Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the present invention relates to a screening method, and has particular reference to a method of screening peripheral blood against a peptide or a library of peptides to identify peptides that may give rise to an enhanced memory T cell response.
• the T cell response is a CD4 + T cell response.
• the invention also provides peptides that can be used to provoke an enhanced memory T cell response for use in the treatment or prophylaxis of a viral infection, especially in patients who are immunologically naive to the virus.
• 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 and other infections. Background to the invention
• influenza 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 $14bilIion 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 predicti ve modelling of strain circulation and the lack of viral coat protein mutation enabling immune evasion during a flu season.
• influenza can extend beyond its usual seasonal impact by shifting its antigenic profile significantly enough to escape from protective unmunity on a global scale. If such pandemic strams carry traits of high virulence and patliogenicity then associated mortality can be devastating, as seen in the 1 18 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 RN from different strains in a common host. The emerging novel pathogen can result i a pandemic where the new flu strain can spread rapidly through communities which lack protective immunity to novel viral proteins.
• 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 (C I) in sero-negative individuals naive to a particular flu strain is not understood, Lee et al (J Clin Invest 118, 3478-3490, 2008) showed thai memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) hi healthy individuals.
• the present inventors have identified a role for memory T cells that recognise particular viral antigens in limiting disease severity in viral infections. This effect led the present inventors to develop a screening method for identifying peptides that can manipulate T cell memory into recognising viral antigens, Peptides having the desired effect have been identified and these peptides may therefore be useful in the preparation of a vaccine composition.
• a first aspect of the present invention is a screening method which may be regarded as an in vitro or ex vivo method of mterrogatmg the immune system to understand what viral antigens are "seen” and responded to by T cells of the immune system during viral infection.
• This screening method enables identification and demonstration of peptides which are important in driving T cell responses. Correlation of those T cell responses with reduction in disease severity allows confirmation of disease protection.
• the present invention provides a screening method for identifying a peptide capable of inducing a T cell response, comprising:
• b) quantifying the response of the T cells to the peptide.
• c) comparing the T cells' response in b) to a response of a control sample comprising T cells obtained from blood from a subject who is not currently infected nor been recently infected with the virus, when contacted with the peptide,
• T cell immunity to the virus means these T cells are capable of reducing symptoms of a viral disease.
• CD4 + T cells induced by influenza peptides are useful in reducing symptoms of influenza. Therefore, a peptide identified as inducing a T cell response can be used to reduce symptoms of a viral infection.
• the first aspect of the present invention also provides a screening method for identifying a peptide capable of inducing a T cell response and inducing T cell immunity comprising:
• a first aspect of the present invention provides a screening method for identifying a peptide capable of inducing a T cell response and inducing T cell immunity to a virus, the method comprising: a) placing a plurality of test subjects who are not currently infected nor recently been infected wit the virus into controlled conditions in isolation,
• suc a correlation indicates a peptide useful in inducing T cell immunity to the virus.
• test sample is obtained at a known time point after inoculation with the virus, for example, 5-28 days, 2-20 days, 3-15 days, 5-10 days, most preferably 7 days post inoculation.
• a second aspect of the present invention is a screening method which may be regarded as an in vitro or ex vivo method of interrogating the immune system to find pre-existing native T cell responses to potential viral antigens, which enable a subject to experience less severe symptoms should they become infected with a virus.
• This screening method also enables identification and demonstration of peptides which are important in driving T cell responses.
• the present invention therefore provides a use of a peptide in a method of screening to identify a peptide capable of ameliorating a viral infection comprising:
• the detection of a T cell response indicates that the peptide is capable of inducing T cell irnmunity to a virus.
• the test sample comprising T cells is obtained from blood from a subject who is subsequently inoculated and infected with the virus, The subsequent inoculation and infection occurs under controlled conditions n isolation.
• the second aspect of the present invention also provides a screening method for identifying a peptide capable of ameliorating a viral infection, comprising:
• an above background response is indicative of a peptide capable of inducing a T cell response and therefore ameliorating a viral infection
• a second aspect of the present invention provides a screening method for identifying a peptide capable of ameliorating infection by a virus, the method comprising:
• Ameliorating a viral infection may be reducing symptoms of viral infection, ameliorating or reducing illness, or reducing or ameliorating a disease caused by a virus.
• test subjects in the first and second aspects of the invention have not been vaccinated against the virus.
• marker of progression of infection is
• a score of severity of symptoms of the viral infection experienced by the subjects and optionally an inverse correlation between symptom scores and T cell responses is identified
• a measure of viral shedding in the test subjects and optionally an inverse correlation between viral shedding and T cell responses is identified
• duration of illness experienced by the test subjects and optionally an inverse correlation between duration of illness and T cell responses is identified
• the peptide is generally about 7 to about 25 amino acids long, optionally 9-25 amino acids long or 10-20 amino acids long and preferably about 15, 16, 17 or 18 amino acids long.
• the level of identity the peptide has with a sequence of a protein of a virus conveniently is at least 70% identity.
• the peptide may have 80%, 90% or 95% identity, in further embodiments the peptide has an identical sequence with a sequence of a viral protein.
• the library may substantially span a protein of a viral proteome.
• the library of peptides substantially spans the conserved proteins of the viral proteome.
• the library of peptides substantially spans the viral proteome.
• the screening methods of the present invention allow selection of one or more peptides from a library of peptides which can induce T cell responses and which therefore may be used to reduce the symptoms of a viral infection.
• the peptide may be synthetic.
• the screening methods of the first and second aspect of the present invention are applicable for the investigation of T cell responses to peptides having a level of identity with any infectious virus.
• the virus may be a respiratory virus, such as an influenza virus, rhmovirus or respiratory syncytial vims.
• the virus may be an influenza virus and can be influenza A.
• the influenza A virus genome encodes eleven proteins. These are hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins Ml and M2, two non-structural proteins NS1 and NEP, PA, and polymerases PB1, PB1-F2, and PB2. HA and NA appear on the virion surface and are highly diverse.
• the core proteins are more conserved between different influenza viruses.
• a peptide of the present invention may have a sequence that is derived from a part of the influenza proteome that is conserved between different strains of influenza A, such as a core protein of influenza.
• Core proteins include NP, Ml, M2, NS 1 , NEP, PA, PB 1 , PB 1 -F2 an d PB2,
• the pep tides of the present invention may be derived from matrix (Ml or M2), nucleoprotein (NP) or polymerase (PB1 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.
• the subject from whom the test sample comprising T ceils is obtained is seronegative for the virus prior to infection.
• the subject has no current or recent viral infection.
• the subject may remain in controlled conditions optionally in isolation. This can be useful to control the viral or other infectious agents with whic the subject comes into contact.
• the test sample is obtained at a known time point after inoculation,
• the severity of the symptoms displayed by the subject in response to infection with the virus may quantified. Depending on the virus concerned a variety of different objective or quasi- objective methodologies are available to the person skilled in the art for this. For instance, in the ease of influenza one or more of the following symptoms may be scored.
• the present inventors have developed a symptom scoring methodology similar to the Jackson score (Jackson et al 1958) and have previously published work based on this (DeVincenzo et al, 2010a; DeVincenzo et al., 2010b; Jackson et al., 1958; Jones et al., 2009; Zaas et al,, 2009).
• twice daily volunteers complete a diary card on which they rate their symptoms on a four point scale from 0 to 3 corresponding to absent to severe.
• the symptoms assessed may be one or more nasal stuffiness, runny nose, sore throat, cough, sneezing, ear ache/pressure, breathing difficulty, muscle aches, fatigue, headache, feverish feeling, hoarseness, chest discomfort and overall discomfort.
• a total symptom score for a day may be obtained by adding the individual symptom scores for a particular day's morning and evening sessions.
• the individual symptoms may be divided into three subgroups: system symptoms (muscle aches, fatigue, headache and fever), upper respiratory symptoms (nasal stuffiness, ear ache/pressure, runny nose, sore throat and sneezing) and lower respiratory symptoms (cough, breathing difficulty, hoarseness and chest discomfort).
• Oral temperatures can also be measured daily, for example 2, 3 or 4 times a day.
• Fever may be considered to be an oral temperature > 37.7°C, Therefore, for any viral infection a subject or the supervising medical practitioner can score one or more symptoms experienced during the infection. The one or more symptoms to be assessed depend on the specific viral infection. Scoring may occur daily, twice daily or at other convenient intervals. Score cards can be generated for the use of the subjects.
• the methods of the first aspect of the present invention can further comprise a step d)of correlating the severity of the symptoms experienced by the subject from whom the test sample was obtained, with the magnitude of the T cell responses quantified in step b). Wherein a correlation between reduced symptom scores and T cell responses indicates that the peptide which caused the T cells response can induce T cell immunity. (Alternatively plirased an inverse correlation between symptom scores and T cell responses indicates that the peptide which caused the T cell response can induce T cell ixrmiumty)
• the methods of the second aspect of the present invention can further comprise a step c)
• the T cell response to the peptide in a plurality of samples from different subjects may be quantified.
• test and control samples comprising T cells are obtained from more than one subject.
• the method requires samples from more than 5, 6, 7, 8, 9, 10, 12, 15 or 20 subjects.
• blood samples from at least 10-20 and preferably samples from 20 to 30 or more subjects are required.
• subjects can be MHC typed prior to inoculation and infection to ensure a spread of MHC subtypes representative of the subject population.
• the present invention seeks to identify peptides that are useful in inducing a T cell response across a population of subjects.
• T cell responses to peptides and optionally correlation to reduced symptom scores allows identification of peptides which reduce symptoms of viral infection across a population of subjects. Therefore it is useful for a vaccine to contain peptides that would be effective for a range of individuals.
• the invention provides a peptide obtainable by the methods described herein.
• This aspect of the invention also provides a peptide having a sequence that is at least 70% identical to a sequence found within the proteome of a vims and which provokes a T cell response in a sample comprising T cells obtained from blood.
• a peptide obtainable by the present methods is capable of reducing symptoms in a subject infected by a virus. Therefore such a peptide is usef l in protecting against a viral disease.
• a peptide of the invention may therefore have a sequence that is at least 70% identical to a sequence found natively in the influenza proteome.
• the influenza A viral proteome that is capable of provoking a CD4 + T cell response
• the peptide may have a sequence that is derived from a pari of the viral proteome that is conserved between different strains of influenza A.
• a peptide of the present invention is capable of reducing symptoms of influenza experienced by a subject.
• the present invention provides a vaccine composition comprising at least one peptide in accordance with the invention for use in medicine.
• the peptide of the invention may be used in a method of treatment or prophylaxis of a viral infection or other condition in a human or non-human animal. Accordingly the invention extends to the use of at least one peptide according to the invention in the preparation of a medicament for the prophylaxis of influenza infection.
• the vaccine composition of the invention may be useful in the prevention (or prophylaxis) of influenza.
• the invention comprehends a method of treatment or prophylaxis of a disease or condition in a human or non-human subject in need thereof, comprising the step of administering a therapeutically or prophylactically effective amount of the vaccine composition of the invention to the subject.
• Said disease or condition may be influenza A.
• This aspect of the invention also 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 mfluenza in the subject.
• prevention and prophylaxis are used interchangeably herein. Prophylaxis of 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.
• a first aspect of the present invention is a screening method which may be regarded as an in vitro or ex vivo method of interrogating the immune system to understand what viral antigens are "seen” and responded to by T cells of the immune system during viral infection to enable a subject to experience less severe symptoms when becoming infected with a virus.
• the first aspect of the present invention is concerned with the T cell responses which are induced during viral infection and which help ameliorate the viral infection.
• a second aspect of the present invention is a screening method which may be regarded as an in vitro or ex vivo method of interrogating the immune system to find pre-existing T cell response potential to viral antigens, which enable a subject to experience less severe symptoms when becoming infected with a virus.
• the second aspect of the present invention is concerned with the pre-existing T cell responses which help ameliorate the viral infection.
• Other studies have obtained T cells from volunteers who were suffering or had recently suffered from a viral infection and investigated the responses of the T cells in vitro to viral peptides.
• these approaches have drawbacks. Due to the complexity of the human immune system, identifying in vitro T cell responses caused by contacting T cells with a specific peptide does not necessarily identify an antigenic peptide that is effective in humans to induce an immune response and reduce severity of disease.
• the complexity of the human immune system for example, means that in vivo certain peptides can be processed preferentially to others and this may not be detected in in vitro experiments.
• studies using animal models necessarily give clinically useful information about human immune responses to antigenic peptides because of the differences between animal arid human immune systems.
• the present invention also links T cell responses to a peptide identified in vitro with the severity of disease symptoms experienced by the subject from whom T cells were obtained.
• the present invention may also link T cell responses to peptides identified in vitro with how ill subjects are during viral infections, for example, the severity of disease symptoms experienced by the subject from whom T cells were obtained. This linkage is possible because the methods of the present invention require T cells to have been obtained from a subject whose time of inoculation and infection is known and whose disease progression is monitored. The disease progression is monitored by obtaining a marker of disease progression, such asscoring the severity of their symptoms. Comparing the T cell responses to a peptide which were detected in vitro, with the how ill a subject felt provides an extra layer of knowledge that has not been achieved before.
• the methods of the present invention may provide a further advantage in that the responses elicited and identified are those which are raised in and experienced by a subject as in a natural infection. Vaccination of subjects during or prior to a study can skew results so that T cell responses identified are likely to be directed to the peptides present in a vaccine rather than directed to the peptides to which the immune system naturally responds,
• Those identified peptides or viral antigens can be used to induce cell mediated immunity to that viral infection. Additionally peptide sequences that are similar (for example having 70% identity or greater) to those identified viral peptide sequences may also be useful to induce cell mediated immunity to viral infection.
• the screening method itself can employ peptides which are similar (for example having 70% identity or greater) to viral peptides and are capable of inducing cell mediated immunity.
• the peptide of the third aspect of the in vention may be obtainable by the screening method of the first or second aspects.
• Cell mediated immunity is an immune response that does not involve antibodies, but instead involves the activation of macrophages, natural killer cells (NK), 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.
• 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 viral peptides reduce the severity and duration of a viral illness.
• the screening methods of the present invention are for identifying peptides which induce a T cell response.
• a T cell response is indicative of inducing T cell immunity.
• T cells of use in the present invention can be CD3+ T cells. 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 CD3+ T cells, including 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.
• 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 preferred T cell response can be from CD8 ⁇ T cells, or from a combination of CD4+ and CD8+ T cells.
• a peptide for use in the first aspect of the invention may have a length in the range of from about 5 to 50 ammo acids, typically from about 5 to 40, more typically from about 8 to 30 and more typically from about 9 to 22 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 5, 6, 7, 8, 9 or 10 amino acids up to 11, 12, 13, 14, 15, 16, 17 or 18 consecutive amino acids.
• the peptide to be screened can be a member of a library of peptides.
• a library of peptides is also referred to interchangeably herein as a peptide library.
• a library of peptides contains a large number of peptides, for example many hundreds or thousands of peptides, for example from 100 to 10000 peptides, typically from 200 to 5000 peptides, more typically from 500 to 1000 peptides, for example 550 to 600 peptides and these peptides typically have a systematic combination of amino acids.
• the library of peptides may be a library of peptides in which each peptide has a sequence that is at least 70% identical to a respective sequence taken from the same at least one viral protein.
• the peptide may be at least 80% or 90% identical to the native sequence; and in some embodiments the peptide may have at least 95% identity to the corresponding sequence in the viral protein.
• the peptide may have the same sequence as in the viral protein.
• Identity as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the ar 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 FASTA (Atschul et al, J. Molec. Biol. 215, 403 (1990)).
• 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 be 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 determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
• Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
• PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules ( d.).
• the default parameters of the respective programs e.g., XBLAST and NBLAST
• the default parameters of the respective programs e.g., XBLAST and NBLAST
• the amino acid sequences of each peptide for use in the invention may have at least 70% identity, using the default parameters of the BLAST computer program (Atschul et ah, 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. More typically, the amino sequence may have at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the amino acid level to the sequence found in the viral protein. Typically, such amino acids retain the function of the original peptide, i.e. the function of generating T cell responses.
• the peptide may therefore be a variant of the respective sequence that is found in a viral protein.
• variant relates to peptides which have a similar amino acid sequence and/or which retain the same function.
• variant encompasses peptides that include one or more amino acid additions, deletions, substitutions or the like, in the present invention, variants of 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 amino acids of a peptide or protei can often be substituted by one or more other such amino acids without eliminating a desired activity of that peptide or protein.
• amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
• amino acids having aliphatic side chains amino acids having aliphatic side chains.
• glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, ieueine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).
• amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tsyptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains): asparagine and gliitaniine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).
• 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 can he 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.
• amino acid substitutions or insertions within the scope of the present invention ca 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- amino acids are present.
• the peptides of the peptide library may be sourced from the same one or two or more viral proteins.
• the viral proteins may be taken from the same strain of virus.
• the peptides of the library may be derived from the matrix (M), nucleoprotein (NP) or polymerase (PB1 or PB2) proteins, Peptides derived from the matrix (M) or nucleoprotein (NP) are particularly preferred.
• the library of peptides may span the at least one viral protein. In one embodiment the library of peptides may span more than one viral protein, for example two or three viral proteins.
• the library of peptides may span the entire viral proteome, A proteome is the entire set of proteins expressed by a genome, cell, tissue or organism.
• the library of peptides may contain a series of peptides that collectively cover a major portion (e.g. greater than 25%, 50%, 75% or 90%) or substantially all (e.g. greater than 99%) of the at least one protein or the entire proteome of the virus, with each peptide having at least the degree of identity to the corresponding native sequence mentioned above.
• the series of peptides may start with a peptide covering the first few amino acids of the at least one protein or proteome of the virus, and may include peptides which in total cover the major portion or all or substantially all of the at least one protein or proteome of the vims.
• the library of peptides in some embodiments may be devised such that they are contiguous; together covering the sequence of at least one protein or the proteome of the virus, in one embodiment, the peptides cover the at least one protein or proteome of the virus sequentially, in other words comparing the sequence of the library of peptides against the viral protein or proteome shows complete single coverage.
• the first peptide covers amino acids 1 to 15, the second peptide covers amino acids 1 to 30, the tliird amino acid covers amino acids 1 to 45 and so on.
• the library of peptides may be devised such that each peptide has a sequence overlap with an adjacent peptide.
• the first peptide covers amino acids 1 to 15, the second peptide covers amino acids 10 to 20, the tliird peptide covers amino acids 15 to 25 and so on.
• the peptides are from 16 to 20 amino acids, typically 18 amino acids, long. In one embodiment, the peptides overlap by 8 to 12 amino acids, typically 10 amino acids.
• the library of peptides may be devised such that comparing the peptides against the viral protein or proteome reveals sequence gaps between one peptide and the next.
• the peptide and the library of peptides may typically be synthetic peptides, Tims, the peptides for use in the invention 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 FeptGen and- synthesised using various commercially available platforms, for example using the proprietary PEPscreen technology from Sigma-Aldrich. Peptides can alternatively be produced recombinantly.
• Peptides for use in the invention are typically in a purified form.
• Peptides for use in the invention may include one or more non-natural amino acids.
• Peptides may be conjugated to one or more further moieties, for example polyethylene glycol (PEG) (Veronese F.M. (2001) Biomaterials 22 pp 4Q5-417).
• PEG polyethylene glycol
• the person skilled in the art would have no difficulty in providing a library of peptides to be screened in accordance with the invention, where each peptide is at least 70% identical (or more as described above) to a respective sequence taken from the same at least one viral protein, and where the peptides in the library optionally span part or all of at least one known viral protein, for instance an mfluenza A protein.
• the peptide is suitably a member of a library of peptides that are derived from one or more proteins - or the entire proteoxne - of the virus with which the subject has been inoculated and infected. In this way many peptides derived from the viral proteome may be screened simultaneously to identify the one or ones which induce a T cell response.
• the screening methods of the first and second aspect of the invention involve contacting a peptide with a test sample comprising T cells obtained from blood from a subject who has been infected with the virus. This step is typically carried out in vitro or ex vivo, The step of taking a blood sample may not therefore form part of the invention.
• the test 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 test sample comprising T cells comprises CD4+ T cells and CD8+ T cells, in certain embodiments the test sample comprises CD4+ T cells.
• test sample comprising T cells has been obtained has been inoculated and becomes infected with a virus prior to the test sample comprising T cells being taken.
• the subject from whom the test sample comprising T cells has been obtained has not been vaccinated against the virus.
• the subject has not been vaccinated against the virus within the preceding 12 months.
• the subject from whom the test sample comprising T cells has been obtained has not been vaccinated against the virus with a vaccine designed to elicit a T cell response.
• the subject has not been vaccinated against the virus with a vaccine designed to elicit a T cell response within the preceding 12 months.
• the subject When the sample comprising T cells is obtamed, the subject is currently infected with the virus or has recently been infected with replicating virus. This can include subjects recently infected with the virus and where symptoms of the infection have subsided.
• the sample comprising T cells is obtained 0 to 28 days from inoculation and infection with the virus, Therefore a subject currently infected or recently infected with the virus is a subject in whom viral replication has been detected within the past 28 days.
• Viral replication is detected from a nasal wash or swab with one positive sample by tissue culture or two positive samples by PCR. The methodologies described herein are advantageous because the exact time of inoculation is known. Test samples may be obtained at known time points after inoculation.
• a test sample may be obtained at approximately 12, 24, 36 or 48 hours after inoculation.
• a test sample may be obtained between 1 and 28 days after inoculation such as at 2, 3, 4, 5, 6 or 7 days after inoculation up to 10, 14, 15, 20, 21 or 28 days after inoculation.
• Multiple test samples may be obtained at differing but known time points after inoculation,
• inoculated is meant the placement of something into the human or animal body that will grow or reproduce, typically to produce or boost immunity to a particular disease.
• the word “inoculation” is sometimes used to mean “vaccination” and therefore the word “inoculated” is also used herein to mean “vaccinated”.
• inoculation may be the non-surgical intra-nasal introduction of inoculum.
• the subject from whom the test sample comprising T ceils has been obtained typically lacks virus-specific antibody responses, in other words lacks antibodies to the virus with which the subject has been inoculated and has become infected. This can also be described as lacking humoral immunity to the particular vims, or the subject being seronegative for a particular virus. Determination of whether or not a subject has antibodies to a particular vims can be carried out by any suitable means, for example using a hemagglutination assay.
• the subject is free from other infection, in some embodiments the subject has been free from infection for the preceding 2 weeks or more, such as 1 month or 2 months, or 3 months, or 6 months.
• each subject is coniined to an isolation unit and therefore any contact with pathogens, viruses or bacteria, can be controlled.
• the subject may be in isolation to control and preferably prevent infection with any other infectious agent other than the particular virus. Isolatio may commence prior to infection, isolation may continue until a test sample has been obtained,
• the screening methods of the present invention are applicable to all viruses. This is because the scientific community is not aware of any viral infection where T cells do not play a role. T cells function in the human response to a huge and disparate range of viral pathogens.
• the screening methods of the present invention investigate how the immune system raises a T cell response to a virus and how this affects illness.
• the virus with which the subject has been inoculated and become infected in the methods of the present invention may be a virus causing acute self-limiting viral infection.
• the virus with which the subject has been inoculated and become infected in the methods of the present invention may be a virus which is safe for use in a human viral challenge model.
• the virus with which the subject has been inoculated may be an attenuated virus or a modified virus, optionally the virus is attenuated or modified so that infectio is self- limiting.
• the virus may be a respiratory virus, an enteric virus, a mucosal virus or a virus that infects by the mucosal route, or a blood borne virus, in embodiments the virus may be a respiratory virus, an enteric viras or a mucosal virus.
• the virus may be a respirator)' virus.
• Viruses which may be of use in the screening methods of the present invention can include: Oithomyxoviridae (including influenza A, B, C discussed further below),
• Paramyxoviridae including respiratory syncytial virus (RSV), metapneumovirus, measles, mumps and Parainfluenza types 1, 2, 3 and 4
• RSV respiratory syncytial virus
• metapneumovirus including measles, mumps and Parainfluenza types 1, 2, 3 and 4
• Picomaviridae including rbinoviruses and enteroviruses
• Caliciviridae including noroviruses
• Flaviviridae including Hepatitis virus C, Dengue virus, Nile West virus.
• Reoviridae (including rotavirus).
• the viras with which the subject has become infected may be an enteric virus, such as noro virus.
• the virus with which the subject has become infected may be a respiratory virus, such as an influenza virus, rmnovirus (HRV), respirator syncytial virus (RSV), human metapneumovirus, adenovirus, coronavirus, boca virus, or other acute respiratory virus.
• a respiratory virus may be influenza virus, rhmovirus or respiratory syncytial virus.
• the virus is an influenza virus.
• the influenza virus is typically influenza A.
• the influenza A may be of subtype H1N1 or H3N2.
• influenza commonly referred to as the flu
• influenza 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, musckjgairis, severe headache, coughing, weakness/fatigue and general discomfort.
• influenza viruses make up three of the five genera of the family Orthomyxoviridae. Of these, influenza A virus is mos 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 virus may be influenza A.
• the influenza A virus can be subdivided into different serotypes or subtypes based on the antibody response to these viruses. The subtypes that have been confirmed in humans are H1N1. H1 2, H2N2. H3N2. H5N1, H7N2. H7N3. H7N7. H9N2 and H10N7.
• influenza A may suitably be of subtype HlNl or H3N2.
• the subject is typically a human subject, but the method of the invention also finds use when the subject is a veterinary subject, i.e. an animal.
• the subject may have been inoculated and become mfected with the virus immediately prior to the test sample being taken.
• the test sample may suitably have been taken some time after inoculation of the subject with the virus, to give the subject's immune system time to react to the virus and for a T cell, response to have been raised.
• the sample may have been taken from the subject from 0 to 28 days after inoculation, or around 1 to 21 days after inoculation of the subject with the virus.
• the sample may have been taken from the subject from 2 to 14 days, more typically from 3 to 10 days, more typically from 4 to 8 days, for example 7 days after inoculation of the subject with the virus,
• the sample is taken at a known number of days post inoculation.
• the screening methods of the first and second aspect of the invention involve quantifying the T cell response to the peptide in the test sample. This step may be carried out ex vivo or in vitro,
• quantifying the T ceil response is meant quantifying any response of T ceils to said peptide.
• the T cell response that is quantified may be the production of one or more cytokines, for example ⁇
• the T cell response for example the production of one or more cytokines, can be quantified using any suitable means.
• 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 mdividual 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.
• the ELISPOT assay may be an IFNy ELISPOT assay.
• 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 .
• the T cell response is quantified.
• T cells can be either CD4+ or CD8-K 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 I ' cell response can be attributed to CD4 + T cells. This 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. This may be useful when studying certain viral infections, for example influenza.
• the screening methods of the first aspect of the invention involve comparing the T cell response to the peptide in a test sample from a subject who has been inoculated and infected with the virus to the T cell response to the peptide in a control sample.
• the control sample comprising T cells has been obtained from a subject who has not been infected with the vims. Therefore the control sample comprising T cells was obtained from a subject who was not raising a T cell response to the virus.
• the control sample may have been taken from a subject prior to viral challenge. Such a subject is also referred to herein as a control subject.
• the control subject typically also lacks virus-specific antibody responses, in other words lacks antibodies to the virus with which the subject is inoculated. This can also be described as lacking humoral immunity to the particular vims, or the subject being seronegative for a particular virus. Determination of whether or not a subject has antibodies to a particular virus can be carried out by any suitable means, for example using a haemagglutmation assay.
• control sample may have been taken from the same subject as the test sample; that is the control sample may have been taken from the subject prior to inoculation and infection with the virus, while the test sample has been taken from the subject after inoculation and infection.
• test sample is obtained at a known number of days post infection. Quantifying the responses of T ceils obtained from blood from a subject who has not been inoculated with the vims and is not currently or recently been infected with the virus is typically carried out in vitro or ex vivo.
• the peptide is contacted with the control sample comprising T cells that have been obtained from the control subject.
• the step of taking the control blood sample may not form part of the invention.
• the control sample comprising T cells can be the same type of sample described above in relation to the test sample or can be different.
• the control sample comprising T cells may be a whole blood sample, a fraction of whol e blood or typically a sample of PBMCs.
• the control sample comprising T cells generally comprises CD4+ T cells and CD8+ T cells, in certain embodiments the control sample comprises CD4+ T cells, for example when studying influenza.
• Quantifying the responses of T cells in the control sample to the peptide may be quantified as described above for the test sample.
• the meihod of the first aspect of the invention may also involve the steps of contacting the peptide with the control sample comprising T cells and quantifying the T cell response to said peptide in the control sample comprising T cells prior quantifying contacting the peptide with the test sample comprising T cells and quantifying the T cell response to said peptide in the test sample. Therefore the order of the steps of the screening methods of the present invention may vary.
• the method of the first aspect of the invention for screening a peptide from a library of peptides for T cell reactivity comprises the following steps:
• step (e) comparing the T cell response in step (b) to the T cell response in step (d);
• step (b) wherein an increased T cell response in step (b) compared to the T cell response to the peptide in the control sample in step (d) is indicative of the peptide having T cell reactivity.
• indication that a peptide has T ceil reactivity may mean that peptide is useful in the preparation of a vaccine composition.
• test and control samples comprising T cells are obtained from a plurality of subjects i.e. more than one subject.
• the methods require samples from more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 subjects.
• samples from 20 to 30 or more subjects are required.
• more than 25, 30, 40 or 50 subjects are required.
• subjects can be MHC typed prior to inoculation and infection to ensure a spread of MHC subtypes representative of the subject population.
• the screening methods of the present invention comprehend obtaining a marker of progression of infection.
• the marker of progression of infection is a sign, symptom or aspect of the viral infection which is detectable and which can vary from one subject to another.
• the present invention advantageously links how ill a subject feels with the in vitro or ex vivo information obtained from analysing T cell responses to peptides.
• the marker of progression of infection may be a score of the severity of symptoms of the viral infection experienced by a subject. Therefore the methods of the invention include scoring of symptoms experienced during mfection by the subject who has been inoculated and becomes infected with the virus and from whom the test sample was obtained
• a score of the severity of the symptoms experienced by a subject can include recording how ill the subject felt or how noticeable the symptoms of the viral infection were.
• the severity of the symptoms displayed by the subject in response to infection with the virus may be quantified.
• a variety of different objective or quasi-objective methodologies are available to the person skilled in the art for this.
• mfluenza one or more of the following symptoms may be scored
• the present inventors have developed a symptom scoring methodology similar to the Jackson score (Jackson et al 1 58) and have previously published work based on this (DeVincenzo et a!., 2010a; DeVincenzo et al., 2010b; Jackson et al., 1958; Jones et al, 2009; Zaas et al., 2009).
• twice daily volunteers complete a diary card on which they rate their symptoms on a four point scale from 0 to 3 corresponding to absent to severe.
• the symptoms assessed may be one or more nasal stuffiness, runny nose, sore throat, cough, sneezing, ear ache/pressure, breathing difficulty, muscle aches, fatigue, headache, feverish feeling, hoarseness, chest discomfort and overall discomfort.
• a total symptom score for a day may be obtained by adding the individual symptom scores for a particular day's morning and evening sessions.
• the individual symptoms may be divided into three subgroups: system symptoms (muscle aches, fatigue, headache and fever), upper respiratory symptoms (nasal stuffiness, ear ache/pressure, runny nose, sore throat and sneezing) and lower respiratory symptoms (cough, breathing difficulty, hoarseness and chest discomfort).
• Oral temperatures can also be measured daily, for example 2, 3 or 4 times a day.
• Fever may be considered to be an oral temperature > 37.7°C.
• For any viral infection a subject or the supervising medical practitioner can score one or more symptoms experienced during the infection.
• the one or more symptoms to be assessed and scored depend on the specific viral infection. Optionally two, three, four or five or more different symptoms are scored.
• Score cards can be generated for the use of the subjects.
• a score or recording of the symptoms of the viral infection can be taken at the time the test sample is obtained.
• a score or recording of the symptoms of the viral infection can be taken every other day, daily, twice a day, three times a day, every 8 hours, ever ⁇ ' 6 hours, every 4 hours, every 3 hours, every 2 hours, ever hour, such as hourly during waking hours following inoculation with the virus.
• Scoring can be quasi-objective, objective, or a combination of both. Quasi-objective scoring can be for example with a score on a scale from 0 to 3, or on a scale from 0 to 10 corresponding from absent to severe being used. Scoring can be objective with a recording of one or more of temperature, mucus production volume, lung function, percentage swelling or number of sneezes being used.
• reduced symptom scores can mean lower scores, i.e. that a subject feels less ill and/or has fewer or less severe symptoms, than usually attributed to infection with the virus under investigation, in embodiments involving a plurality of subjects reduced symptom scores can mean reduced scores in comparison with another test subject.
• the marker of progression of infection can be viral shedding or viral load. Therefore the screening methods of the present invention can include obtaining a measure of viral shedding or viral load. Viral shedding or viral load can be assessed via nasal washes from samples derived via, for example, nasal washes, nasal swabs, nasopharyngeal swabs, bronchial alveolar lavage or other techniques of collecting virus containing samples.
• the marker of progression of infection can be duration of illness experienced by a subject. Therefore the screening methods of the present invention can include recording the duration of illness. Duration of illness can be measured from the start of a symptom of the viral infection until cessation of the symptom of the viral infection. For some viruses duration of illness can be measured in days. Alternatively, for other viral infections the duration of illness can be measured in hours.
• the marker of progression of infection can be a measure of a biomarker modulated in a subject during infection with a virus.
• a plurality of biomarkers modulated during infection may be used.
• the biomarker selected may be one which is typically increased during infection with a virus.
• the biomarker selected may be one which is typically decreased during infection with a virus.
• the screening methods of the present invention can include obtaining a measure of more than one marker of progression of infection.
• both symptom scores and viral shedding information may be used.
• symptom scoring and illness duration may be selected.
• viral shedding and illness duration may be assessed.
• symptom scoring, viral shedding and illness duration may provide useful information. It may be regarded that a peptide to which the subject's T cells respond in the screening methods of the first aspect of the present invention, is a viral peptide (or a peptide with a suitably similar sequence) "seen" by the subject's immune system during infection and to which an immune response was raised.
• a peptide of the invention provokes a T cell response, preferably a CD4+ T cell response, with a magnitude that correlates inversely with the severity of symptoms associated with a viral infection.
• the screening methods of the present invention may require a step of identifying a correlation between a marker of progression of infection and T cell responses, optionally the magnitude of the T cell responses.
• the correlation may be performed using one or more of the methodologies available to investigate a relationship between two variables.
• the marker of progression of infection is symptom scores
• the screening methods can identify a correlation between lower symptom scores and the magnitude of the T cell responses.
• the screening methods can include identifying an inverse correlation between symptom, scores and T cell responses. This can identify a peptide able to ameliorate infection.
• the marker of progression of infection is duration of illness
• the screening methods can identify a correlation between shorter duration of illness and the magnitude of the T cell responses.
• the screening methods can include identifying an inverse correlation between illness duration and T cell responses. This can identify a peptide able to ameliorate infection,
• the screening methods can identify a correlation between lower viral shedding and the magnitude of the T ceil responses.
• the screening methods can include identifying an inverse correlation between viral shedding and T cell responses. This can identify a peptide able to ameliorate infection.
• the marker of progression of infection is a biomarker typically increased during infection with a virus
• the screening methods can identify a correlation between biomarker levels and the m agnitude of the T cell responses.
• the screening methods can include identifying an inverse correlation between biomarker level and T cell responses.
• the screening methods can identify a correlation between biomarker levels and the magnitude of the T cell responses.
• the screening methods can include identifying a positive correlation between biomarker level and T cell responses, This can identify a peptide able to ameliorate infection.
• the screening methods of the second aspect of the present mvention involve contacting a peptide with a test sample comprising T cells obtained from blood from a subject who is subsequently infected with the virus.
• the step is typically carried out in vitro or ex vivo.
• the step of taking a blood sample may not therefore form part of the invention.
• test sample comprising T cells may be described as above.
• test sample may be referred to as a TO test sample.
• the subject from whom the test sample comprising T cells has been obtained has not yet been inoculated or become infected with a virus.
• the subject from whom the test sample comprising T cells has been obtained is typically seronegative for a particular virus. Determination of whether or not a subject has antibodies to a particular virus can be carried out by any suitable means, for example using a haemagglutination assay.
• the virus of interest in the second aspect of the present invention is as described above with respect to the first aspect of the present invention.
• the subject is typically a human subject, but the method of the invention also finds use when the subject is a veterinary subject, i.e. a non-human animal.
• the peptide is suitably a member of a library of peptides that are derived from one or more proteins - or the entire proteome of the virus. In this way many peptides derived from the viral proteome may be screened simultaneously to identify the one or ones which induce a T cell response.
• the screening methods of the second aspect of the invention involve quantifying the T cell response to the peptide in the test sample. The step may be earned out as described above.
• the screening methods of the second aspect of the present invention may involve the subject from whom the test sample comprising T cells is obtained subsequently being inoculated and becoming infected with the virus, it follows that if the subject experiences a milder infection as determined by a marker of progression of infection such as reduced symptom scores, as described above, then this may be the result of memory T cells.
• Peptides identified as inducing T cell responses in samples comprising T cells obtained from subjects who subsequently experience a milder infection may be peptides capable of conferring T cell immunity and being valuable in protection against disease.
• indication that a peptide induces a response may mean that peptide is useful in the preparation of a vaccine composition.
• test samples comprising T cells are obtained from more than one subject.
• the method requires samples from more than 5, 6, 7, 8, 9, 10, 12, 15 or 20 subjects, Preferably samples from 20 to 30 more subjects are required. This is for the reasons described above.
• the screening method further comprises the step of testing the effectiveness of the identified peptide in inducing T cell immunity to the particular virus.
• a subject seronegative for the particular virus is vaccinated with the identified peptide.
• the subject is infected with the particular virus and symptoms of the infection are scored and compared against symptom scores of a subject seronegative for the particular vims who was not vaccinated with the identified peptide prior to infection with the particular virus.
• inoculation of a subject with a vims is via intra-nasal introduction of a virus.
• the methods of the first and second aspect of the invention have been used to identify peptides according to the third aspect of the invention that are derived from a strain of influenza A virus and provoke a CD4 + T cell response in a sample comprising T cells from a subject who was seronegative for that strain of the virus.
• the magnitude of the CD4 + T cell response was found to correlate inversely to the severity of symptoms suffered by a subject who was initially seronegative for a strain of virus when inoculated with that strain of the virus.
• the screening methods of the first and second aspect of the present invention identify a peptide capable of inducing a T cell response and inducing T cell immunity and/or ameliorating a viral infection.
• the methods can further include steps which determine whether the identified peptide is cross-reactive and is therefore also valuable in inducing a T cell response and inducing T cell immunity and/or ameliorating a viral infection against more than one virus or more than one strain or subtype of a virus.
• a peptide can be capable of inducing a T cell response and inducing T cell immunity and/or ameliorating a viral infection to more than one strain or subtype of influenza.
• the screening methods of the first and second aspects of the present invention can further include a step of testing whether T cells which respond to the identified peptide can also respond to an equivalent peptide from a second virus or a second strain or subtype of the virus. If T cells can demonstrate a capability to cross-recognise an equivalent, peptide from a second virus or a second strain or subtype of the same virus, both peptides are valuable as they can be used to induce T cell responses to both strains or subtypes of the virus. In other words the peptides are cross-reactive. Either peptide can he used to vaccinate against both viruses or both viral strains or subtypes.
• the screening methods of the first and second aspects of the present invention may therefore include a step of
• an above background response is indicative of cross-recognising T cells and therefore indicative of the identified peptide and the equivalent peptide being cross- reactive.
• an equivalent peptide generally refers to a peptide of similar sequence to the identified peptide.
• the differences in sequence between the identified peptide and the equivalent peptide may derive from the differences in protein sequence between different strains or subtypes of a virus,
• an equivalent protein refers to the protein having the same designation.
• the NP protein of a first influenza A strain or subtype can be the equivalent protein to the NP protein from a second influenza A strain or subtype.
• the M protein of a first influenza A strain or subtype can be the equivalent protein to the M protein from a second influenza A strain or subtype.
• a level of identify is as described above.
• the methods of the first and second aspect of the invention may be used to screen for peptides according to the third aspect of the invention that are derived from viruses other than influenza and give rise to a similar T cell response in patients infected with such a virus. Tills is because T cell responses are mounted by the immune system to all viruses. Therefore, since T cells are an integral part of the immune response, T cell responses are a valuable part of the immune system's defences against any viras and the methods of the present invention are applicable to identifying peptides expected to induce T cell immunity to any viral infection.
• a third aspect of the present invention provides a peptide that is at least 70% identical to a sequence found within the proteome of a viras and which provokes a T cell response.
• the peptide is therefore predicted to be capable of inducing T cell immunity to a virus.
• the third aspect of the present invention encompasses peptides obtainable by the screening method of the first and second aspect of the present invention described above,
• the present inventors used a human challenge model of influenza infection, as described in the Example below, to identify certain peptides which induce a T cell response
• the T cell response is preferably a CD4+ T cell response, Therefore the peptides are predicted to be capable of inducing T cells immunity.
• the sequences of the peptides identified by the inventors are shown in Table I below together with their SEQ ID NOs.
• the peptides having the sequences of SEQ ID NOs: 6-12, 14-15 and 18-20 were identified from an H3N2 subtype of influenza A and the peptides having the sequences of SEQ ID NOs: 23-26 and 29-31 were identified from an H1N1 subtype of influenza A.
• the present invention also encompasses peptides comprising sequences at least 70% identical to a sequence listed above, optionally the peptide may be at least 80% or 90% or 95% identical to a sequence listed above.
• the present invention also encompasses fragments of such peptide sequences of lengths as described above. Additionally the present invention provides peptides consisting of the sequences identified in Table 1. 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 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 be used to denote any amino acid.
• Preferred variants of the peptides for use in the present invention include one or more conservative substitutions as defined herein.
• a fourth aspect of the present invention is a vaccine comprising one or more pepti des capable of inducing a T cell response.
• the peptides are capable of inducing T cell immunity to a virus.
• 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 peptides of use in a vaccine are obtainable by the screening method of the first aspect of the present invention and may include the peptides of the second aspect of the invention.
• the vaccine can comprise one or more, two or more, optionally three or more peptides having SEQ ID NO 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 19, 20, 23, 26, 29, 30 or 31 (corresponding to SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 in the sequence listing), or peptides at least 70% identical thereto or a fragment thereof.
• the vaccine composition of the invention can he formulated for use by any convenient route.
• the vaccine composition of the invention may be a pharmaceutical 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, excipient, diluent, adjuvant, vehicle, buffer or stabiliser in addition to one or more peptides of the invention as the therapeutically or prophylaetieally active, ingredient.
• Such carriers include, but are not limited to, saline, buffered 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 intradermal 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 know in the art of pharmacy, for example by admixing the peptides with the carrier(s) or excipient(s) under sterile conditions.
• the vaccine composition is adapted for administration by the subcutaneous, intramuscular, intravenous or intradermal route, typically by injection.
• 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, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueoiis 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.
• 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 (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
• sterile liquid carried, for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions can be prepared from 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,
• excipients which can be used include for example water, polyols and sugars.
• oils e.g. vegetable oils
• oil-in- water or water in oil suspensions can be used.
• 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.
• 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.
• 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.
• the composition may be applied as a topical ointment or cream.
• the active ingredient When formulated in an ointment, the active ingredient can he employed with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-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, solubilising 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 lyophilised.
• 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 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 Qui! 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, IL-15, IL-6, GM-C8F, IFNy or other cytokines promoting T cell responses. This can be done in addition to conventional adjuvant, as described above.
• 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, IL-15, IL-6, GM-C8F, IFNy or other cytokines promoting T cell responses.
• 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.
• the daily dosage of the active agent will be from lj,ig kg to lOmg/kg body weight, typically around li ⁇ 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 vaccine 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. Timing of administration of the vaccine ma 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-human 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 "prophylactically 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,
• the present invention provides a kit of parts comprising a vaccine composition 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 vaccine composition of the invention and one or more cytokines and/or adjuvants for separate, subsequent or simultaneous administration to a subject.
• a fifth aspect of the present invention is a peptide capable of inducing T cell immunity for use in a method of treatment or prophylaxis of influenza.
• the fifth aspect of the present invention is the use of a peptide capable of inducing T cell immunity for the manufacture of a medicament for the treatment or prophylaxis of influenza.
• the peptide of can be a peptide obtainable by the first or second aspect of the present invention.
• the peptide can be a peptide of the second aspect of the present invention.
• the peptide can be a peptide having SEQ ID NO 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 19, 20, 23, 26, 29, 30 or 31 , (corresponding to SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 or 17 in the sequence listing), or a peptide at least 70% identical thereto or a fragment thereof.
• a fifth aspect of the present invention also provides a method for the treatment or prophylaxis of influenza comprising administering to an individual in need thereof a peptide capable of inducing T cell immunity.
• the peptide is obtamable by the first or second aspect of the present invention.
• the peptide can be a peptide having SEQ ID NO 6, 7. 8, 9, 10, 11, 12, 14, 15, 18, 19, 20, 23, 26, 29, 30 or 31, (corresponding to SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 in the sequence listing), or a peptide at least 70% identical thereto or a fragment thereof.
• Figure 1 shows an Elispot layout of experimental influenza A infection in humans
• H3N2 challenge study (A Wiseonsm/67 05) T cell Elispot layout
• H1N1 challenge study (A B.risbane/59/07) T cell Elispot layout.
• Freshly isolated 300.000 PBMC were put into each well and stimulated with peptide pool at 2 ⁇ ' ⁇ for 18-24 hours.
• Figure 2 shows viral shedding in nasal wash, seroconversion and symptom development in seronegative healthy volunteers experimentally infected with influenza A.
• 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 four 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
• FIG. 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- ⁇ 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 5 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 MP 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
• 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) H1N1 (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.
• 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.
• SEB Staphylococcus enterotoxin B
• Figure 8 a Human parenchymal (i) and (ii) and bronchial tissue stained (iii and iv) for MHC ⁇ (HLA-BR) 2mm sequentially cut sections and immunostained using i subtype 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 counterslam is shown in blue. Size bar represents 50um.
• the study confirmed that pre-existing cell mediated immuriiiy to a virus (in other words the existence of memory T cells within a subject, which respond to peptide antigens of a virus) results in a reduction n disease symptoms, duration of disease and viral shedding, when the subject is infected with that virus.
• the cognate peptide antigens for the pre-existing memory T cells were identified. These peptides can be used to induce T cell immunity to the virus.
• the study demonstrates the effectiveness of the screening method of the invention for identifying peptides which correspond to antigens inducing T cell responses during immune response to viral infection. These peptides can be used to induce T cell immunity in a virus.
• H3 2 challenge study a total of 17 healthy adult volunteers, which are haemagglutination-inhibition (HI) titres less than 1:8 to influenza A/Wisconsin/67/05, were enrolled in the study.
• HlNl 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/TCH/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.
• 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 vims 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
• Group 4 received low dose of virus.
• Nasopharyngeal swab were collected daily from baseline day 0 during the quarantine period for virus isolation. Serum samples were taken daily 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 time point day 3 was taken for H1N1 study.
• 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, sneezing, earache/pressure, breathing difficult ⁇ ', 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 respiratory symptoms (nasal stiffness, ear ache/pressure, runny nose, sore throat, and sneezing) and lower respiratoxy symptoms (cough, breathing difficulty, hoarseness and chest discomfort).
• Viral load in the nasopharyngeal samples were determined by TCII1 ⁇ 2 assay as described by the WHO manual of Animal influenza:
• H3N2 A Brisbane 59/2004 (H1N1), A New York 388/2005 (H3 2) (surface proteins), and A/New York 232/2004 (H3N2) (internal proteins).
• 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 H1N1 and H3N2 were 554 and 601 respectively.
• Ex vivo IFNy ELISPOT assays were used to identity T cells which respond to stimulation with a specific peptide and therefore secrete IFNy.
• Peptides were used at a final concentration of 2 ⁇ g/m ⁇ each.
• the putative peptide from each positive- response well could be deconvoluted 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.
• PBMC Peripheral mononuclear cells
• each peptide in each well was 2 ag/ l, for both peptide pools and individual peptides. All ELISPOT assays were performed using the human IFN- ⁇ 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 ⁇ ). The spots on each well were counted using an automated ELISPOT reader and AID ELISPOT 3.1.1 HR software (Autoimmune Diagnostika).
• T cells were CD4 or CDS
• 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.
• Elispot assay response greater than 10 SFC/million PBMC was considered positive after background substraction and when T ceil lines could be generated from respective peptides and tested positive again with ICS.
• CD38+ and proliferating (Ki67+) cells in freshly isolated PBMC were stained by 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 CD 107a (clone H4A3, BD) and IFN- ⁇ (clone X G1.2, BD) in both CD4 and CDS memory cells were also studied ex vivo using frozen PBMC as described previously (Li et aL, J.
• PBMC peripheral blood mononuclear cells
• 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 immunostained 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 CQunterstain is shown in blue.
• PBECs Primary bronchial epithelial cells
• influenza A virus strain X31 was supplied at a concentration of 4 x 10 7 pfu/ml (a kind gift of 3 VBiosciences). Inactivated virus (UVX31) was prepared by exposure to an ultra-violet (UV) light source for 2 h.
• UVX31 Ultra-violet
• 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 > 2 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 3 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 % CO? in 0.5ml of BEBM-ITS. Cells were dispersed by trypsinisation and prepared for flow cytometric analysis as previously described.
• BEBM Bronchial Epithelium Basal Media
• BEBM+ITS insulin, transferrin and selenium
• Allophycocyanin-Cyanine 7 (APC-Cy7)-conjugated anti-HLA-DR (BD Biosciences, Oxford, UK) or appropriate isotype control (IgG2a BD Biosciences Oxford, IJK).
• intracellular staining for viral nueleoprotem (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).
• CMI cell mediated immunity
• the H1N1 challenge group did not exhibit reliable viral shedding - ⁇ a recognised phenomenon with this egg grown vims (Steel J, et al. J Virol 2009 Feb;83(4): 1742-53).
• 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)).
• Antibody a3 ⁇ 4d T ceil res onses of infected volunteers All volunteers enrolled were screened to ensure they were sero-negative for antibodies to the challenge virus. However, the antibody responses (HA1 titre) were detectable after 7 days post challenge (Fig 5a), at which time the viruses were completely cleared as indicated in Figure
• 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 4).
• Fig 4 At baseline, in the H3N2 group, 11 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- ⁇ response, in the H1N1 challenge group, 8 out of 9 (89%) 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 matrix protein (6/9, 67%).
• the body dramatically responds to viral peptides during mfection raising T cell responses and the present methods allow identification of those peptides.
• the total memory T cell response had returned to baseline levels ( ⁇ 1000 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 14 infected subjects (50%) were T cell positive, with the average number of proteins recognized reduced to 1 (range 1-2). In the HlNl 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).
• T cells 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 Gmta ML et al Immunol Cell Biol. 85, 85-92 (2007)) was studied.
• a correlation test (Spearman rank correlation test, Prism 5) was run to see if the magnitude of flu-specific CD4 or CDS 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.
• MHC Class II Expression 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 II on the respiratory epithelium - the target of influenza infection.
• PBECs primary bronchial epithelial cells in culture

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