEEIRRIWRQ),

SEQ ID NO: 28 (GSTLPRRSGAAGAAVKGV), and

SEQ ID NO: 32 (S STGLKNDLLENLQ A YQ ) .

or a fragment thereof of at least 9 contiguous amino acids. it is expected that induced T cells may respond to peptides in which 1, 2 or a few amino acids differ from the peptide which induced that T cell line. Conveniently the peptide comprises a sequence having at least 87%, 88%, 89%, 90%, 93%, 94%, 95% or 99% identity with a peptide selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 13, 16. 17, 21, 22, 24, 25, 27, 28 and 32 or a fragment thereof of at least 9 amino acids.

Optionally the peptide comprises a sequence selected from SEQ ID NOS: 1, 2, 3, 4, 5, 13. 16, 17, 21, 22, 24, 25, 27, 28 and 32, or a fragment thereof of at least 9 amino acids,

Preferably the peptide is 9 to 50 amino acids n length, optionally 9 to 40, 9 to 30 and preferably 9-25, 1 -25 or 15-20 amino acids in length.

Advantageously the fragment of SEQ ID NOS: 1, 2, 3, 4, 5, 13, 16, 17, 21, 22, 24, 25, 27, 28 or 32 is at least 10, 11, 12, 13, 14, 15, 16, or 17 amino acids in length.

In some embodiments the peptide is selected from SEQ ID NOS: 1, 2, 3, 4, 5, 13, 16, 17, 21, 22, 24, 25, 27, 28 and 32.

Optionally the peptide is capable of inducing a CD4+ T cell response when contacted with a sample comprising T cells. Induction of a CD4⁺ T cell response indicates that the peptide can be used to reduce symptoms and/or limit the severity of an influenza infection. A second aspect of the present invention provides a vaccine comprising a peptide according to the present invention. In some embodiments the vaccine comprises at least 2, 3, 4, or 5 of the peptides of the present invention, In view of the ability of peptides of the present invention to provoke a CD4⁺ T cell response, having a magnitude that correlates inversely to the severity of symptoms associated with an influenza infection, as disclosed below, and in view of the T cells' ability to tolerate a small degree of sequence variation in. the peptide antigens to which they may respond, a peptide of the invention may be used to induce T cell immunity to influenza or used in a method of treatment or prophylaxis of an influenza infection in a human or non-human animal.

Therefore a third aspect of the present invention provides a peptide or a vaccine according to the present invention for use in therapy. Conveniently a peptide or vaccine according to the present invention may be for use in a method of inducing T cell immunity to influenza virus. The T cell immunity induced is effective against at least one influenza strain or subtype or serotype. Optionally the T cell immunity induced is effective for more than one influenza virus strain or subtype or serotype. Preferably the T cell immunity induced is effective for at least influenza virus strains H3N2 and H1N1. Further optionally the T cell immunity induced is effective for more than one strain of a subtype or serotype of influenza virus. Preferably the T cell immunity induced Is effective for at least seasonal and pandemic strains of H1N1 influenza.

Conveniently a peptide or vaccine according to the present invention may be for use in a method of treating or preventing influenza infection or symptoms of influenza infection. It has been found that the peptide or vaccine is cross-protective and is useful for treating or preventing influenza infection caused by more than one influenza strain or subtype or serotype. Preferably the peptide or vaccine of the invention is useful for treating or preventing influenza infection caused by more than one influenza A strain. For example, at least influenza virus strains H3N2 and. H1N1 can be treated or prevented, alternatively at least influenza virus strains, seasonal H1N1 and pandemic H1N1 can be treated or prevented. In some embodiments the present invention provides a method for inducing T cell immunity to influenza infection comprising administering a therapeutically effective amount of a peptide according to the present invention or a vaccine according to the present invention to a subject in need thereof.

In other embodiments the present invention provides a method for treating or preventing influenza infection or symptoms of infl uenza infection comprising administering a therapeutically effective amount of a peptide according to the present invention or a vaccine according to the present invention to a subject in need thereof.

Advantageously the subject is a human subject, optionally selected from the elderly, the young, individuals regularly exposed to influenza virus.

Generally the peptide or vaccine of the invention is cross-protective meaning that it is useful for inducing T cell immunity in which the T ceils are cross-reactive and are valuable in treating or preventing influenza infection caused by more than one influenza strain or subtype or serotype.

The present inventors have also developed screening methods to identify peptides according to the present invention.

The invention comprehends generating an immune response against influenza in a human or non-human animal subject by administering to said subject a prophylactically effective amount of the vaccine composition of the invention. The immune response may be a prophylactic immune response that either prevents the subject from developing influenza altogether or at least reduces the severity of the symptoms of influenza in the subject.

The terms "prevention" and "prophylaxis" are used interchangeably herein. Prophylaxis includes both the complete prevention of any disease symptoms developing and the development of milder symptoms of the disease than would otherwise have been the case without the vaccination. The vaccine composition of the invention can therefore be used for example to cause a less severe influenza illness than would have been the case without the vaccination. The vaccine composition of the invention can in other words be said to immunise a subject against influenza.

Detailed description of the invention

The present invention is concerned with peptides and vaccines containing such peptides which can he used to induce T cell immunity to influenza.

Influenza (commonly referred to as the flu) is an infectious disease caused by RNA viruses of the family Orthomyxoviridae (the influenza viruses) that affects birds and mammals. The most common symptoms of the disease are chills, fever, sore throat, muscle pains, severe headache, coughing, weakness/fatigue and general discomfort.

The influenza viruses make up three of the five genera of the family Orthomyxoviridae. Of these, influenza A vims is most common in humans. Influenza B and C also infect humans but are less common. The type A viruses are the most virulent human pathogens amongst the three types of influenza and cause the most severe disease.

The influenza A virus can he sub divided into different serotypes or subtypes based on the antibody response to these viruses. The sub types that have been confirmed in humans are H1N1 , H1N2, H2N2, H3N2, H5NL H7N2, H7N3, H7N7, H9N2 and H10N7. Of these, H1N1 was responsible for the 1918 influenza pandemic and swine flu in 2009 and H5N1 caused avian flu (or bird flu) which siarted in Hong Kong in 1997 and there have been further subsequent outbreaks. it is an aim of the present invention to provide peptides, and vaccines containing such peptides, which are useful in inducing T cell immunity to more than one strain or serotype or subtype of influenza, in particular, the present invention provides peptides, and vaccines containing such peptides which are useful in inducing T cell immunity to several, many or substantially all influenza A viruses.

Cell mediated immunity is an immune response that does not involve antibodies, but instead involves the activation of macrophages, natural killer cells ( K), antigen-specific cytotoxic T-lymphocytes (T cells), and the release of various cytokines in response to an antigen. Activated antigen-specific cytotoxic T cells can induce apoptosis in body cells displaying epitopes of foreign antigen on their surface, such as virus-infected cells.

Following a virai infection, memory T cells, a subset of infection fighting T cells, persist. At a subsequent encounter with the same virus, pre-existing memory T cells play a key role in the immune response to the virus. Memory T cells enable a faster and stronger immune response to be mounted, resulting in an infection which is of shorter duration and with less severe and/or with a reduced number of symptoms. The inventors have demonstrated (see below) that pre-existing memory T cells responding to influenza viral peptides reduce the severity and duration of an influenza infection. The screening methods described below are for identifying peptides which induce a T cell response. A T cell response is indicative of inducing T cell immunity. Therefore a peptide which induces a T cell response may be useful for inclusion in a vaccine against the virus from which they are derived.

T cells which respond to peptide antigens can be CD4+ and/or CD8+ T cells. After a viral infection a subset of the activated T cells will persist as memory T cells. Therefore the memory T cells can be CD4÷ and/or CD8+ T cells. When the virus in question is influenza, the preferred T cell response is from CD4+ T cells and pre-existing memory CD4+ T cells may be more effective than pre-existing memory CD8+ T cells in reducing symptom severity in an influenza infection.

The influenza A virus genome encodes eleven proteins. These are haemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins Ml and M2, two non-structural proteins NS1 and NEP, PA, and polymerases FBI, PB1-F2, and PB2. HA and NA appear on the virion surface and are highly diverse. Th core proteins are more conserved between different influenza viruses. Suitably a peptide of the present invention may have a sequence that is derived from a part of the influenza protsome that is conserved between different steins of influenza A, such as a core protein of influenza. Core proteins include NP, Ml , M2, NS1, NEP, PA, PB1, PB1-F2 and PB2. The peptides of the present invention may be derived from matrix (Ml or M2), nucleoprotein (NP) or polymerase (FBI or PB2) proteins, because these proteins are subject to less mutation than the proteins of the viral coat. Peptides derived from matrix (Ml or M2) or nucleoprotein (NP) are particularly preferred because they noted to be the most highly conserved core proteins between different strains, serotypes or subtypes of influenza. The conserved nature of these proteins means the same or substantially similar peptide antigens may be found in many, most, or even substantially all influenza A strains.

Therefore the peptides of the present invention may induce cell mediated immunity in which T cells are cross-reactive between different influenza strain subtypes or serotypes because the peptide antigens of the different influenza strains are identical or have a high level of identity. A vaccine comprising peptide antigens corresponding to influenza viral coat proteins is expected to be effective only against the strain of influenza from which the antigen was derived and closely related influenza strains. It is not expected that such a vaccine will be effective against distantly related strains of influenza. Furthermore, it is also expected that over time, the antigenic draft of viral surface epitopes will reduce the effectiveness of this type of vaccine, even against the strain of virus from which the peptide antigens were initially derived. Therefore the present inventors have focussed on more conserved core proteins of the influenza virus to derive their peptide antigens. Additionally, the present inventors have demonstrated (as described below) the valuable of the peptides of the present invention are cross-protective.

The present inventors have used a human influenza challenge model to follow influenza infection through symptom monitoring and viral shedding analysis. The study may also be regarded as testing which influenza peptide antigens are "seen' arid responded to by the immune system, in this case T cells, during the influenza infection. The T cells were then further studied to identify whether they would "see" and respond to corresponding peptide antigens derived from different strains, subtypes or serotypes of influenza.

More specifically a human challenge model of influenza infection was used by the inventors, as disclosed in the Example below, to identify certain peptides of the H1N1 and H3N2 subtypes of the influenza A virus that were recognised by CD4+ T cells and which were correlated to limiting disease severity in healthy volunteers who lack established humoral immunity to the challenge strains. The CD4+ T cells were further tested to determine whether corresponding peptides from different influenza strains or subtypes were recognised by them. Positive results show these peptides induce immunity that is cross-protective between different influenza A virus subtypes (heterosubtypic immunity), Such peptides may therefore be useful in the preparation of a vaccine composition for the prevention of influenza infection. Such vaccine compositions may be effective in the prophylaxis of influenza infection.

The present invention provides a peptide comprising a sequence having at least 85% identity to a peptide selected from SEQ ID Nos: 1, 2, 3, 4, 5, 13, 16, 17, 21 22, 24, 25, 27, 28 and 32, the sequences of which are shown Table 1 below, or a fragment thereof of at least 9 contagious amino acids. The sequences having SEQ ID No: 1, 2, 3, 4, 5, 13, 16, 17, 21 22, 24, 25, 27, 28 arid 32 are derived from conserved, core proteins NP and Ml or M2 of influenza A.

Peptides of the present invention, and shown in Table 1 have been demonstrated to be cross protective against different strains or serotypes or subtypes of influenza because they induce cross-reactive T cell responses. Consequently, these peptides and vaccines comprising such peptides can be used to induce cell mediated immunity against a broad spectrum of different influenza viral infections,

In Table 1 above, and throughout this specification, the amino acid residues are designated by the usual IUPAC single letter nomenclature. The single letter designations may be correlated with the classical three letter designations of amino acid residues as follows;

A = Ala G = = Gly M -^■ Met S - ^■ Ser

C = Cys H = ^::: His N ^~ Asn T = ^: Thr

D = Asp 1 = lie P = Pro v = = Val

E = Glu K = = Lys Q = G!n = Trp

F = Phe L - = Leu R = Arg Y = = Tyr

The full names of the amino acids are as follows: alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu). phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or lie), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), Threonine (T or Thr), tryptophan ( W or Trp), tyrosine (Y or Tyr) and valine (V or Val), Where a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. References to aspartic acid include aspartate, and references to glutamic acid include glutamate, unless the context specifies otherwise. The symbol X may he used to denote any amino acid. As used herein, the term "peptide" refers to a short sequence of amino acids and includes oligopeptides and polypeptides. These terms are therefore used interchangeably herein, A peptide of the invention may have a length, in the range of from about 9 to 50 amino acids, typically from about 9 to 40, more typically from about 9 to 30 and more typically from about 9 to 25 amino acids, for example from about 10 to 20 amino acids, although these lengths are not intended to be limiting. In some embodiments the peptide may have a length of from 8, 9 or 10 amino acids up to 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids. A peptide according to the present invention may typically be a synthetic peptide. Thus, the peptides may be obtained synthetically, for example by the production of synthetic DNA and expression there from. Methods for the production of synthetic peptides are well known in the art. Peptides can be designed using software, for example the Los Alamos National Library web-based software PeptGen

(http://www.fflv.lanl, govfe^ and synthesised using various commercially available platforms, for example using the proprietary PEPscreen technology from Sigma-Aldrich, Peptides can alternatively be produced recombinanily. Peptides for use in the invention are typically in a purified form. Using these techniques, the person skilled in the art would have no difficulty in providing peptides in accordance with the invention.

As used herein the term "identity" is as known in the art and is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs, Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FAST A (Atschul et ctl, J. Molec. Biol. 215, 403 (1990)).

One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention. The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can he introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions/total number of positions x 100), The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of arlin and Aitschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Aitschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Aitschul et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score ^::: 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes. Gapped BLAST can be utilised as described in Aitschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.rihn.nm.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABiOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosei, 10 :3-5: and. FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8, Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. Typically, the amino acid sequences of each peptide of the invention may have at least 85% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403-410 (1990)) provided by HGMP (Human Genome Mapping Project), at the amino acid level, to the native amino acid sequences of influenza. More typically, the amino sequence may have at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the amino acid level to a sequence found in the viral protein.

A peptide according to the invention may therefore he a variant of the respective sequence that is found in a viral protein. As used herein the term "variant" relates to peptides which have a similar arnino acid sequence and/or which retain the same function. For instance, the term "variant" encompasses peptides that include one or more amino acid additions, deletions, substitutions or the like. The peptides of the invention retain the function of generating T cell responses.

An example of a variant of the present invention is a peptide that is the same as the native peptide, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such ammo acids of a peptide or protein can often be substituted by one or more other such amino acids without eliminating a desired activity of that peptide or protein.

Thus the amino acids glycine, alanine, valine, leucine and isoleucme can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred mat glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucme are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginme and histidine (amino acids having basic side chains); aspartate and glutamate (ammo acids having acidic side chains); asparagine and glutamtne (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Substitutions of this nature are often referred to as "conservative" or "semi- conservative" amino acid substitutions.

Amino acid deletions or insertions can also be made relative to the native sequence in the viral protein. Thus, for example, amino acids which do not have a substantial effect on the activity of the peptide, or at least which do not eliminate such activity, can be deleted. Such deletions can be advantageous, particularly with longer polypeptides since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.

Amino acid insertions relative to the sequence of the native peptide can also be made. This cars be done to alter the properties of a peptide for use in the present invention (e.g. to enhance antigenicity).

Amino acid changes can be made using any suitable technique e.g. by using site-directed mutagenesis or solid state synthesis.

It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- aniino acids are present. it should also be appreciated that the peptides of the present invention may be conjugated to one or more moieties such as polyethylene glycol (PEG) (Veronese F.M. (2001) Biomaterials 22, pp 405-417).

Preferred variants of the peptides for use in the present invention include one or more conservative substitutions as defined herein.

In some embodiments, fragments of peptides with SEQ ID Nos: 1 , 2, 3, 4, 5, 13, 16, 17, 21, 22, 24, 25, 27, 28 and 32 may be used. Fragments may be of at least 9 amino acids in length. This is because this is believed to be at the lower range of the peptide antigen sizes that are presented to CD4^T T cells by MHC class II molecules on APCs. In further embodiments fragments of these peptide sequences may be 9-mer fragments, 10-mer fragments, l l-mer fragments, 12-mer fragments, 13-mer fragments, 14-mer fragments, 15-mer fragments, 16- nier fragments, 17-mer fragments or 18-mer fragments of consecutive amino acids.

For example, for SEQ ID NO: 4, which has the sequence TRPILSPLT GILGFVF and is 17 amino acids in length, preferred fragments will be of 9, 10, 11, 12, 13, 14, 15 or 16 consecutive amino acids of SEQ ID NO: 4. It can be seen that 9-mer fragments of SEQ ID NO: 4 will be as follows: TRPILSPLT

RPILSPLTK PILSPLTKG ILSPXjTi GI

LSPLTKGIL SPLTKGILG PLTKGILGF LTKGILGFV TKGILGFVF 10-mer, 1 1-mer, 12-mer, 13-mer, 14-mer, 15-mer and 16-mer fragments can be constructed in the same way.

For example, for SEQ ID NO: 13, which has the sequence LYDKEEIRRIWRQANNGEDA and is 20 amino acids in length, preferred fragments will be of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 1 consecutive amino acids of SEQ ID NO: 1 . It can be seen that 9-mer fragments of SEQ ID NO: 13 will be as follows:

LYDKEEIRR YDKEEIRRI DKEEERRIW KEEIRRIWR EE!RRIWRQ

EIRRiWRQA IRRIWRQAN

RRIWRQANN

RIWRQA NG IWRQA NGE WRQANNGED RQANNGEDA

IG-mer, 11-mer, 12-raer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-msr and 19-mer fragments can be constructed in the same way.

In some embodiments the peptides of the present invention may comprise additional amino acids flanking the peptide sequences having identity with SEQ ID Nos: 1, 2, 3, 4, 5, 13, 16. 17, 21, 22, 24, 25, 27, 28 or 32 or fragments thereof such that the peptides of the present invention may be longer than these sequences.

When considering the sequence variants, shorter fragments or additional flanking amino acids the sequences of the invention can vary from exact matches with SEQ ID No: 1, 2, 3, 4, 5, 13, 16, 17, 21, 22, 24, 25, 27, 28 or 32.

Preferably an amino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 1.

Preferably an amino acid sequence may have more than 89% or more than 90% identity with SEQ ID No: 2.

Preferably an amino acid sequence may have more than 83% or more than 85% identity with SEQ ID No: 3.

Preferably an arnino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 4.

Preferably an amino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 5.

Preferably an amino acid sequence may have more than 80% or more than 90% identity with SEQ ID No: 13.

Preferably an amino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 16. Preferably an amino acid sequence may have more than 90% or more than 95% identity with SEQ ID No: 17.

Preferably an amino acid sequence may have more than 85% or more than 90% identity with SEQ ID No: 21.

Preferably an amino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 22.

Preferably an amino acid sequence may have more than 85% or more than 90% identity with SEQ ID No: 24.

Preferably an amino acid sequence may have more than 85% or more than 90% identity with SEQ ID No: 27.

Preferably an amino acid sequence may have more than 85% or more than 90% identity with SEQ ID No: 28.

Preferably an amino acid sequence may have more than 95% or more than 99% identity with SEQ ID No: 32.

The second aspect of the present invention is a vaccine comprising a peptide of the first aspect of the present invention. In some embodiments, the vaccine may comprise 2, 3, 4 or 5 or more of these peptides.

The vaccine composition of the invention can be formulated for use by any convenient route. The vaccine composition of the invention may be a phamiaceiitical composition. The vaccine composition of the invention can alternatively simply be referred to as a composition. The vaccine composition of the invention may suitably include a pharmaceutically acceptable carrier, exeipient, diluent, adjuvant, vehicle, buffer or stabiliser in addition to one or more peptides of the invention as the therapeutically or prophylactically active ingredient. Such carriers include, but are not limited to, saline, bufiered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol and combinations thereof.

The vaccine composition may be in any suitable form depending upon the desired method of administering it to a patient. The vaccine composition can be adapted for administration by any appropriate route, for example by the parenteral (including subcutaneous, intramuscular, intravenous or intrademial or by injection into the cerebrospinal fluid), oral (including buccal or sublingual), nasal, topical (including buccal, sublingual or transdermal), vaginal or rectal route. Such a compositio can be prepared by any method known in the art of pharmacy, for example by admixing the peptides with the carrier(s) or excipient(s) under sterile conditions. Typically, the vaccine composition is adapted for administration by the subcutaneous, intramuscular, intravenous or intradermal route, typically by injection. Alternatively, the vaccine composition may be adapted for oral or nasal administration.

A pharmaceutical composition adapted for parenteral administration may be an aqueous and non-aqueous sterile injection solution which can contain anti-oxidants, buffers, baeteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. Excipients which can be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The composition can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophiHzed) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared fro sterile powders, granules and tablets.

A pharmaceutical composition adapted for oral administration, can be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions)

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which can be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) can be used to provide oil-in- water or water in oil suspensions, A pharmaceutical composition adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. A suitable composition wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, may comprise an aqueous or oil solution of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists that can be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

A pharmaceutical composition adapted for transdermal administration may be presented as a discrete patch intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient can be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research. 3{6):318 (1986).

A pharmaceutical composition adapted for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil. For infections of the eye or other external tissues, for example mouth and skin, the composition may be applied as a topical ointment or cream. When formulated in an ointment, the active ingredient can be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oi -in-water cream base or a water-in-oil base. A pharmaceutical composition adapted for topical administration to the eye may comprise eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. A pharmaceutical composition adapted for topical administration in the mouth may comprise lozenges, pastilles or mouth washes.

The pharmaceutical composition may contain preserving agents, solubiiising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention can themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. The vaccine composition of the invention may also contain one or more other prophylactically or therapeutically active agents in addition to the at least one peptide as defined herein.

The peptide for use in the vaccine compositions of the invention may or may not be lyopliilised.

The vaccine composition of the invention may also include a pharmaceutically acceptable adjuvant in addition to the peptide(s) as defined herein. Adjuvants are added in order to enhance the immunogenicity of the vaccine composition.

Suitable adjuvants for inclusion in a vaccine composition are known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminium hydroxide), alum plus Bordatella pertussis and immune stimulatory complexes (ISCOMs, typically a matrix of Quil A containing viral proteins).

The vaccine composition of the invention may also include or be co-administered with one or more co-stimulatory molecules, such as B7, and/or cytokines, such as an interferon or an interleukin, that can promote T cell immune response such as 11-2, 1I.-15, TL~6, GM-CSF, IFNy or other cytokines promoting T cell responses. This can be done in addition to conventional adjuvant, as described above.

Dosages of the vaccine composition of the present invention can vary between wide limits, depending upon the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

This dosage can be repeated as often as appropriate. For example, an initial dose of the vaccine may be administered and then a booster administered at a later date, For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from lHg kg to lOmg/kg body weight, typically around lC^g/kg to lmg kg body weight. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependent on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention. The vaccme composition of the invention can be administered by any convenient route as described herein, such as via the intramuscular, intravenous, intraperitoneal or oral routes or by injection into the cerebrospinal fluid,

The vaccine composition of the invention can be administered to patients felt to be in greatest need thereof, for example to children or the elderly or individuals exposed to influenza virus. Timing of administration of the vaccine may be important; for example a vaccination strategy can be put in place once an outbreak of influenza has been identified, in order to prevent the spread of the virus in a community. The vaccine composition can be used in particular subsets of patients, for example those who have not already suffered from a particular strain of influenza, for example seasonal flu.

The method of prophylaxis can be of a human or non-hitman animal subject and the invention extends equally to uses in both human and/or veterinary medicine. The vaccine of the invention is suitably administered to an individual in a "prophyiactically effective amount", this being sufficient to show benefit to the individual.

The vaccine composition of the invention can be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit, Such a kit would normally (although not necessarily) include instructions for use. It can include a plurality of said unit dosage forms.

Accordingly, in yet another aspect, the present invention provides a kit of parts comprising a vaccine composi tion of the invention and one or more cytokines and/or adjuvants in sealed containers.

In yet another aspect, the present invention provides a kit of parts comprising a vaccme composition of the invention and one or more cytokines and/or adjuvants for separate, subsequent or simultaneous administration to a subject. The peptides of the present invention were obtained or confirmed by a screening method developed by the inventors which may be regarded as having 3 stages. 1) An in vitro method of interrogating the immune system to understand what influenza viral antigens are "seen" and responded to by T cells of the immune system during influenza infection. 2) Correlating the T cell responses to influenza viral antigens to reduce system severity during influenza infection. 3) Determining whether the same T cell lines respond to corresponding influenza viral antigens from different strains or subtypes.

The in vitro interrogation methods involve detecting T cell responses to peptide antigens via the Elispot assay sample comprising T cells may be a whole blood sample, a fraction of whole blood or typically a sample of peripheral blood mononuclear cells (PBMCs). Such a sample can be obtained, for example, by separating PBMCs from whole blood by density gradient centrifugation. The blood can be heparinised prior to such separation. PBMCs include any blood cell having a round nucleus. Cell types include for example lymphocytes, monocytes or macrophages, These are the blood cells providing a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population consists of T cells (CD4 and CDS positive -75%), B cells and NK cells (-25% combined). The PBMC

population also includes basophils and dendritic cells. The sample comprising T cells comprises CD4+ T cells and CD8+ T cells, in certain embodiments the sample comprises CD4+ T cells.

By "T cell response" is meant detecting and generally quantifying any response of T cells to said peptide. Typically, me T cell response that is quantified may be the production of one or more cytokines, for example IFNy.

The T cell response, for example the production of one or more cytokines, can be quantified using any suitable means. For example, the response can be quantified using an ELISPOT (enzyme-linked immunosorbent spot) assay. The ELISPOT assay is based on the ELISA immunoassay and allows visualisation of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides information both on the type of protein produced by a particular cell and the number of reactive cells. Typically, the ELISPOT assay may be an IFNy ELISPOT assay. Suitably, the ELiSPOT assay may be carried out in 96-well plates. A variety of other methods of quantifying the T cell response will be known to the person skilled in the art. In one embodiment, the T cell response is quantified. As noted above, T cells can be either CD4+ or CD8÷. In another embodiment, the T cell response of both CD4⁺ and CD8 ^! T cells may be quantified at the same time, and then a second assay may be carried out to determine what proportion of the T cell response can be attributed to CD4⁺ T cells. TMs may be done, for example, by depletion of CD8⁺ T cells and then carrying out a further ELISPOT assay for the same cytokine, for example an IFNy ELISPOT assay.

Preferred features described with respect to an aspect of the present invention apply for other aspects mutatis mutandis. Following is a description by way of example only with reference to the accompanying drawings of embodiments of the invention. In the drawings: Brief description of the drawings. Fig re I shows an Elispot layout of experimental influenza A infection in humans, (a)

H3N2 challenge study (A^/Wisconsin/67/05) T cell Elispot layout, (b) H1N1 challenge study (A/Brisbatie/59/07) T cell Elispot layout. Freshly isolated 300,000 PBMC were put into each well and stimulated with peptide pool at 2 μ^τηΐ for 18-24 hours. Fi ure 2 shows viral shedding in nasal wash, seroconversion and symptom. development in seronegative healthy volunteers experimentally infected with influenza A. (a) Volunteers infected with cell grown H3N2 (WS/67/05) virus or egg- grown H1N1 (BR/59/07) virus. The virus in the nasal sample was titrated by ΤΟΒ₅₀ assay. Presence of flu-specific antibody was measured by haemagglutination inhibition assay, (b) Correlation of total symptom scores against the peak nasal virus shedding in H3N2 infected subject by Spearman rank correlation test, (c) Mean symptom scores and oral temperatures of volunteers infected with H3N2 virus, (d) Mean symptom scores and oral temperatures of volunteers infected with H1N1 vims. Symptom assessments were performed by the volunteers twice daily on a four-point scale (absent to severe), The score for each symptom group was obtained by adding the total individual symptom scores for that particular group on that particular day. Oral temperatures were determined fo r times a day for the duration of the study and the highest temperature was represented.

Figure 3 shows symptom scores in each infected volunteer infected with influenza A.

(a) Total symptom scores (y axis) in volunteers infected with H3N2 (WS/67/05) (x axis showing days after H3N2 challenge infection).

(b) Upper respiratory symptom scores (y axis) in volunteers infected with H3N2 (WS/67/05) (x axis showing days after H3N2 challenge infection).

(c) Lower respirator}.' symptom scores (y axis) in volunteers infected with H3N2 (WS/67/05) (x axis showing days after H3N2 challenge infection).

(d) Systemic symptom scores (y axis) in volunteers infected with H3N2 (WS/67/05) (x axis showing days after H3N2 challenge infection).

(e) Total symptom scores (y axis) in volunteers infected with H1 1 (BR/59/07) virus (x axis showing days after HiN I challenge infection).

(f) Upper respirator}_'- symptom scores (y axis) in volunteers infected with H1N1 (BR/59/07) virus (x axis showing days after H1N1 challenge infection).

(g) Lower respiratory symptom scores (y axis) in volunteers infected with H1N1 (BR/59/07) virus (x axis showing days after H1 1 challenge infection).

(h) Systemic symptom scores (y axis) in volunteers infected with H1 1 (BR/59/07) virus (x axis showing days after H1N1 challenge infection). Figure 4 shows T cell responses in seronegative healthy volunteers experimentally infected with influenza A virus. Flu-specific T lymphocyte responses were measured from freshly isolated PBMC ex vivo from each volunteer by IFN-γ release after stimulation with corresponding peptide pools spanning the entire challenge influenza proteome. Each bar represented the total T cell responses to entire influenza proteome and each colour box represented the response to each protein. X axes denote subject number. Figure § shows antibody and T cell responses in seronegative healthy volunteers experimentally infected with influenza A virus, (a) Presence of flu-specific antibody was measured by haemagglutination inhibition assay, (b) Plot of proportion of infected subjects demonstrating positive T cell responses to I P and M flu proteins at baseline. The Y-axis represents the proportion (%) of subjects from both challenge studies with positive response to NP and M proteins and their CD4 and CDS dependency, (c) Activated and proliferating cells (CD38+K167+) could be detected ex vivo in HlNl-infected subjects by flow cytometry (d) Changes in the proportion of activated and proliferating (CD38+Ki67+) T cells in both CD4 and CDS population of freshly isolated PBMC from volunteers infected with H1N1 virus by flow cytometry, (e) Correlation between proportion of CD38⁺Ki67⁺ T cells on day 7 of H1N1 infected subjects with their magnitude of Elispot response by Spearman rank correlation test.

Figure 6 shows correlations between flu-specific total T and CD4 T cell responses to internal proteins and measure of influenza severity (viral shedding, symptom severity or illness duration) in volunteers infected with (a) H3N2 (WS/67/05) or (b) HIN! (BR/59/07). Correlations between total symptom scores or length of illness duration against flu-specific total T cell responses or CD4 flu-specific T cells specific to internal proteins including nucleoprotein and matrix of challenge virus. All tests were run by spearman rank correlation test.

Figure 7 shows phenotypic and functional studies of CD4 and CDS cells at baseline and day 7. (a) Expression of ΪΡΝγ and CD 107a in memory T cells of a representative H3N2 infected subject after stimulation with peptide pools to influenza proteins. PBMC from baseline and day 7 samples were stimulated with different peptide pools (Flu, NP, M) for 6 hours and the ex vivo response was measured by FACS staining. Both memory CD4 and CDS responses in the same sample were measured. Staphylococcus enterotoxin B (SEB) was used as positive control (b) Killing function of CD4+ T cell lines from the same baseline sample upon recognition of autologous target cells pulsed with peptides was measured by chromium Release Assay. Perforin- dependent cytotoxicity was measured by sensitivity to concanamycin. Figure 8 Cross-reactivity study of CD4^÷ memory cells from H1 1 infected subjects to peptides from pandemic H1N1, PBMC from different donors were stimulated with decreasing concentration of peptides and the response to each peptide was measured by IFN-γ ELISPOT. Each plot represented the results from one donor to autologous peptide (closed circle) and heterologous peptides (open circles).

Figure 9 a). Human parenchymal (i) and (ii) and bronchial tissue stained (iii and iv) for MHC ΪΙ (HLA-DR) 2mm sequentially cut sections and inununostained using isotype control monoclonal antibodies (i) and (iii) or antibodies specific for HLA-DR (ii) and (iv) at the same concentration. Signal was amplified using the ABC system, and colour developed using DAB stain, Specific staining is shown in brown, haematoxylin counterstain is shown in blue. Size bar represents 50um. b) (i) Representative histograms showing specific staining of HLA-DR expression on primary bronchial epithelial cells (PBECs) by flow cytometry using cells incubated in the presence or absence of HLA-DR APCCy7 or IgG2a APCCy? (isotype). (ii) Graph of mean fluorescence intensity of HLA-DR expression on PBECs using flow cytometry. NT - non treated control, X31 influenza infected cells, UVX31- UV inactivated viral control. HLA-DR is constittitively expressed on primary respiratory epithelial cells, there is a small rise in expression following in vitro infection of these cells with influenza virus which was significant in comparison to stimulation with UV -treated (inactivated) virus. This confirms that respiratory epithelial cells are potential target cells for cytotoxic CD4+ T cells.

Study deslga

Between October, 2008 and October, 2009, two separate prospective, randomised, and double blinded, parallel group clinical studies of experimental human influenza A infections were undertaken in a single site in Cambridge, UK. The two studies were carried out 9 months apart. An H3N2 challenge study was carried out between 24 October and 24 November, 2008 whereas an ΗΓΝ1 challenge study was carried out between 18 August and 18 September, 2009. Healthy, non-pregnant adults between the ages 18 and 45 were eligible for the enrolment. Exclusion criteria included health care workers, history of acute respiratory illness, chronic illness or medications. In H3N2 challenge study, a total of 17 healthy adult volunteers, which are haemagglutination-inhibition (HI) titres less than 1:8 to influenza A/W-sconsin/67/05, were enrolled in the study. Whereas, in H1N1 challenge study, a total of 24 healthy adult volunteers with HI titres less than 1 :8 to influenza A/Brisbane/59/07 were enrolled in the study. Both studies were conducted in compliance with Good Clinical Practice guidelines (CPMP/iCH 135/95) and declaration of Helsinki. The protocols were approved by East London and City and the Southampton and Southwest Hampshire ethics review committees. Written informed consent was obtained from each participant with an ethics committee approved form. No medications, except acetaminophen for treatment of severe symptoms, were permitted. Subjects were compensated for their participation of the study.

Study outline

Screening assessments began within 45 days of the scheduled viral inoculation, Volunteers were confined to individual rooms in an isolation unit 2 days before the day of inoculation, and remained in isolation for 7 days thereafter.

Therefore, contact with any pathogens such as viruses or bacteria is completely controlled. Isolation, and monitoring of subjects allows study of infection and symptoms of the infection. Inoculation occurs under clinical conditions so that the exact time of inoculation is known. Therefore samples obtained from the subject can be taken at known time points after inoculation.

The subjects were randomised into 4 groups and each group of the participants were inoculated intra-nasally with different doses of influenza A virus on day 0. The dose of the virus was designated as 1:10 (high), 1:100 (medium-high), 1:1000 (medium-low) and 1 :10,000 (low) from the original virus stock. Group 1 received high dose, Group 2 received medium-high dose, Group 3 received medium-low and Group 4 received low dose of virus. Nasopharyngeal swab were collected daily from baseline day 0 during the quarantine period for virus isolation. This al lowed analysis of viral shedding.

Serum samples were taken dail for serum cytokine and biomarker study, Fresh whole blood for cellular assays was taken on day -2 or 0, 7 and day 28, An additional lime point day 3 was taken for H1N1 study. Samples comprising T cells, for example PBMC samples, allowed analysis of activated and responding T cells. information about symptom severity was recorded, Oral temperatures were measured four times daily. Fever was defined as an oral temperature >37.7°C. Symptom assessments were performed by the volunteers twice daily on a four-point scale (0-3 corresponding to absent to severe) (Hayden et al., J. Clin. Invest. 101(3), 643-649, 1998), The symptoms assessed were nasal stuffiness, runny nose, sore throat, cough, sneering, earache/pressure, breathing difficulty, muscle aches, fatigue, headache, feverish feeling, hoarseness, chest discomfort, and overall discomfort. The total symptom score for each day was obtained by adding the individual symptoms scores for that particular day including morning and evening sessions. The individual symptoms contributing to the total symptoms scores were divided into three subgroups: systemic symptoms (muscle aches, fatigue, headache, and fever), upper respirator symptoms (nasal stuffiness, ear ache/pressure, runny nose, sore throat, and sneezing) and lower respiratory symptoms (cough, breathing difficulty, hoarseness and chest discomfort).

Viruses

In both challenge studies, GMP grade viruses were manufactured and processed by GlaxoSmithKl e, UK. The stock virus were diluted to four different inoculum titres and prepared in individual aliquots intended for single use and then administered. The titr e of the stock virus was 10⁷ TCID₅₀ infectious dose. They were ten-fold diluted and the titre were ranged from high titre (1 : 10), medium-high (1 : 100), 3 medium-low titre (1 : 1 ,000) and low titre (1 : 10,000). Subjects were observed for potential allergic reactions for 30 ruin following inoculation. In H3N2 challenge study, tissue culture grown A Wisconsin/67/05 virus was used. In H1N1 challenge study, egg grown A''Brisbane/59/2007 virus was used. Virus titration by TCID₅₀ (Tissue Culture Infections Dose 50%) assay

Viral load in the nasopharyngeal samples were determined by TCID₅₀ assay as described by the WHO manual of Animal influenza

(http://www.whoint/vace^

diagnosis_and_surveillance _2GG2__5.pdf), Serial ten-fold dilutions of virus-containing samples were inoculated into 96-well microtitre plates seeded with Madin-Darby canine kidney (MDCK) cells, and incubated for 5-6 days at 37°C. Cytopathic effects in individual wells were determined via light microscopy. 'litre greater than 1:5 was considered positive, Hemagglutination Inhibition (HI) Assay

Haemaggluiinin-specific antibody titers against HIN1 (A Brisbane/59/2007) or H3N2

(A/Wisconsin 67/05) in the serum^' samples were determined by HI assay using chicken erythrocytes as described in WHO manual

(http://www.who.mt/vaccmejresearcb^

diagnosis and surveillance 2002 5.pdi).

Synthetic peptides

1.8-rtier peptides overlapping by 10 amino acid residues and spanning the full proteome of the HlNl and H3N2 influenza A viruses were designed using the Los Alamos National Library web-based software PeptGen (http://www.hiv.lanl.gov/content/sequence-

/PEPTGEN/peptgen.html) and synthesized (purity >70%; PEPscreen; Sigma-Aldrich) using the sequences of the following strains: A/Brisbane 59 2004 (HlNl), A/New York 388/2005 (H3 2) (surface proteins), and A/New York 232/2004 (H3N2) (internal proteins). In H3N2 peptides, the amino acid sequence homology between challenge Wisconsin strain and New York strain was greater than 99%. The total numbers of peptides used in detecting antigen- specific responses for Hl l and H3N2 were 554 and 601 respectively.

Identifying peptides "seen" by T eells of immsrae system

Ex vivo XFNv Elispot assays were used to identify T cells which respond to stimulation with a specific peptide and therefore secret IFNy.

In the each influenza Elispot assay, all overlapping peptides in each individual were simultaneously tested using 2-dimensional matrices with a total of 50 pools (1^st D = 25 pools; 2^nd D ==^: 25 pools; up to 25 peptides/pool) so that each peptide was present in two different pools (see Figure 1 for Elispot layout). Peptides were used at a final concentration of 2 μg/πύ each. The putative peptide from each positive response well could be deconvolved from a 2- dimensional matrix system where each peptide only appeared once in each dimension. The putative peptides were then confirmed individually in the second Elispot assay with the same input cell number per well.

Ex vivo IFN-γ ELISPOT assay

Peripheral mononuclear cells (PBMC) were separated from 50 ml heparinised blood by density gradient eentrifugation using Lymphoprep (Axis-Shield, Norway) and Leucosep tube (Greiner, UK) within 3-6 hour upon each bleed (Li et al, J. Immunol. 181, 5490-5500 2008). To detect influenza-specific effector memory cells (CD45RO+), PBMC were added into 96-well Elispot Multiscreen plates (MAIPS4510. Millipore) at 300,000 cells/well and cultured with peptide pools for 18-24h incubation at 37°C and 5% CO_?.. The end concentration of each peptide in each well was 2 fig/ml, for both peptide pools and individual peptides. All ELISPOT assays were performed using the human IFN-γ ELISPOT kit (Mabtech) according to the manufacturer's instructions. The internal negative control was no peptide in quadriplicates, and positive controls were EC (a mixture of EBV and CMV T cell epitope peptides) or PHA (10 μ^'ηιΐ). The spots on each well were counted using an automated ELISPOT reader and AID ELISPOT 3.1.1 HR software (Autoimmune Diagnostika). In pool responses, wells containing spot numbers greater than the mean 4- 4 SD of three negative control wells (no peptide) were regarded as positives in each individual, provided that the total was greater than 50 spot forming cells (SFC)/million PBMC, to rule out false positives where background was very low. In all assays, values of no peptide control wells were 1.8 ± 4.6 SFC/million PBMC for 150 healthy subjects and 2 ± 5.7 SFC/million PBMC for 150 influenza-exposed subjects. Values of T cell responses were all background subtracted and presented in SFC/million PBMC. To determine whether T cells were CD4 or CDS, in the second ELISPOT assay, cell depletion was also conducted by Dynal CDS beads, as described in the manufacturer's instructions (Invitrogen, UK), before the ELISPOT assay. Undepleted PBMC served as positive controls. For single peptide confirmation Elispot assay, response greater than 10 SFC/million PBMC was considered positive after background substraction and when T cell fines could be generated from respective peptides and tested positive again with ICS. Gen ration of sSi ri-ierm T cell aes

Short-term. T cell lines were generated to confirm influenza peptides and the CD4 or CD8⁺ property of each peptide by ICS and flow cytometry, as described previously (Li et al._s J, Immunol. 181, 5490-5500, 2008). In brief, frozen samples of PBMC were thawed and rested for 2h before stimulating with 10 μ /ηι1 of each peptide at final concentration for 1 h. Cells were cultured in RPMi 1640 supplemented with 10% human serum (National Blood Services, UK) and 25 ng/ml IL-7 (PeproTech) for 3 days, and then 100 U of IL-2/ml (Proleukin, Novariis UK) was added every 3 to 4 days thereafter. On day 14, cells were washed three times with sterile PBS and then rested in fresh RAB-10 for 25 to 35 h at 37°C, 5% C0₂.

FACS staining assa

Activated (CD38+) and proliferating (Ki67+) cells in freshly isolated PBMC were stained with mAbs against human Ki67-FITC (Clone B56, BD Biosciences), DR-PE (clone TU36, BD) CD38-APC (clone HB7, BD), CD4-pacific blue (Clone MT130, DakoCytomation), and CD8-PE-Cy5 (Clone SKI, BD). Cytotoxicity as measured by expression CD107a (clone H4A3, BD) and IFN-γ (clone XMG1.2, BD) in both CD4 and CDS memory cells were also studied ex vivo using frozen PBMC as described previously (Li et al., J. Immunol. 181 , 5490-5500 2008). PBMC (1 million per stimulation) were stimulated with peptide pools for 6 hours in the presence of brefeidin A and monensin. For each stimulation condition, at least 500,000 total events were acquired using LS ii (BD immunocytometry Systems, San Jose, California). Data analysis was performed using FlowJo (version 8.8.4; TreeStar, Ashland, Oregon). Response greater than 3 times background was considered positive.

Chromium Release Assay

A standard ⁵⁵Cr release assay was used as described previously (McMichael A J et al, N Engl J Med 309, 13- 17, 1983). T cell lines generated from PBMC samples were used as effector cells and their autologous EBV-transformed B cell lines were used as target cells. Inhibition of perforin-mediated cytotoxicity was obtained by incubating the CD4+ T cells for 2h with 100 nM concanamycin (Sigma). Specific ^S!Cr release was calculated from the following equation: ([experimental release-spontaneous release]/[maximum release-spontaneous release])x 100%.

Testing for cross-reactive T ceils and cross-protective peptides

Peptides which induced a T cell response were identified using the Elispot assay. The magnitude of the T ceil responses preferably showed an inverse correlation with the severity of influenza infection symptoms in the subjects from whom the T cells had been obtained, indicating that those peptides may induce cell mediated immunity. Subsequently, those responding T cell lines were re-tested, using the Elispot assay, for responses to corresponding peptides from different influenza strains. The corresponding peptides from different influenza strains may be identical or may have a high level of identity to the initial peptides to induce iFNy secretion and identification of a T cell response. T cells secreting ΪΡΝγ in response to peptides derived from corresponding proteins of different influenza strains were considered cross-reactive T cells. Those peptides initially activating the T cell lines were considered cross-protective peptides.

Epithelial Cell MHC Class II Expression Immimohistochemistry.

Lung explants were harvested from lung tissue recovered from patients undergoing routine thoracic surgery under additional consent, Human parenchymal and bronchial tissue was fixed in acetone prior to embedding in GMA resin. Two millimetre sections were cut sequentially and immimostained using isotype control monoclonal antibodies or antibodies specific for MHC II (HLA-DR) at the same concentration.. Signal was amplified using the ABC system, and colour developed using DAB stain, Specific staining is shown in brown, haematoxylin counterstain is shown in blue.

Flow cytometry

Primary bronchial epithelial cells (PBECs) were obtained from subjects undergoing research bronchoscopies in the Wellcome Tmst Clinical Research Facility at Southampton General Hospital. Bronchial brushings were cultured in Bronchial Epithelium Growth Media (BEGM), (Lanza, Wokingham, UK) in collagen coated flasks (PureCol™, Inamed Biomaterials, California, USA) and incubated in a humidified atmosphere at 37 °C, 5 % C0₂, The collection and use of these samples was approved by the Southampton and South West Hampshire Research Ethics Committee (SEC No: 06/Q1701/98 & 08/H0504/138). influenza A virus strain X31 was supplied at a concentration of 4 x 10 pfu rni (a kind gift of 3 VBiosciences). Inactivated virus (UVX31 ) was prepared by exposure to an ultra-violet (UV) light source for 2 h.

PBECs were seeded at 1 x 10^s cells per well onto a collagen-coated 24 well plate and left at 37 °C, 5 % C(¾ for 24 h. Cells were then growth media starved for 24 h in 0.5ml Bronchial Epithelium Basal Media (BEBM) supplemented with 1 mg ml BSA, insulin, transferrin and selenium (BEBM+iTS). Cells were incubated for 2 h with no virus, or 2 x 10' pfu of X31 or UVX31. Cells were then washed three times with BEBM+ITS and incubated for a further 20 h at 37 °C, 5 % CC in 0.5ml of BEBM-ITS. Cells were dispersed by trypsinisation and prepared for flow cytometric analysis as previously described.

Samples were incubated on ice in the dark for 30 rnin with Allophycocyarm-Cyanine 7 (APC-Cy7)-conjugated anti-HLA-DR (BD Biosciences, Oxford, UK) or appropriate isotype control (IgG2a BD Biosciences Oxford, UK), After washing, intracellular staining for viral nueleoproteim (NP)-1, was performed using BD Cytofix Cytoperm kit according to manufacturer's instructions, and AlexFluor 488 (AF488)-conjugated anti-NP-1 antibody (HB~ 65, a kind gift of 3VBiosciences). Flow cytometric analysis was performed on a FACSAria using FACSDiva software v5.0.3 (all BD),

Statistics

All graphs were presented by GraphPad Prism (version 5) and statistical analysis was done by GraphPad Prism and SPSS. Magnitude of T cells response was presented by SFC/million PBMCV and breadth of T cell response was defined by the number of proteins recognized by each subject. To study the role of T cell in the virus shedding (viral control) and symptom development (rrnmunopathology), correlation was run between pre-existing T cells and measures of infection and illness (virus litre, symptom assessments, temperature) by Spearman rank correlation analysis. Correlation analysis was based on data collected from all infected (culture positive and or fourfold or greater rise in HI antibody titre) individuals. Results

Human Influenza infection Model

In order to study the impact of existing CMI on influenza infection, an experimental infection model was established using live influenza A virus in human volunteers (Oxford JS et al Expert Rev And Infect Ther. 3, 1-2 (2005)). A total of 41 healthy volunteers aged between 19 and 41 were inoculated intra-nasally with serial 10 fold dilution of influenza A viruses; a cell grown H3N2 WS/67/05 and an egg grown HlNl BR/59/07. Subjects were studied prospectively from inoculation in a clinical isolation facility with measures of viral shedding, symptom development, cellular and humoral immune responses for the first 7 days and again at day 28. In the H3N2 challenge study, 8 out of 17 (47%) volunteers were female and the median age was 26.5 yr (range 22-41) (TaMe 2). in HlNl challenge study, 7 out of 24 (29%) were female and the median age was 24 yr (range 19-35).

Tabe ^&eiri¾^apte

0-40 52-76 5- .i 0-9 0-22 3-65 0-31 0-38

One subject was unavailable for D28 visit. **Two subjects were unavailable for D28 visit.

All volunteers selected were seronegative to the challenge strain and virus PCR negative in nasal lavage at the iixne of challenge. The overall infection rate was defined by evidence of virus shedding and/or seroconversion by day 28. This was higher in subjects (14/17, 82%) challenged with H3N2 virus than subjects (9/24, 38%) challenged with H1N1 virus, in the H3N2 challenge group, virus shedding persisted in individuals for as long as 7 days but most subjects (8/14, 57%) cleared the virus completely by day 4. (Fig. 2a). The H1N1 challenge group, did not exhibit reliable viral shedding - a recognised phenomenon with this egg grown virus (Steel J, et al J Virol. 2009 Feb;83(4): 1742-53).

In the H3N2 challenge infection, total symptoms closely tracked peak viral load (Fig. 2b, r™0.6977, p^~0.0055, Spearman coefficient). Similar symptom profiles were observed between the two challenge cohorts and were comparable to wild type infections in this population (Newton DW at al Am J Manag Care. 6, 265-75 (2000)). In the H3N2 challenge group, ί 1 out of 14 (79%) infected subjects developed one or more symptoms, and as a group, exhibited symptom scores that peaked on day 3 and returned to normal by day 7 after viral inoculation (Fig. 2c). 3 out of 14 subjects (21%) developed fever (oral temperature>37.7 'C) and the highest temperatures were detected on day 2. In the H1N1 group, 8 out of 9 (89%) infected subjects developed one or more symptoms and showed mean symptom scores that peaked on day 4 and returned to normal by day 7 after viral inoculation (Fig. 2d). Also, 1 out of 9 infected subjects (11 %) developed fever and the highest temperatures were detected on day 2. In both challenge groups, the total symptoms were dominated by upper respiratory illness as defined by the presence of symptoms such as ramiy nose and sore throat, occurred in 10/14 (71%) subjects in H3N2 group and 8/9 (89%) subjects in H1N1 group. Lower respiratory symptoms such as cough and hoarseness were much milder in severity and occurred in 3/14 (21%) in H3N2 group and 2/9 (22%) in H1N1 group. Scores for systemic symptoms such as muscle aches and fatigue were also present in 6/14(43%) in H3N2 group and 5/9(56%) in H1N1 group. For more details on the distribution of symptoms of each infected subject, see Fig, 3, Antibody ¾nd T cel. responses of infected volunteers

All volunteers enrolled were screened to ensure they were seronegative for antibodies to the challenge virus. However, the antibody responses (HAI titre) were detectable after 7 days post challenge (Fig 5a), at which time the viruses were completely cleared as indicated in Figure 2a.

Prior to viral challenge the nature of pre-existing T cell memory from previous infection exposure was determined, T cell responses to proteins expressed by the challenge virus were present in most volunteers in both studies prior to challenge despite the absence of detectable antibodies to the same strains. The size of total T cell responses was below 1000 SFC/million PBMC in all subjects studied at baseline (Fig 4b). At baseline, in the H3N2 group, 1 1 out of 14 (79%) infected subjects showed memory T cell responses recognizing one or more H3N2 proteins, with an average of two proteins recognized (range 1-5). The most immunodominant proteins were nucleoprotein (8/14, 57%) and matrix proteins (7/14, 50%), which are highly conserved across strains, based on the number of subjects and the magnitude of IFN-y response. In the H1N1 challenge group, 7 out of 9 (78%) infected subjects showed memory T cell responses that recognized one or more proteins at the baseline, with an average number of one protein recognized (range 1-3), The most immunodominant protein was matri protein (6/9, 67%). These results show that conserved viral peptide sequences from nucleoprotein and the matrix proteins are important in cell mediated immunity and T cell responses, and the present methods allowed identification of these peptides.

On day 7 after challenge infection, both the breadth and magnitude of memory T cell responses increased dramatically in the peripheral blood by an average of 10 fold in both study groups (Fig 4b), In the H3N2 group, 14 out of 14 (100%) of infected subjects demonstrated positive T cell responses with an average of five proteins recognized (range 1~ 8). Pre-existing T cell responses against each protein were expanded in addition to new responses that had not been detected at baseline. In the H1N1 challenge group, 9 out of 9 (100%) infected subjects were T cell positive responding to an average of five proteins (range 2-7). No significant changes in the T cell responses against known CDS epitopes of CMV and EBV were found in control wells, suggesting bystander activation was minimal (data not shown). Therefore the body dramatically responds to viral peptides during infection raising T cell response and the present methods allowed identification of those peptides.

On day 28, the total memory T cell response had returned to baseline levels (< 100(3 SFC/million PBMC) in both challenge groups. Immunodominant protein responses such as NP and M persisted at a baseline level whereas most newly generated responses against other proteins had vanished after the acute phase of infection, in the H3N2 challenge group, 7 out of 10 infected subjects (70%) were T cell positive, with the average number of proteins recognized reduced to 1 (range 1 -2). In the HINl challenge group, 8 out of 9 infected subjects (89%) were T cell positive, with the average number of proteins recognized reduced to 2 (range 2-4). However, 4 out of 9 (44%) newly generated HA responses persisted at lower levels (average 60 SFC/million PBMC). In addition, epitope mapping and whether they were mediated by CD4 or CDS T cells was determined for responses to the immunodominant proteins (NP and M) on all baseline samples from both challenge groups. T cell response against immunodominant proteins were predominantly CD4 T cell mediated in both groups (CD4 vs CDS 56% vs 44% for H3N2, and 71% vs 28% for HINl) (Fig, 5b), consistent with a previous report (Lee LY et a! J Clin Invest. 1 18, 3478-3490 (2008)).

To understand better the kinetics of T cell responses the functional status of both CD4 and CDS cells during the course of infection with HINl virus was studied. Activated (CD 8⁺) and proliferating cells (Ki67⁺) of both CD4 and CDS cells from freshly isolated PBMC were undetectable before the challenge (Fig. 5c). Both markers were present on the greatly expanded T ceil population on day 7 before reraming to baseline level on day 28. (Fig 5d). The number of Ki67⁺CD38^÷ T cells correlated with the frequency of SFC by Elispot on day 7 (Fig, 5e, r=0.9, p===0.002, Spearman coefficient).

Impact of Pre-existing T eell responses OH viral shedding and symptom scores in experimental influenza infection

The role of T cells in controlling virus shedding (viral control) (Li IW et al. Chest. 137,759-68 (2010)) and symptom development (immunopathology) (La Grata NL et at Immunol Cell Biol, 85, 85-92 (2007)) was studied. The relationship between pre-existing T cells responding to total and immunodominant influenza proteins (NP + M), virus shedding, total symptom scores and illness duration was analysed. A correlation test (Spearman rank correlation test, Prism 5) was run to see if the magnitude of flu-specific CD4 or CD8 cells were correlative in virus shedding and disease severity as indicated by total symptom scores and length of illness duration in both H3N2 and H1N1 challenge studies. The results clearly showed that the magnitude of CD4 response against nmunodominant nucleoproteia (NP) and matrix (M) proteins was inversely correlative to peak virus shedding, symptom scores and illness durations (Table 2), This demonstrates that pre-existing T cell immunity to a virus can ameliorate subsequent infection with a virus. This also shows that the present methods allow determination of the peptides which induce a response. As shown in Figure 6 and Tables 3a and 3b, the magnitude of total pre-existing T cells was strongly correlated with illness duration in H3N2 challenge study (Table 3a, Fig 6a, r— 0.5740, ρ==Ό.0318, Spearman coefficient) and total symptom scores in H1N1 challenge (Table 3b, Fig 6b, r=-0.7113,_p=0.0369₅ Spearman coefficient,).

When the T cell responses to the hnmunodominant proteins (NP and M) were examined in detail, it was observed that these protective T cell responses were mediated by pre-existing CD4 (Fig 6a, right panel; Fig 6b top right), but not CDS T cell responses. The observed correlation was independent of the size of flu-specific CDS response in that the magnitude of pre-existing CD4, but not CDS, cells against the internal proteins NP and M were inversely associated with total symptom scores in both challenge groups. More importantly, virus shedding of H3N2 was predominantly controlled by the level of pre-existing CD4 responses to internal proteins NP and M (r=-0.6Q87, p=0.0209, Spearman coefficient) but this was not the case for CD8 cells (r=-0.0127, p=0.9657, Spearman coefficient).

To determine the relationship between the acutely expanding T cell population and illness metrics, the relationship between peak T cells on day 7, viral load and symptom severity was determined. The size of the developing acute T cell response correlated positively with viral shedding and illness severity for both models. These findings suggests that pre-existing memory CD4 T cells are the key in the CM! response in limiting illness that once illness is established acutely expanding cell populations tracked peak viral load and thus symptoms.

Phenotypes of pre-existing T cells agaiast NP and M Flu proteins

Pre-existing T cell responses against internal protein NP and M as measured by IFN-γ responses were largely CD4 T cell mediated in both H1N1 and H3N2 study groups. I the H3N2 challenge group, 9 subjects had NP and M responses at baseline and 8/9 (89%) had their peptides identified at a single peptide level (Table 4a). For the M protein, 7 out of 10 (70%) peptide responses were CD4 T cell mediated and for the NP protein, 8 out of 1 1 (73%) peptide responses were CD4 T cell mediated. In the H1N1 challenge group, 7 subjects which were positive with NP and M at baseline and 7/7 (100%) had their peptides identified at a single peptide level (Table 4b). For the M protein, 5 out of 5 (100%) peptides were seen by CD4. T ceils and for the NP protein, 3 out of 6 (50%) peptides were seen by CD4 T cells.

Phenotypes of induced T eells against NP and M t½ proteiss

In the day 7 antigen-specific T cell response to NP and M proteins most of the response was by CD4 T cells (Table 5). Upregulation of CD107a expression on memory CD4 T cells was observed following ex vivo stimulation of peptide pools to NP or M proteins (Fig 7a). Their cytotoxic function was further examined by a Cr-release assay using short-term T cell lines generated from baseline PBMC and on the killing of peptide pulsed autologous B cell lines. As shown in Figure 7b, these CD4 T cells killed autologous target cells in a peptide specific manner. The killing was sensitive to concanamycin, suggesting cytotoxicity was dependent on the perforia''granzyme pathway. Therefore, these memory CD4 T eelis possess a cytotoxic activity as described previously (Cazazza JP et al J. Exp Med, 203,2865-77 (2006)),

Table 4a - T ceil peptide responses m H3N2 challenge stady sub jects

celi

able 5 - T eel! esponses Hl l .e¾¾ltejage study

Heterosubtypic Immunity of pre-existing memory CD4 T cells

Because the sequence of NP and M proteins among human influenza A viruses is highly conserved, it. was next tested whether these memory CD4 T cells from HlNl or H3N2 challenge subjects could cross-recognise equivalent peptides from pandemic HlNl or from HlNl or H3N2 proteins respectively on ex vivo ELISPOT. The peptides tested were found to be highly cross-reactive to the corresponding peptides in a similar manner to their cognitive peptides despite several alternate amino acid changes in the internal proteins (Table 6).

Heterosubtypic immunity of pre-existing memory CD4⁺ T cells to novel Pan emic HlNl proteins

We next tested whether these memory CD4⁺ T cells from HlNl challenge subjects could cross-recognise equivalent peptides from pandemic HlNl proteins on ex vivo ELISPOT, Five out of six seasonal HlNl peptides tested demonstrated that T cells were found to be highly cross-reactive to the corresponding pandemic HlNl peptides in a similar manner to their cognate peptides despite several amino acid changes in the internal proteins (Fig, 8).

MHC Class II Expressio on Respiratory Epithelium and Changes during Infection

A role for cytotoxic CD4⁺ cells in limiting viral infection would implicate the need for expression of MHC class 11 on the respiratory epithelium - the target of influenza infection. To investigate this we analysed the constitutive expression of the MHC class II molecule, HLA-DR, in explanted lung tissue and on primary bronchial epithelial cells in culture (PBECs) and the effect of in vitro influenza infection on expression in PBECs. We found significant constitutive expression of this molecule in both lung tissue and cultured. PBECs with a rise in HLA-DR expression after infection of PBECs compared to cells treated with UV-inactivated vims (data in Fig, 9a₅b).

Claims

Claims

1. A peptide comprising a sequence having at least 85% identity to a peptide selected from the group consisting of:

SEQ ID NO 4 (TRPILSPLTKGILGFVF),

SEQ ID O 21 (KGILGFVFTLTVPSERGL),

SEQ ID NO 22 (DPNNMDRAVKLYRKLKRE),

SEQ ID NO 24 (REITFHGAKEIALSYSAG),

SEQ ID NO 1 (MSLLTEVETYVLSIV),

SEQ ID NO 2 (EVETYVLSIVPSGPLKA),

SEQ ID NO 3 (EALMEWLKTRPILSPLTK),

SEQ ID NO 5 (LTKGILGFVFTLTVPSER),

SEQ ID NO 13 (LYDKEEIRRIWRQANNGEDA),

SEQ ID NO 16 (ELIRMVKRGINDRNFWR),

SEQ ID NO 17 (RMCNILKGKFQTAAQRAM),

SEQ ID NO 25 (ARQMVQAMRAIGTHPSSS),

SEQ ID NO 27 (VRELVLYDKEEIRRIWRQ),

SEQ ID NO 28 (GSTLPRRSGAAGAAVKGV), an

SEQ ID NO 32 (SSTGLK DLLENLQAYQ ), or a fragment thereof of at least 9 amino acids,

2. A peptide according to claim 1, wherein the peptide comprises a sequence having at least 90%>, 95% or 99% identity with a peptide selected from the group consisting of SEQ ID NOS: 4, 21, 22, 24, 1, 2, 3, 5, 13, 16, 17, 25, 27, 28 and 32, or a fragment thereof of at least 9 amino acids.

3. A peptide according to claim 1, wherein the peptide comprises a sequence selected from SEQ ID NOS: 4, 21, 22, 24, 1. 2, 3, 5, 13, 16, 17, 25, 27, 28 and 32, or a fragment thereof of at least 9 amino acids.

4. A peptide according to any one of claims 1 to 3, wherein the peptide is 9 to 50 amino acids in length, optionally 9 to 40, 9 to 30 and preferably 9 to 25 amino acids in length.

5. A peptide according to any one of claims 1 to 4, wherein the fragment of SEQ ID NOS: 4, 21, 22, 24, 1 , 2, 3, 5, 13, 16, 17, 25, 27, 28 or 32 is at least 10, 11, 12, 13, 14, 15, 16, or 17 amino acids in length.

6. A peptide according to claim 1, wherein the peptide is selected from SEQ ID NOS: 4, 21 , 22, 24, 1, 2, 3, 5, 13, 16, 17, 25, 27, 28 and 32.

7. A peptide according to any one of claims 1 to 6, wherein the peptide is capable of inducing a CD4+ T cell response when contacted with a sample comprising T cells

8. A vaccine comprising a peptide according to any one of the preceding claims.

9. A vaccine according to claim 8, wherein the vaccine comprises at least 2, 3, 4, or 5 of the peptides.

10. A peptide according to any one of claim 1 to 7, or a vaccine according to claim 8 or 9 for use in therapy. 11. A peptide according to any one of claim 1 to 7, or a vaccine according to claim 8 or 9 for use in a method of inducing T cell immunity to influenza virus.

12. A peptide or vaccme according to claim 1 1 , wherein the T cell immunity induced is effective for more than one influenza virus strain, preferably the T cell immunity induced is effective for more than one influenza A virus strain,

13. A peptide or vaccine according to claim 12, wherein the T cell immunity induced is effective for at least influenza virus strains H3N2 and H1N1. 14. A peptide or vaccme according to claim 12, wherein the T cell immunity induced is effective for at least influenza virus strains seasonal H1N1 and pandemic H1N1. ί 5. A peptide according to any one of claims 1 to 7 or a vaccine according to claim 8 or 9, for use in a method of treating or preventing influenza infection, or symptoms of influenza infection. 16. A peptide or vaccine according to claim 15, wherein the peptide or vaccine is cross- protective and is useful for treating or preventing influenza infection or symptoms of influenza infection caused by more than one influenza strain.

17. A peptide or vaccine according to claim 16, wherein at least influenza virus strains H3N2 and H1 1 can be treated or prevented.

18. A method for inducing T cell immunity to influenza infection comprising

administering a therapeutically effective amount of a peptide according to any one of claims 1 to 7 or a vaccine according to claims 8 or 9 to a subject in need thereof.

19. A method for treating or preventing influenza infection or symptoms of influenza infection comprising administering a therapeutically effective amount of a peptide according to any one of claims 1 to 7 or a vaccine according to claim 8 or 9 to a subject in need thereof. 20. A method according to claim 18 or 19, wherein the subject is a human subject, optionally selected from the elderly, the young, individuals at risk of exposure to influenza virus.

21. A method according to claim 18 or 1 , wherein the peptide or vaccine is cross- protective and is useful for inducing T cell immunity to, or treating or preventing influenza infection caused by, more than one influenza strain.