WO2015033137A1

WO2015033137A1 - Biological methods and materials for use therein

Info

Publication number: WO2015033137A1
Application number: PCT/GB2014/052668
Authority: WO
Inventors: Ajit Lalvani; Saranya Sridhar; Shaima BEGOM
Original assignee: Imperial Innovations Limited
Priority date: 2013-09-04
Filing date: 2014-09-04
Publication date: 2015-03-12

Abstract

There is provided methods of determining prognosis of an influenza infection in an individual comprising: (i) providing a sample comprising T-cells; (ii) exposing the sample of (i) to one or more influenza antigens; (iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-y without secreting IL-2; and optionally (iv) calculating the cells identified in (iii) as a percentage of those provided in (i); wherein the identification of cells in (iii) and/or the percentage of T-cells calculated in (iv) correlates to the risk of the individual developing symptomatic disease following an influenza infection. There are also provided compositions and kits for use in such methods.

Description

BIOLOGICAL METHODS AND MATERIALS FOR USE THEREIN

The present invention relates to methods for determining prognosis of an influenza infection, methods of determining influenza immunity status and methods of identifying compounds that induce influenza immunity as well as compositions and kits for use in such methods.

Influenza remains a significant public health problem affecting an estimated 15% of the global population and causing between 250,000-500,000 global deaths every year (World Health Organisation (WHO) 2009).

Protective immunity to influenza infection is mediated primarily through antibodies to the surface proteins of the influenza virus. However, the virus is constantly changing its surface proteins to evade this antibody-mediated immunity. When this antibody- mediated immunity is circumvented as in seasonal epidemics and during pandemics, individuals and populations become susceptible to infection. At such times, limiting disease severity and symptoms become important. But, what immune response correlates with protective immunity against disease severity has remained unclear. Current influenza vaccines are designed to induce antibodies that can prevent influenza infection. However, as the influenza virus constantly mutates to evade this protective antibody-mediated immunity these vaccines need to be changed every year to keep up with viral evolution and match the circulating virus strain in that year. Thus, there is a need for a "universal influenza vaccine" that is protective against all influenza virus strains which does not require annual updating.

One way to develop a universal influenza vaccine is to target virus proteins that are conserved across all strains and to induce T-cells that are more likely to recognise such highly conserved virus proteins. Further, T-cells specific for such highly conserved viral proteins have been shown to provide protective immunity against symptomatic disease in animal models of influenza and in experimental challenge models in humans. However, there has been no evidence or identification of a T-cell correlate that is associated with protection against symptoms following natural infection with influenza.

McMichael ef a/. (N Engl J Med, 1983. 309(1): p. 13-7) showed using a human experimental challenge study that CD8+ T-cells were associated with reduced symptom severity. Wilkinson et al. (Nat Med, 2012. 18(2): p. 274-80) showed using a human experimental challenge study that CD4+ T-cells were associated with reduced symptom severity. Forrest et al. (Clin. Vaccine Immunol., 2008. 15(7): p. 1042- 1053). showed that higher post-vaccination IFNg secreting T-cells were correlated with protection against influenza infection. McElhaney et al. (J Immunol, 2006. 176(10): p. 6333-9) showed that in elderly adults, higher Granzyme B levels secreted by T-cells was correlated with protection against infection. Wagar et al. (2011) Plos One 6(8) report the increase in late effector (KLRG1 +CD57+) T-cells in older people who are at greater risk of severe influenza.

Thus, there are a number of different types of T-cells that have been implicated in protection against influenza. However, a particular subtype of T-cells or the viral proteins targeted by these T-cells has not previously been identified. Further, no previous work has correlated T-cells with protection against influenza symptoms in humans following natural infection.

There is a need to identify a T-cell correlate of protection that can be used to guide T- cell universal influenza vaccine development. WO 2005/015207 describes immune diagnostic assays based on stimulating T-cells with pathogen-specific antigens. WO 2013/037804 describes methods of determining if a T-cell response to a virus is capable of being mounted. WO 2013/093514 describes influenza peptide antigens that induce CD4+ T-cells. WO 2013/1 13092 describes methods of determining an immune response to an immunostimulatory composition focussed on measuring a T-cell subset expressing the biomarkers granzyme B and perforin.

Sridhar et al. (Eur. J. Immunol. 42: 2913-2924 (2012)) describe pre-existing heterosubtypic CD8 T cells that recognise pH1 N1 virus. Sridhar et al. discuss the predominance of the IFN-gamma-only-secreting functional T cell subset and possible reasons for it (e.g. repeated exposure to influenza virus antigens over many influenza seasons). Sridhar et al. do not suggest that this specific IFN-gamma-only-secreting functional subset mediates protection. Sheible et al. (Vaccine 29: 2159-2168 (2011 )) identify significantly higher influenza- specific single-cytokine secreting IFN-y-only and TNF-a-only cells than multiple- cytokine (more than one of IL-2, IFN-γ, and TNF-a) secreting cells in healthy adults. Sheible et al. teach that T cells secreting more than one of the three cytokines tested (IL-2, IFN-γ, and TNF-a) have been associated with better prognosis in a number of chronic virus infections when compared to single cytokine-positive cells but do not suggest a protective effect of single-cytokine producing cells.

There is no established cellular correlate of protection against influenza. Research groups have previously measured different aspects of the T-cell response and different phenotypes of T-cells. The most commonly measured T-cell type has been only defined by CD8+IFNg+.

The inventors have now combined simultaneous measurement of both cellular phenotype and function to give highly detailed data on the influenza-specific T-cell response and shown for the first time, that when influenza-specific T-cell phenotype and function are combined with the use of a particular set of T-cell antigens, it correlates with protection against developing symptoms and the severity of illness episode following influenza infection.

The inventors provide a method based on the first cellular correlate of protection against symptomatic influenza after natural infection in humans. The inventors have now found that CD8+ T-cells secreting IFNg+ but not IL-2 are associated with protection against developing severe symptoms following influenza infection.

The use of this novel T-cell correlate of protection can help in developing and evaluating new candidate T-cell inducing vaccines to influenza. This new cell phenotype can be measured to evaluate whether the candidate vaccine is likely to be efficacious. Secondly, vaccines can be designed to specifically induce this T-cell population. Furthermore, a kit used to measure this correlate will allow risk stratification of individuals most likely to develop severe influenza disease. This will allow targeting of current vaccines to these individuals to protect them against infection.

For the first time a set of peptides that are conserved across different viral strains of influenza were used to specifically measure CD8+ T-cells and in combination with simultaneous measurement of IFNg and IL-2 secreting cells.

The methods described herein identify and quantify IFNg+IL-2-CD8+ cells as well as correlate them to live influenza virus or a set of peptides. This correlates with the risk of developing symptomatic influenza. The higher the proportion of these cells, the less likely the risk of developing severe symptoms following influenza infection. This subset of T-cells will also allow design of vaccines to induce this specific immune response and determination of whether a vaccine can generate these protective cells which will indicate increased likelihood of protection against symptomatic disease.

In a first aspect of the invention there is provided a method of determining prognosis of an influenza infection in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

(iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-γ without secreting IL-2; and optionally

(iv) calculating the cells identified in (iii) as a percentage of those provided in (i);

wherein the identification of cells in step (iii) and/or the percentage of cells calculated in (iv) correlates to the risk of the individual developing symptomatic disease following an influenza infection.

The inventors have also found that a particular subset of CD8+ T-cells secreting IFN- Y but not IL-2 and having a CCR7- CD45RA+ combination of cell surface markers is associated with protection against developing severe symptoms following influenza infection.

Therefore, the methods described herein may also quantify the proportion of IFNy +IL-2-CD8+ cells that express CD45RA but not CCR7 to live influenza virus or a set of peptides. This also correlates with the risk of developing symptomatic influenza. The higher the proportion of these cells, the less likely the risk of developing severe symptoms following influenza infection. This subset of T-cells will also allow design of vaccines to induce this specific immune response and determination of whether a vaccine can generate these protective cells which will indicate increased likelihood of protection against symptomatic disease.

The method may therefore optionally include the following additional steps:

a) identifying those cells of step (iii) which are also CCR7 negative and CD45RA positive; and optionally b) calculating the cells identified in step (a) as a percentage of those identified in step (iii);

wherein the identification of cells in step (a) and/or the percentage of cells calculated in (b) correlates to the risk of the individual developing symptomatic disease following an influenza infection, and wherein steps (iii) and (a) can be carried out either sequentially or simultaneously.

In certain embodiments of the invention, the markers CD45RO, CD26L, Ki-67, KLRG1 and/or CD107 are also measured as part of the methods of the invention.

In certain embodiments of the invention, the T-cells are identified in step (iii) of the methods of the invention by identifying those cells that are CD3 positive.

"Symptomatic disease" can be defined clinically by the presence of one or more symptoms.

In certain embodiments the symptomatic disease is associated with one or more of fever; cough; nasal congestion; runny nose; body aches; fatigue; headache; muscle pain (myalgia); watering eyes; reddened eyes, skin, mouth, throat and/or nose; rash; abdominal pain; and diarrhoea.

In preferred embodiments the symptomatic disease is associated with one or more of fever, cough, headache, myalgia or sore throat, diarrhoea and runny nose. In certain embodiments the symptomatic disease is associated with viral shedding.

In certain embodiments of the invention, the symptomatic disease is severe symptomatic disease. Non-severe symptomatic disease may be determined through any one or more of fever (temperature >38C), fever with cough and sore throat, fever with >1 of cough, sore throat, runny nose, myalgia, arthralgia; whereas severe symptomatic disease may be determined through one or more of pneumonia, necessity for mechanical ventilation, death as well as the use of a symptom scoring system.

Symptom scoring systems are well known in the art and are described further in the examples section. The correlation with quantitative symptom score as shown in the examples indicates at least as potent a protective effect of the T-cells identified in step (iii) or (a) of the method in limiting severe symptom illness and its associated mortality.

In certain embodiments of the invention the individual is at increased risk of developing symptomatic disease following an influenza infection if no cells are identified at step (iii) or (a). In other words, if no cells are identified at step (iii) or (a), the individual is at increased risk of developing symptomatic disease following an influenza infection.

For the avoidance of doubt, where "step (iii) or (a)" is referred to throughout the specification, it is intended that step (iii) is being referred to unless the additional step

(a) is being performed, in which case step (a) is instead being referred to. For example, with reference to the above paragraph, if step (a) is not being performed, no cells identified at step (iii) means that the individual is at increased risk of developing symptomatic disease following an influenza infection, whereas, if step (a) is being performed, no cells identified at step (a) instead means that the individual is at increased risk of developing symptomatic disease following an influenza infection.

In certain embodiments the risk of the individual developing symptomatic disease following an influenza infection is inversely proportional (negatively correlated) to the percentage value calculated in step (iv) or (b) of the method. In other words, a higher percentage value calculated in step (iv) or (b) indicates a reduced risk of the individual developing symptomatic disease following an influenza infection.

For the avoidance of doubt, where "step (iv) or (b)" is referred to throughout the specification, it is intended that step (iv) is being referred to unless the additional step

(b) is being performed, in which case step (b) is instead being referred to. For example, with reference to the above paragraph, if step (b) is not being performed, a higher percentage value calculated in step (iv) indicates a reduced risk of the individual developing symptomatic disease following an influenza infection, whereas, if step (b) is being performed, a higher percentage value calculated in step (b) instead indicates a reduced risk of the individual developing symptomatic disease following an influenza infection. The percentage value calculated in step (iv) or (b) which will indicate if there is an increased risk of the individual developing symptomatic disease following influenza infection may differ based on biological variability due to ethnicity, age, other coexisting diseases, infecting strain of influenza virus, country or seasonality of influenza circulation.

Exemplary percentage values are provided as follows:

In certain embodiments the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (iv) is less than or equal to 50 cells per 1 million (or 0.005%) to 200 cells per 1 million (or 0.020%). Preferably wherein the percentage calculated in step (iv) is less than or equal to 0.006% to 0.018%, 0.007% to 0.016%, 0.008% to 0.014%, 0.009% to 0.012%.

In preferred embodiments the percentage calculated in step (iv) is less than or equal to 0.02%, 0.019%, 0.018%, 0.017%, 0.016%, 0.015%, 0.014%, 0.013%, 0.012%, 0.011 %, 0.010%, 0.009%, 0.008%, 0.007%, 0.006%, or 0.005%. Most preferred is less than or equal to 100 cells per 1 million (or 0.010%)

In certain embodiments the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (b) is less than or equal to 30% to 75%. In certain embodiments the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (b) is less than or equal to 31% to 70%, 32% to 65%, 33% to 60%, 34% to 55%, 35% to 50%, 36% to 45%, 37% to 43%, 38% to 42%, or 39% to 41%. In certain embodiments the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (b) is less than or equal to 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61 %, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%. 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30%. Preferably the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (b) is less than or equal to 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, or 35%. Most preferably the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (b) is less than or equal to 40%.

By "influenza" we refer to influenza (or "flu") as caused by any of the influenza viruses A, B or C. Preferably, the influenza is caused by influenza virus A. Examples of particular serotypes of influenza virus A to which the methods of the invention may relate are H1 N1 , H2N2, H3N2, H5N1 , H7N7, H1 N2, H9N2, H7N2, H7N3, H10N7 and/or H7N9. Preferably, the influenza is caused by influenza virus A, serotype H1 N1. Even more preferably, the serotype H1 N1 is pandemic H1 N1 (pH1 N1).

By "individual" we refer to any organism capable of being infected with influenza. Preferably the individual is a mammal or bird. More preferably the individual is a mammal such as a mouse, rabbit, goat, sheep, dog or human. Most preferably the individual is human.

By "influenza antigens" we refer to any influenza viral protein capable of eliciting an immune response. Examples of influenza antigens include PB1 , NP, M1 , NS1 , NS2, PB1 F2, HA, NA, M2 and PB2 proteins. A selection of particular influenza antigens that are CD8 epitopes are listed in Table 5.

T-cells identified in step (iii) of the methods of the invention may be identified as live T-cells by any method known in the art, such as the use of either a dead cell marker or a live cell marker. Such markers are well known in the art (e.g. LIVE/DEAD® fixable dead cell stain kits, PI, Trypan blue, Annexin V, Zombie Yellow, BrDU,7-AAD).

In preferred embodiments of the invention, steps (iii) and (a) of the methods of the first aspect of the invention are performed by multi-parameter flow cytometry and/or Fluorescence-immunospot.

The particular subsets of T-cells as specified in steps (iii) and (a) can be identified by any suitable technique known in the art. However, a preferred technique is that of multi-parameter flow cytometry. This technique is well known in the art as a method which can be used to determine the amount of cells of a certain type in a sample by criteria such as cell phenotype and/or cell function and using a cocktail of visually tagged antibodies specific for multiple markers of cell phenotype or function. The gating strategy as shown in Figure 11 may be followed in order to identify the subsets of T-cells.

A preferred method of multi-parameter flow cytometry will involve reduction of the number of fluorophore detectors required. It is estimated that the method could be simplified to a 5 or 6 colour flow cytometry assay to only identify the following distinct markers - CD8, IFNg, IL-2, CD45RA, CCR7 (and optionally CD3). This is a realistic target for a diagnostic assay in a clinical setting given the flow cytometry assays currently performed in patients with blood dyscrasias e.g. lymphoma.

In a second aspect of the invention there is provided a method of determining influenza immunity status in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

wherein the identification of cells in step (iii) and/or the percentage of cells calculated in (iv) correlates to influenza immunity status of the individual.

The method may optionally include the following additional steps:

a) identifying those cells of step (iii) which are also CCR7 negative and CD45RA positive; and optionally

b) calculating the cells identified in step (a) as a percentage of those identified in step (iii);

wherein the identification of cells in step (a) and/or the percentage of cells calculated in (b) correlates to influenza immunity status of the individual, and wherein steps (iii) and (a) can be carried out either sequentially or simultaneously.

In a third aspect of the invention there is provided the use of the method of the second aspect in a method of identifying one or more compounds that induce influenza immunity in an individual. By "compound" and "test compound" we refer herein to any compound that might be tested to determine if it is able to induce influenza immunity in an individual. Preferably the compound is a peptide.

In one embodiment the compound is a candidate vaccine.

In certain embodiments the suitability of a test compound for inducing influenza immunity is indicated by the identification of one or more cells at step (iii) or (a). In other words, if cells are identified at step (iii) or (a), the test compound is suitable for inducing influenza immunity.

In certain embodiments the suitability of a test compound for inducing influenza immunity is proportional (positively correlated) to the percentage value calculated in step (iv) or (b) of the method.

In other words, a higher percentage value calculated in step (iv) or (b) indicates an increased suitability of a test compounds for inducing influenza immunity. The percentage value calculated in step (iv) or (b) which will indicate if the test compound is suitable for inducing influenza immunity may differ based on biological variability due to ethnicity, age, other co-existing diseases, infecting strain of influenza virus, country or seasonality of influenza circulation. Exemplary percentage values are provided as follows:

In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (iv) is at least 50 cells per 1 million (or 0.005%) to 200 cells per 1 million (or 0.020%). Preferably a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (iv) is at least 0.006% to 0.018%, 0.007% to 0.016%, 0.008% to 0.014%, 0.009% to 0.012%.

In preferred embodiments the percentage calculated in step (iv) is at least 0.02%, 0.019%, 0.018%, 0.017%, 0.016%, 0.015%, 0.014%, 0.013%, 0.012%, 0.011 %, 0.010%, 0.009%, 0.008%, 0.007%, 0.006%, or 0.005%. Most preferred is at least 100 cells per 1 million (or 0.010%). In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 30% to 75%. In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 31 % to 70%, 32% to 65%, 33% to 60%, 34% to 55%, 35% to 50%, 36% to 45%, 37% to 43%, 38% to 42%, or 39% to 41 %. In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 75%, 74%, 73%, 72%, 71 %, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%. 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, or 30%.

Preferably a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35%.

Most preferably a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 40%.

In a fourth aspect of the invention there is provided a method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(i) providing a sample comprising T-cells wherein the sample derives from an individual previously administered with one or more test compounds;

(ii) exposing the sample of (i) to one or more influenza antigens;

(iv) calculating the cells identified in (iii) as a percentage of those provided in

0);

wherein the identification of cells in step (iii) and/or the percentage of cells calculated in (iv) correlates to the ability of the one or more test compounds to induce influenza immunity in an individual.

The method may optionally include the following additional steps: a) identifying those cells of step (iii) which are also CCR7 negative and CD45RA positive; and optionally

wherein the identification of cells in step (a) and/or the percentage of cells calculated in (b) correlates to the ability of the one or more test compounds to induce influenza immunity in an individual, and wherein steps (iii) and (a) can be carried out either sequentially or simultaneously.

In certain embodiments a period of time is allowed to elapse between the previous administration of the one or more test compounds and the sample provision in step (i). In other words, step (i) does not occur immediately after the individual is administered with one or more test compounds. Preferably the period of time allowed to elapse between the previous administration and sample provision is selected from 3 days to 6 weeks.

For example, the period of time allowed to elapse between the previous administration and sample provision is 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

In a fifth aspect of the invention there is provided a method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more test compounds;

The method may optionally include the following additional steps:

In certain embodiments a period of time is allowed to elapse between steps (ii) and (iii). In other words, step (iii) does not occur immediately after step (ii). Preferably the period of time allowed to elapse between step (ii) and step (iii) is selected from 6 hours to 1 week.

For example, the period of time allowed to elapse between step (ii) and step (iii) is 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

In certain embodiments the suitability of a test compound for inducing influenza immunity is indicated by the identification of one or more cells at step (iii) or (a) of the method the fourth or fifth aspects. In other words, if cells are identified at step (iii) or (a), the test compound is suitable for inducing influenza immunity.

In certain embodiments the suitability of a test compound for inducing influenza immunity is proportional (positively correlated) to the percentage value calculated in step (iv) or (b) of the methods of the fourth or fifth aspects.

In other words, a higher percentage value calculated in step (iv) or (b) indicates an increased suitability of a test compounds for inducing influenza immunity.

The percentage value calculated in step (iv) or (b) which will indicate if the test compound is suitable for inducing influenza immunity may differ based on biological variability due to ethnicity, age, other co-existing diseases, infecting strain of influenza virus, country or seasonality of influenza circulation.

Exemplary percentage values are provided as follows:

In preferred embodiments the percentage calculated in step (iv) is at least 0.02%, 0.019%, 0.018%, 0.017%, 0.016%, 0.015%, 0.014%, 0.013%, 0.012%, 0.011%, 0.010%, 0.009%, 0.008%, 0.007%, 0.006%, or 0.005%. Most preferred is at least 100 cells per 1 million (or 0.010%).

In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 30% to 75%.

In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 31% to 70%, 32% to 65%, 33% to 60%, 34% to 55%, 35% to 50%, 36% to 45%, 37% to 43%, 38% to 42%, or 39% to 41 %.

In certain embodiments a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 75%, 74%, 73%, 72%, 71 %, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51 %. 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41 %, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31 %, or 30%.

Preferably a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35%. Most preferably a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (b) is at least 40%.

In certain embodiments the method comprises an additional step (v) of determining if the one or more test compounds increase the percentage calculated at step (iv) or (b) in comparison to a control value. In other words, when the sample is exposed to the one or more test compounds the percentage calculated at step (iv) or (b) is higher than a control value. In certain embodiments wherein the sample derives from an individual previously administered with one or more test compounds, the control value is obtained by performing the method but using a sample of cells at step (i) obtained from the individual prior to their being administered with one or more test compounds.

In certain embodiments wherein the sample is exposed to one or more test compounds in step (ii), the control value is obtained by performing the method without exposing the sample to one or more test compounds at step (ii) and/or exposing the sample to one or more influenza antigens instead of the one or more test compounds at step (ii).

In certain embodiments the one or more influenza antigens specified in step (ii) comprise at least one influenza antigen that is conserved across multiple (or all known) influenza A strains.

In certain embodiments all of the one or more influenza antigens are conserved across multiple (or all known) influenza A strains. In certain embodiments the one or more influenza antigens comprise one or more of B1 , NP, M1 , NS1 , NS2, PB1 F2, HA, NA, M2 and PB2 proteins

In a preferred embodiment the one or more influenza antigens comprise one or more of the peptides described in Table 5. In one embodiment the one or more influenza antigens comprise all of the peptides described in Table 5.

In certain embodiments of the invention the sample provided in step (i) of the methods of the invention is a blood sample, preferably a peripheral blood mononuclear cell (PBMC) sample.

In certain embodiments of the methods of the invention an additional step is first performed in order to determine that the sample of cells is either infected with influenza or is obtained from a subject infected with influenza. Tests for determining that the sample of cells is either infected with influenza or is obtained from a subject infected with influenza are well known in the art (e.g. Rapid Influenza Diagnostic Tests, RT-PCR (Centers for Disease Control and Prevention. Lab Diagnosis of Influenza - http://www.cdc.gov/flu/professionals/diagnosis)). In a sixth aspect of the invention there is provided a vaccine composition comprising one or more compounds identified by any of the methods of the third, fourth or fifth aspects of the invention.

In certain embodiments the vaccine composition additionally comprises a pharmaceutically acceptable excipient, diluent or carrier. Examples of such compositions and methods for their administration are described in Example 5. In a seventh aspect of the invention there is provided a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen- binding fragments thereof that are individually specific for each of CD8, CD45RA and CCR7.

In other words, the composition as a whole is able to bind to each of CD8, CD45RA and CCR7 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, CD45RA and CCR7. In certain embodiments the plurality of antibodies or antigen-binding fragments thereof additionally binds to each of IFN-γ and IL-2 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for each of IFN-Y and IL-2. In other words, the composition as a whole is able to bind to each of CD8, CD45RA, CCR7, IFN-Y and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, CD45RA, CCR7, IFN-γ and IL-2.

In an eighth aspect of the invention there is provided a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-Y and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2.

In other words, the composition as a whole is able to bind to each of CD8, IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, IFN-γ and IL-2. In a ninth aspect of the invention there is provided a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen- binding fragments thereof that are individually specific for each of CD45RA and CCR7.

In other words, the composition as a whole is able to bind to each of CD45RA and CCR7 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD45RA and CCR7.

In a tenth aspect of the invention there is provided a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of IFN- Y and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of IFN-γ and IL-2.

In other words, the composition as a whole is able to bind to each of IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of IFN-γ and IL-2. In certain embodiments, the composition of the seventh, eighth, ninth or tenth aspects is as a whole additionally able to bind to one or more of CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107 and the plurality additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for the one or more of CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107.

In a preferred embodiment, the composition of the seventh, eighth, ninth or tenth aspects is as a whole additionally able to bind to CD3 and the plurality additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3.

In an eleventh aspect of the invention there is provided a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2. In other words, the composition as a whole is able to bind to each of CD8, IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, IFN-γ and IL-2. In certain embodiments, the composition of the eleventh aspect is as a whole additionally able to bind to one or more of CD45RA, CCR7, CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107 and the plurality additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for the one or more of CD45RA, CCR7, CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107.

In a preferred embodiment, the composition of the eleventh aspect is as a whole additionally able to bind to CD3 and the plurality additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3. The antigen-binding fragment may be selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)₂ fragments), single variable domains (e.g. VH and V_ domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).

Also included within the scope of the invention are modified versions of antibodies and an antigen-binding fragments thereof, e.g. modified by the covalent attachment of polyethylene glycol or other suitable polymer. Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833- 3837; Winter et al., 1991 , Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler et al., 1975. Nature 256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81 :31 -42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et ai, 1984. Mol. Cell. Biol. 62:109-120).

Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982).

Antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manuaf, Cold Spring Harbor Laboratory, New York). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

In certain embodiments the plurality of antibodies or antigen-binding fragments thereof is fixed to a solid support.

In certain embodiments the antibodies or antigen-binding fragments thereof with a particular specificity are separately detectable to those with a different specificity.

In a preferred embodiment the antibodies or antigen-binding fragments thereof are visually detectable.

The antibodies or antigen-binding fragments thereof may be labelled in order to allow their detection. For example, they may be labelled with a fluorescent label (e.g. a fluorophore) or with a radio label.

Multiple examples of suitable labels such as fluorophores are well known in the art.

Fluorophores of interest include, but are not limited to fluorescein dyes (e.g. fluorescein dT, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4', 1 ,4,-tetrachlorofluorescein (TET), 2',4',5',7',1 ,4-hexachlorofluorescein (HEX), and 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE)), cyanine dyes such as Cy5, dansyl derivatives, rhodamine dyes (e.g. tetramethyl-6-carboxyrhodamine (TAMRA), ATTO dyes (such as ATTO 647N) and tetrapropano-6-carboxyrhodamine (ROX)), DABSYL, DABCYL, cyanine, such as Cy3, anthraquinone, nitrothiazole, and nitroimidazole compounds, or other non-intercalating dyes. Particularly preferred fluorophores include, but are not limited to PE-CF594, AF700, QDot605, Qdot655, PE-Cy7, PerCP-Cy5.5, APC-Cy7, BV570, V450, PE, FITC, APC, Biotin, PE-Cy5. As used herein, "fluorophore" (also referred to as fluorochrome) refers to a molecule that, when excited with light having a selected wavelength, emits light of a different wavelength.

In a twelfth aspect of the invention the composition of the seventh, eighth, ninth, tenth or eleventh aspects are for use in diagnosing individuals at risk of developing symptomatic disease following influenza infection.

In a thirteenth aspect of the invention there is provided a kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, CD45RA and CCR7;

(ii) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of IFN-γ and IL-2; and

(iii) instructions for use. In other words, the composition (i) of the kit is a composition which as a whole is able to bind to each of CD8, CD45RA and CCR7 and comprises antibodies or antigen- binding fragments thereof that are individually able to bind only one of CD8, CD45RA and CCR7; and the composition (ii) of the kit is a composition which as a whole is able to bind to each of IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of IFN-γ and IL-2.

In a fourteenth aspect of the invention there is provided a kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2; (ii) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD45RA and CCR7; and

(iii) instructions for use.

In other words, the composition (i) of the kit is a composition which as a whole is able to bind to each of CD8, IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, IFN-γ and IL-2; and the composition (ii) of the kit is a composition which as a whole is able to bind to each of CD45RA and CCR7 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD45RA and CCR7.

In a fifteenth aspect of the invention there is provided a kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, CD45RA, CCR7, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, CD45RA, CCR7, IFN-γ and IL-2; and

(ii) instructions for use.

In other words, the composition (i) of the kit is a composition which as a whole is able to bind to each of CD8, CD45RA, CCR7, IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one CD8, CD45RA, CCR7, IFN-γ and IL-2.

In a sixteenth aspect of the invention there is provided a kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2; and

(ii) instructions for use. In other words, the composition (i) of the kit is a composition which as a whole is able to bind to each of CD8, IFN-γ and IL-2 and comprises antibodies or antigen-binding fragments thereof that are individually able to bind only one of CD8, IFN-γ and IL-2. In certain embodiments of the kit of the thirteenth, fourteenth, fifteenth or sixteenth aspects, the composition of (i) is as a whole additionally able to bind to one or more of CD45RA, CCR7, CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for the one or more of CD45RA, CCR7, CD3, CD45RO, CD26L, Ki-67, KLRG1 and/or CD107.

In a preferred embodiment of the kit of the thirteenth, fourteenth, fifteenth or sixteenth aspects, the composition of (i) and/or (ii) is as a whole additionally able to bind to CD3 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3.

In a preferred embodiment of the kit of the thirteenth, fourteenth, fifteenth or sixteenth aspects, the composition of (i) and/or (ii) is as a whole additionally able to bind to dead cells and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for dead cells.

In certain embodiments the kit of the thirteenth, fourteenth, fifteenth or sixteenth aspects additionally comprises one or more influenza antigens (as defined previously), a live and/or dead cell discriminator, a positive control and/or a negative control.

Example dead cell discriminators include LIVE/DEAD® fixable dead cell stain kits, PI, Trypan blue, Annexin V, Zombie Yellow, BrDU,7-AAD. An example of a negative control is media alone. Example positive controls include one or more of phytohaemagglutinin, ionomycin and phorbol myrsitate acetate.

In a tenth aspect of the invention there is provided the use of a kit of the ninth aspect of the invention in the method of any of the first, second, third, fourth or fifth aspects of the invention. Definitions and abbreviations

By "CD3" we refer to cluster of differentiation 3. By "CD8" we refer to cluster of differentiation 8. By "CCR7" we refer to C-C chemokine receptor 7 By "CD45RA" we refer to cluster of differentiation 45 RA isoform.

By "IFN-γ" we refer to interferon-gamma or "IFNg".

By "IL-2" we refer to interleukin-2. By "+" we mean positive. In other words, the particular immunological molecule/marker the + is associated with is present.

By "-" we mean negative. In other words, the particular immunological molecule/marker the - is associated with is absent.

By "antibody" we include substantially intact antibody molecules, as well as chimaeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen binding fragments and derivatives of the same.

By "antigen-binding fragment" we mean a functional fragment of an antibody that is capable of binding to a particular antigen.

Examples embodying aspects of the invention will now be described with reference to the following figures in which:

Figure 1 shows the study outline and timeline in context of 2009-10 and 2010- 11 pandemic in UK.

Study outline (upper panel) in the context of timeline of the evolving pH1 N1 pandemic (lower panel). Lower bar chart shows UK influenza virological surveillance data from WHO Flunet (http://www.who.int/influenza/qisrs laboratory/flunet/en/), highlighting the study recruitment and follow-up time-points for seasons 1 and 2 in relation to influenza activity in the UK during 2009 - 201 1. Bars indicate influenza A detected by virological national surveillance. Week number refers to the week of the year. Healthy adults were recruited just after the 1st wave of the pandemic had passed in the UK and followed over two influenza seasons with PBMCs and serum samples collected prior to and at the end of each influenza winter season. During each influenza season, symptom questionnaires were emailed to participants every three weeks with automated weekly reminders. Nasal swabs were collected by participants if symptomatic and returned to the laboratory. Infection was defined by detection of pH1 N1 virus in returned nasal swab or a four-fold rise in pH1 N1 haemagglutination inhibition titre in paired serum samples. Arrows between boxes denote longitudinal progression of individuals during the study.

Figure 2 shows the study flow chart of sample selection for analysis of heterosubtypic T cell correlate of protection against clinical outcomes of infection.

A total of 342 participants were enrolled in the study. During the study period, 51 individuals were infected (identified by sero-conversion or nasal self-swabbed pH1 N1 RT-PCR+) of whom 43 individuals were included in the final analysis. Individuals were excluded from the analysis because of: non-availability of paired serum samples for diagnosis of infection (n = 94), being sero-positive for pH1 N1 at baseline indicating previous encounter with pH1N1 virus and thereby preventing analysis of heterosubtypic T cell responses (n = 68), remaining uninfected through the study (n = 128), not returning any symptom survey (n = 6) and non-availability of viable stored PBMCs (n = 3). Of these 43 individuals, definitive clinical outcomes were ascertained in 25 individuals. As per the analysis plan, the 25 infected individuals were grouped based on an increasing severity of symptomatic outcome during influenza-illness episode. Shaded boxes represent the different groups compared in the analysis. Light grey represent individuals with asymptomatic or symptomatic influenza infection, mid grey represents those with fever versus those without fever during illness episode and dark grey individuals with fever and cough or sore-throat (a CDC- defined symptomatic illness). Those individuals who shed or did not shed virus were also compared (dark grey right hand side boxes). Increasing symptomatic severity of influenza illness episode of individuals with defined clinical outcome at a known timepoint during the influenza season⁰ n=25

Illness with any symptoms (Individuals with asymptomatic infection (no illness episode reported) n=3. Individuals with symptomatic infection (reported symptomatic illness episode) n=22).

Illness with Fever (Individuals without fever during illness episode (documented temperature >38°C or history of severe feverishness) n= 12; Individuals with fever during illness episode (documented temperature >38°C or history of severe feverishness) n= 12).

Illness with fever and cough/sore throat (Individuals with Fever or cough/sore throat not both, during illness episode (CDC-defined ILI symptoms) n=15; Individuals with Fever and cough/sore throat during illness episode (CDC-defined ILI symptoms) n=10)

Comparison of shedding

Viral shedding Nasal swab returned (n=15) (Individuals not shedding during illness episode (pH1 N1 virus negative by RT-PCR in nasal swab) n=4. Individuals shedding during illness episode (pH1 1 virus negative by RT-PCR in nasal swab) n=1 1 )

Figure 3 shows frequencies of pre-existing cross-reactive T-cells are inversely associated with illness severity in infected individuals

Responses to live pH1 N 1 virus stimulation (A,D,E), summed responses to conserved CD8 epitopes from PB1 , M1 and NP proteins (B,F,G) and CMV lysate (control antigen) (C,H, I) of total cytokine-secreting cells and IFN-Y+I L-2- IFN-Y-I L-2+, IFN- Y+IL-2+ dual cytokine-secreting cells quantified by fluorescence-immunospot. Total cytokine-secreting T cells represent the summated frequencies of the three functional subsets, I FN-Y+IL-2-, IFN-γ— IL-2+ and IFN-y+IL-2+. Cellular immune response to live pH1 N1 virus, CD8 conserved influenza epitopes and CMV lysate in individuals (n = 25) developing an illness with fever versus no fever and fever with cough or sore throat versus those without fever and cough or sore throat, p values estimated by Mann-Whitney non-parametric test. In the box plots, the box represents the third centile (75%) and first centile (25%) with the horizontal line in the box representing the median (50%). The whiskers represent 1.5 times the interquartile range with outliers shown. The scatter plots represent the frequency of cellular response for each individual.

Figure 4 shows inverse correlation of cross-reactive T-cells and symptom score.

Correlation between frequency of total T (A,B,C) and IFN-v+IL-2- (D,E,F) cellular responses to live virus (A,D), summed response to conserved CD8 epitopes from PB1 , M1 and NP proteins (B,E) and CMV lysate (control antigen) (C,F) quantified by fluorescence-immunospot and symptoms score. Symptom score was defined by totalling scores for each of the following symptoms: fever, sore throat, cough, headache, myalgia, r indicates the Spearman rank correlation coefficient.

Figure 5 shows inverse correlation of pre-existing cross-reactive late effector CD8+IFN-Y+IL-2- cells and symptom score.

Phenotypic characterisation using multi-parameter flow cytometry of the different memory subsets of influenza virus-specific CD8+ IFN-v+IL-2- cells based on CCR7 and CD45RA surface expression following overnight stimulation of PBMCs with live pH1 N1 virus in pH1 N1-infected individuals (n=22; in 3 of 25 infected individuals, samples were of insufficient quantity to undertake flow-cytometry). Proportion of CD8+ IFN-Y+IL-2- secreting cells that were effector memory (CD45RA-CCR7-), late effector (CD45RA+CCR7-), central memory (CD45RA-CCR7+) or naive (CD45RA+CCR7+) phenotype (A). Correlation between the proportion of pre-existing CD3+CD8+ IFN-Y+IL-2- cells of the late effector CD45RA+CCR7- subset and total symptom score (B). r indicates the Spearman rank correlation coefficient. In individuals with influenza-specific late effector CD45RA+CCR7-CD8+ IFN-v+IL-2- cells (n = 17), functional characterisation of these cells for expression of CD107A/B, CCR5 and TNF-a was undertaken with multi-parameter flow cytometry. Symbols represent proportion of CD45RA+CCR7-CD8+ IFN-v+IL-2- cells expressing CD107A/B, CCR5 and TNF-a for each individual with the line depicting the median response (C).

Figure 6 shows pre-existing influenza-specific T cell responses at baseline.

Baseline cytokine secreting T cells to live virus (A) and summed total of CD8 epitopes from M1 , PB1 and NP (B) for each individual measured by fluorescence- immunospot. Frequencies of antigen-specific cells were calculated by subtracting the average number of Spot Forming Cells (SFCs) in negative no peptide control wells from SFCs in antigen-containing test wells. Pie charts represent the average of the relative proportions of the total cytokine-secreting cellular response contributed by the three distinct cytokine-secreting subsets. The frequency of pH1 N1 live virus- specific T cells (median 152 SFCs/million PBMCs; IQR: 28-236; range 4-1308) was higher (p<0.001) than frequencies of T cells to conserved CD8 epitopes from PB1 , NP or M1 proteins (median 56 SFCs/million; IQR: 20-104; range 0-268). The majority of the live virus-specific response on flow-cytometry was from CD3+ T cells (data not shown).

(in order shown in figure 6 (left to right) F421 ; F197; F291 ; F151 ; F161; F169; F175; F207; F059; F238; F075; F093; F094; F376; F172; F004; F087; F171 ; F019; F041 ; F061 ; F071 ; F018; F214; F196; F179; F413; F021 ; F216; F399; F278; F221 ; F162; F190; F089; F155; F385; F058; F070; F229; F181 ; F156)

Figure 7 shows pre-existing influenza-specific total-cytokine-secreting T-cells is not different between individuals who develop infection versus age and gender-matched controls that remain uninfected.

Comparison of the baseline total-cytokine-secreting T cells to live virus measured by fluorescence-immunospot between individuals who subsequently develop infection (n=43) and age and gender-matched individuals who remain uninfected (n=34). The line represents the median response for each group.

Figure 8 shows risk of developing influenza episode with fever as a function of frequency of pre-existing heterosubtypic influenza-specific cells (odds ratio for total cells 0.14, 95% CI 0.02 - 0.94; odds ratio for IFN^+IL-2- 0.16, 95% CI 0.02 - 0.99).

A logistic model was estimated for the probability of developing a fever during illness episode for each ten-fold increase in the number of SFCs/million PBMCs enumerated by fluorescence-immunospot in response to live virus stimulation for each cytokine- secreting subset. Each 10-fold increase in the frequency of pH1 N1 virus-specific T- cells was associated with a 7-fold decrease in risk of developing influenza illness with fever (odds ratio for total cytokine-secreting T-cells: 0.14, 95% CI: 0.02-0.94 and for IFN-Y+IL-2- T-cells: 0.16, 95% CI - 0.02 - 0.99) . Frequencies of IFN-y+IL-2+ and IFNy-IL-2+ T-cells did not predict risk (odds ratio for IFN-y+IL-2+ cells 0.53,95% CI 0.19 - 1.44; odds ratio for IFNy-IL-2+ cells 0.55, 95% CI 0.23 - 1.33). Despite fewer infected individuals than estimated in our sample size calculation, this substantially greater-than-estimated effect size and a higher prevalence of cross-reactive T-cells enabled us to detect a difference.

Figure 9 shows responses to CD8 conserved epitopes are from CD8+ T-cells PBMCs undepleted or depleted of CD8+ T-cells by labelling with CD8 microbeads and passing through a LD column (MACS Miltenyi) were stimulated overnight with CD8 conserved epitope pools (Supplementary table 3). The purity of the depletion in each case was > 98% by flow cytometry staining. Frequencies of different cytokine secreting T-cell subsets were enumerated using fluorescence-immunospot (n=3) and responses were abrogated when CD8 depletion was undertaken. Bars show mean and SEM.

Figure 10 shows heterosubtypic T-cell responses associated with prevention of viral shedding.

Responses to live pH1 N1 virus stimulation (A) were measured by fluorescence- immunospot allowing enumeration of IFN-v+IL-2-, IFN-y-IL-2+ and IFN-y+/IL-2+ dual cytokine-secreting cells. Box plots represent responses compared between individuals with viral shedding and those not shedding virus, p values estimated by Mann-Whitney non-parametric t test. The difference in total cytokine secreting cells did not reach statistical significance (p=0.09). In the box plots, the box represents the third centile (75%) and first centile (25%) with the horizontal line in the box represents the median (50%). The whiskers represent 1.5 times the interquartile range with outliers shown. The scatter plots represent the frequency of cellular response for each individual.

Figure 11 shows the gating strategy for flow cytometric analysis of CD8+IFN- Y+IL-2- cells.

Responses to live pH1 N1 virus stimulation were measured by multi-parameter flow cytometry. PBMCs stimulated with live pH1 N1 virus for 18 hours were stained for surface markers of memory, lung homing, degranulation and intracellular cytokines. PBMCs were gated on live, single cells, low forward and side scatter for lymphocytes, a negative dump gate for CD56, CD14 and CD19 (not shown), CD3+ (gate not shown), IFN-y+IL-2- and CD8+CD4-. CD8+ cells of the CD45RA+CCR7- subset were analysed for expression of CCR5, CD107 and TNF-a using Flowjo software. The Fluorescence Minus One (FMO) controls are shown for CCR7, CD45RA,CD107 and CCR5 which were used to set the gates to identify positive populations. Figure 12 shows correlation of pre-existing cross-reactive CD4+IFN-y+IL-2- cells and symptom score.

Phenotypic characterisation using multi-parameter flow cytometry of the different memory subsets of influenza virus-specific CD4+IFN-y+IL-2- cells based on CCR7 and CD45RA surface expression following overnight stimulation of PBMCs with live pH1 N1 virus in pH1 N1 -infected individuals (n=22; in 3 of 25 infected individuals, samples were of insufficient quantity to undertake flow-cytometry). (A) Proportion of CD4+IFN-Y+IL-2- secreting cells that were effector memory (CD45RA-CCR7-), late effector (CD45RA+CCR7-), central memory (CD45RA-CCR7+) or naive (CD45RA+CCR7+) phenotype. (B) Correlation between the proportion of preexisting CD3+CD4+IFN-Y+IL-2- cells of the late effector CD45RA+CCR7- subset and total symptom score, r indicates the Spearman rank correlation coefficient. We found no association of symptom score and the proportion of CD4+ central-memory CD45RA-CCR7+ (r=0.0734; p=0.75) or the effector-memory CD45RA-CCR7- (r=- 0.1306; p=0.56) T-cells.

Figure 13 shows CD45RA and CCR7 expression is not changed by in vitro stimulation.

Phenotypic characterisation using multi-parameter flow cytometry of the different memory subsets of influenza virus-specific CD4+ and CD8+ T-cells based on CCR7 and CD45RA surface expression following overnight stimulation of PBMCs with live pH1 N1 virus (open symbols) or unstimulated controls (closed symbols) in 43 individuals. (A) Proportion of CD3+CD4+ T-cells of effector memory (CD45RA-CCR7-), late effector (CD45RA+CCR7-), central memory (CD45RA- CCR7+) or naive (CD45RA+CCR7+) phenotype. (B) Proportion of CD3+CD8+ T- cells of effector memory (CD45RA-CCR7-), late effector (CD45RA+CCR7-), central memory (CD45RA-CCR7+) or naive (CD45RA+CCR7+) phenotype. Symbols represent responses for each individual with the horizontal line representing the median response. Statistical analysis by one-way ANOVA with Dunn's post-test comparisons showed no statistically significant differences in proportions of the different memory subsets between unstimulated and live virus stimulated donor responses. Methods used in the Examples Study design and cohort

Healthy adult (>18 years) staff and students of Imperial College London were invited to participate. Individuals already vaccinated for influenza or likely to be offered pandemic vaccination (as per the UK government guidelines in September 2009) were ineligible. Written informed consent was obtained for all participants following study approval by the North West London Research Ethics Committee (study reference number 09/H0724/27). Participants were recruited between 13^th September and 6^th November 2009 and followed through the 2009-10 and 2010-1 1 influenza seasons with blood collected at the start and end of each season (Figure 1). Development of any symptomatic illness during each influenza season was recorded by participants on a web-based symptom questionnaire every three weeks. The average response rate for the surveys was 75%. In addition, participants were provided with nasal swab packs, self-swabbing instructions and requested to record temperature, self-sample and return nasal swabs when experiencing any influenza-like symptoms (Ip, Schutten et al. J Infect Dis 205, 631-634 (2012))

Laboratory assays PBMCs were isolated by Ficoll-Paque PLUS (Amersham Biosciences) density centrifugation and cryopreserved in heat-inactivated foetal calf serum supplemented with 10% DMSO (Sigma-Aldrich) at -180°C in liquid nitrogen as previously described (Casey R, Blumenkrantz D, Millington K, et al. PLoS One 2010;5:e15619). All assays were undertaken using cryopreserved PBMCs with >80% viability of cells after thawing. Serum was stored at -20°C.

Haemagglutination Inhibition assay

Antibody responses to the virus strain A/England/195/2009, circulating in the UK during our study (pH1 N1) were measured by the haemagglutination inhibition (HI) assay used for UK national surveillance, (Centre for Infections, Health Protection Agency (London, UK) - Miller, E., et al. Lancet 375, 1100-1 108 (2010)), with seroconversion defined as a fourfold rise in HI titre on paired serum samples taken before and after each influenza season. Briefly, human sera were treated with receptor-destroying enzyme (RDE) II, (Denka Seiken, Japan) for 18 hours followed by heat inactivation for 1 hour at 56°C. Sera were screened in a limiting dilution range using the NIBRG122 virus and incubated with the haemagglutinin (HA) antigen suspension for 1 hour followed by addition of 0.5% RBC suspension (turkey blood). The reaction is left for 1 hour at room temperature before reading. Each sample is titrated in duplicate and individual titres reported did not differ by more than a twofold serial dilution. The serum titre is equal to the highest reciprocal dilution, which induces a complete inhibition of haemagglutination. Suitable control serum samples were included in all analyses, with post-infection ferret serum samples raised against the pH1 N1 virus strain as positive controls; human pooled serum samples from individuals with either high antibody titres to currently circulating influenza H1 , H3, and B strains or from individuals with no antibody titres to these seasonal strains were used as negative controls. Microneutralisation assay

As microneutralisation (MN) assays are more sensitive than HI assays, we additionally undertook MN assays on baseline serum samples to confirm seronegativitiy to pH1 N1. Human sera were heat inactivated for 30 min at 56°C and twofold serial dilutions were produced using virus diluent (DMEM + 2 ig ml^"1 TPCK Trypsin) in a total volume of 100μΙ in immunoassay plates. The diluted sera were mixed with an equal volume of virus diluent containing 100xlD50 influenza H1 N1 A/California/7/09 virus and incubated for 2 h at 37°C in a 5% C0₂ humidified atmosphere, after which 100 μΙ of MDCK cells at 1.5 * 10⁵ cells ml^"1 were added to each well. The plates were incubated for a further 18-20h at 37°C and 5% C0₂. Cell monolayers were washed with PBS and fixed in cold 80% (v/v) methanol for 10 min.

The presence of viral protein was detected by ELISA with a monoclonal primary antibody against influenza A NP (MCA400 Mouse anti influenza a nucleoprotein, AbD Serotec) and a secondary HRP conjugate (Goat polyclonal secondary to Mouse IgG HRP, Abeam ab97023). After staining for 10 minute using o-phenylenediamine dihydrochloride, absorbance was read at 490nm in a plate reader. The mean of two replicates were read and samples were scored as positive for infection if the OD490 was equal to or above a value 50% between the mean OD490 from four virus negative control wells and the mean OD490 from four no sera, virus positive control wells.

The reciprocal serum dilution corresponding to the highest dilution to be scored negative for infection is the 50% neutralising antibody titre. Virus and peptides

Presence of virus in nasal swabs was confirmed by a multiplex real-time RT-PCR assay that is more sensitive (and can detect virus for longer period after symptom- onset) than viral culture (Weinberg, G.A., et al. J Infect Dis 189, 706-710 (2004)), using standard methods by the Health Protection Agency, England (Miller, E., et al. Lancet 375, 1 100-1 108 (2010)) and Scotland (Gunson, R.N. & Carman, W.F. BMC Infect Dis 11 , 192 (2012)).

The influenza A(H1 )v specific assay of the Health Protection Agency (HPA) contains primers and a dual-labelled TaqMan MGB probe (Applied Biosystems) targeting conserved sequences in the HA gene of A(H1 1 )v viruses, and the positive control swine A(H1 N1) virus A/Aragon/3218/2009, in a 1-step TaqMan PCR assay has been previously described in detail (Miller et al. Lancet 2010;375:1 100-8).

Live pH1 1 virus was obtained by growing a recombinant A/England/195/09 strain in MDCK cells. Peptide pools 9-mer peptides representing highly conserved reported class I restricted epitopes in influenza A virus from PB1 , M1 and NP proteins were obtained from the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Peptide Arrays, Peptides for Expected Conserved MHC Class I Epitopes of Influenza Virus A Proteins, NR-2667 (Table 3). For each protein, PB1 , M1 and NP, a separate pool of peptides was made at a concentration of 5Mg ml for each peptide in the pool. Cell culture lysates from cells infected with CMV were also used (East Coast Biologicals Inc., USA).

Case definitions

Influenza (pH1 N1)-infected individuals were defined as pH1 N1-unvaccinated individuals with antibody seroconversion or detection of virus in nasal swabs. Among infected individuals, symptoms reported from the online surveys defined clinical outcomes associated with the influenza infection.

Symptomatic infection was defined as having at least one reported episode of symptomatic illness. Asymptomatic infection, by our stringent definition, necessitated absence of any reported illness episode. A total symptom score for each illness episode was calculated based on individuals assessing severity of symptoms as none, mild (not interfering with normal daily activities) or severe (affecting normal daily activities or requiring medical attention) in addition to reporting recorded temperatures of >38 °C. We attributed a weighted score of 0 for none, 1 for mild and 4 for severe for each of the canonical influenza symptoms (sore throat, cough, headache, myalgia and fever) as used in previous clinical studies (Monto et a/., J. Arch Intern Med 160, 3243-3247 (2000); Zambon et al. Arch Intern Med 161 , 21 16- 2122 (2001)) to create a summed total symptom score. The summed symptom score was designed by totalling the weight for each of the canonical influenza symptoms used in clinical studies with a weight of 0 for none, 1 to mild symptoms and 4 for severe symptoms attributed to each symptom. Mild symptoms were of a severity that did not interfere with normal daily activities while severe symptoms were those that affected normal daily activity or required medical attention.

Table 1 - Symptom score weightings

Symptomatic outcomes associated with the illness episode were measured and categorised in four different ways: illness episode with any symptoms; illness episode with fever (recorded temperature >38 °C or reported fever rated as severe); illness episode with fever plus cough or sore throat (CDC-defined influenza-like-illness definition); and a total symptom score for the illness episode. Ex vivo Fluorescence-immunospot assay (FLISpot)

Fluorescence-immunospot (Mabtech AB, Stockholm, Sweden) to simultaneously measure interferon-gamma (IFN-γ) and interleukin-2 (IL-2) secretion was undertaken as previously described (Casey, R., et al. PLoS One 5, e15619 (2010)), by investigators blinded to clinical data. PBMCs were stimulated with peptide pools (5Mg ml^-1) of conserved class I restricted 9-mer epitopes from PB1 , M1 and NP proteins (n=91 peptides, Table 3), live pH1 N1 virus (A/England/09/195) or CMV lysate as control antigen. The median background frequency of the IFN-γ and IL-2 response was 4 SFCs per million (IQR:0-12) and 16 SFCs per million (IQR:0-32) PBMCs respectively.

Frequencies of antigen-specific cells were calculated by subtracting the average number of Spot Forming Cells (SFCs) in negative control wells from SFCs in antigen- containing test wells for each donor. The frequency of total cytokine-secreting cells was calculated by summating frequencies of the three functional subsets, IFN-v⁺IL-2^_ ,IFN-v1L-2⁺ and IFN-v⁺IL-2⁺.

Flow-cytometry assay

PBMCs were stimulated with media (negative control), phorbol myristate acetate (PMA)/lonomycin (positive control), live pH1 1 virus and CMV lysate for 18 hours to maintain consistency with the FLISpot assay. Cells were stained for surface markers and intracellular cytokines as previously described (Sridhar, S., et al. Eur J Immunol 42, 2913-2924 (2012)) with at least 1 million live cells collected for all samples.

Staining of PBMCs was undertaken as previously described following 18 hours stimulation with media (negative control), phorbol myristate acetate (PMA)/lonomycin (positive control), live pH1 N1 (A/England/09/195) virus (MOI = 5) and CMV lysate (10 μg/mL, control antigen) to maintain consistency with the Fluorescence-immunospot assay. Monesin A (Sigma-Aldrich) was added 2 hours after addition of stimulus. Staining with CD107a (clone H4A3, BD Biosciences) and CD107b (clone H4B4, BD Biosciences) was undertaken at the time of stimulation. Cells were blocked with 10 % human AB serum (Sigma) to prevent non-specific antibody binding prior to staining with amine-reactive viability dye (LIVE/DEAD® Fixable Blue Dead Cell Stain Kit, Invitrogen) as a live/dead marker and surface staining was undertaken using a suitable combination of fluorochrome labelled anti-human CD3 (clone UCHT1 , BD Biosciences), CD4 (clone S3.5, Invitrogen), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone HCD14, Biolegend), CD19 (cloneHIB19, Biolegend), CD56 (clone HCD56, Biolegend), CCR7 (clone 3D12, BD Biosciences), CD45RA (clone MEM-56, Invitrogen) and CCR5 (clone 2D7, BD Biosciences) markers. Intracellular cytokine staining (ICS) was performed with BD Cytofix/Cytoperm Plus kit according to the manufacturer's instructions and cells were stained with IFN-γ (cloneB27, BD Biosciences), IL-2 (clone MQ1-17H12, BD Biosciences) and TNF-a (clone MAb11 , BD Biosciences) antibodies. Fluorescence minus one controls stimulated with live pH1 N1 virus were used for identifying positive populations of CCR7, CD107, CCR5 and CD45RA. In all samples, at least 1 million live events were collected and analysed. Antigen- specific cytokine responses were calculated only if the responses were >0.001% of the parent population. Flow cytometric analyses were performed using a Fortessa (BD Biosciences) and data were analyzed with Flow Jo (Tree Star) software and SPICE software (NIH). Table 2 - Fluorescent markers

Sample size estimation

We estimated that 100 infected individuals with a prevalence of 60% asymptomatic infection would allow detection of a moderate effect size (odds ratio=4) between symptomatic and asymptomatic infection for each ten-fold increase in cross-reactive T-cell frequency (prevalence of 60%) with 80% power at p=0.05 (two-tailed) significance. Based on 30% incidence of infection during the 1 ^st wave of the UK pandemic (Miller, E., et al. Lancet 375, 1 100-1 108 (2010)) , we calculated a sample size of 350 participants to attain 100 infected individuals. Statistical analysis

Our primary objective was to identify whether individuals developing mild or asymptomatic illness, had higher frequencies of cross-reactive CD8⁺ T-cells prior to infection. Our analytical strategy was to correlate frequencies of antigen-specific CD8+ T-cells with symptom score and compare these frequencies across symptom groups. Frequencies of antigen-specific T-cells between predefined groups categorised by an increasing severity of symptoms (Figure 2) were compared using non-parametric Mann-Whitney 2-tailed test. Total symptom score reflecting illness severity as a continuous variable was correlated with frequency of cross-reactive T-cells using Spearman rank correlation. Association of cellular immunity with infectiousness during an influenza episode was assessed by comparing T-cells in individuals with viral shedding versus those without viral shedding. Logistic regression was used to model the relationship between T-cell frequency (SFCs/million PBMC) and risk of clinical outcomes. Statistical analysis was undertaken using Stata version 10 (STATA Corp. Texas, USA).

A logistic model was used to model the relationship between number of cytokine secreting cells (SFCs/million PBMCs) and risk of illness with fever. The probability of illness was modelled as 1/1 + exp(a + βί,) where t is the number of cytokine secreting cells. A curve of probability of illness against number of cytokine secreting cells represents a susceptibility curve (Forrest et al. Clin Vaccine Immunol 2008; 15: 1042- 53). The curves were first estimated with only SFCs/million as the explanatory variable and then with age as a covariate. Goodness of fit of models compared using likelihood ratio chi-squared test showed no significant difference when age was used as a covariate. Example 1 - Study design and cohort; and Incidence of infection and outcomes

We designed a prospective cohort study recruiting 342 eligible participants starting prior to the onset of the second UK pandemic wave and followed through the two consecutive influenza seasons, 2009-10 and 2010-11 , in which pH1 N1 was the predominant circulating strain (Figure 1). 51 individuals lacking neutralising antibodies to pH1 N1 by standard haemagglutination-inhibition and microneutralisation assays (Table 3) developed incident pH1 N1 infection of whom 43 had complete clinical data and viable baseline PBMCs (Figure 2). The median age was 34.5 years [IQR 27-40] (Table 4) and there were no differences in age or gender between the individuals included and excluded from the analysis.

Table 3 - Individual haemagglutination inhibition and microneutraiisation assay titres to pH1N1 virus at baseline for 51 individuals who subsequently developed infection

* Haemagglutination Inhibition assay undertaken as described above. Titre of 8 or

less was considered negative

# Microneutraiisation assay undertaken as described above. TItres of less than 20

considered negative. Table 4 - Demographics of cohort included in the final analysis.

Table 1 Cohort baseline demographics

Number |%)

Demographics |n = 43)

Age In years ". median (IQR| [range] 34.5 (27 - 40) [18 - 64]

Age categories *

18 -25 8 (19.05)

26 -40 24 (57.14)

41-55 8 (19.05)

> 56 years 2(4.76)

Sender

Male 17 (39.53)

Occupational status '

Staff 6 (14.29)

Student 36 (85.71)

Risk Factors for Influenza Infection |n = 43)

Living with children at home

Yes 13 (30.23)

No 30 (69.77)

Self-reported history of Influenza vaccination in 2008*

Yes 2 (4.76)

No 40 (95.24)

DNK 1

Self reported life-time history of influenza vaccination*'

Yes 12 (27.91)

No 31 (72.09)

Of the 43 incident cases included in the analysis, 32 were diagnosed by antibody seroconversion and 11 by detection of virus in nasal swab of whom all except one also had antibody seroconversion.

In 25 of these individuals, we reliably determined the date, symptoms and symptom score for the clinical episode when influenza infection occurred, whereas in the remaining 18 infected individuals the presence of more than one reported symptomatic episode precluded this determination.

These 25 individuals were grouped according to their clinical outcomes for the analysis (see Methods) as depicted in Figure 2. Of the 25, 15 returned nasal swabs during their illness episode with 11 shedding virus (nasal swab RT-PCR-positive) while 4 did not shed virus despite having antibody seroconversion.

Example 2 -Pre-existing cross-reactive T-cells and illness severity

Pre-existing cross-reactive T-cells to pH1 N1 virus were detected in all 43 seronegative individuals (Figure 6) with a predominance of T-cells with an IFN-v⁺IL-2- cytokine-secreting profile as previously reported (Sridhar, S., et al. Eur J Immunol 42, 2913-2924 (2012)). The frequency of cross-reactive T-cells was independent of age. We first assessed the association between pre-existing T-cells and risk of subsequent pH1 N1 infection. As expected, frequencies of pre-existing cross-reactive total cytokine-secreting T-cells or the cytokine-secreting T-cell subsets at baseline did not differ between individuals who became infected (n = 43) and age-and gender- matched individuals who did not acquire pH1 1 infection (n = 34) (Figure 7).

Among pH1 N1 infected individuals, we determined the relationship between the frequencies of cross-reactive total cytokine-secreting T-cells prior to infection with subsequent development of symptomatic outcomes. We assessed the global CD4 and CD8 response to antigens conserved between the pandemic and previously circulating influenza A virus strains by quantifying T-cells responding to live virus. Higher frequencies of pre-existing cross-reactive total T-cells to live pH1 N1 virus (p = 0.03) were detected in individuals who developed illness without fever (n = 12) compared to those whose illness was accompanied by fever (n = 13) (Figure 3A). The quantitative relationship between the frequency of virus-specific T-cells and the risk of developing influenza illness is depicted in figure 8. Three individuals with completely asymptomatic infection had higher frequencies of pre-existing total cross- reactive T-cells to pH1 N1 virus (p = 0.02) than the 22 with symptomatic infection (data not shown).

We also tested if CD8⁺ T-cells to highly conserved viral epitopes mediate a protective response by assessing cellular responses to highly conserved CD8 epitopes from the immunodominant internal PB1 , NP and M1 proteins (Table 5).

Table 5 - Sequences of predicted conserved MHC Class I epitopes from PB1, M1 and NP proteins, collectively denoted as "CD8 conserved epitopes"

Higher frequencies of total cytokine-secreting T-cells to conserved CD8 epitopes were detected in individuals developing an illness without fever (p = 0.02) (n = 12) or symptoms of ILI (p = 0.04) (n = 15) than those with fever (n = 13) or ILI symptoms (n = 10) (Figure 3B). CD8⁺ T-cell depletion abrogated the response to these epitopes confirming that responses were mediated by CD8⁺ T-cells (Figure 9).

Total symptom score during the illness episode correlated inversely with the frequency of pre-existing total cytokine-secreting T-cells specific for live virus (r = - 0.39, p = 0.05) (Figure 4A) and more strongly for conserved CD8 epitopes of immunodominant core proteins (r = -0.50, p = 0.01 ) (Figure 4B). Further enumeration of the 3 key functional T-cell subsets by fluorescence-immunospot identified only the IFN-y⁺IL-2~ T-cell subset as associated with reduced risk of developing a more severe influenza infection (Figure 3 D-G). The strongest inverse correlation was between total symptom score and the frequency of I FN-y⁺IL-2~ T-cells specific for conserved CD8 epitopes (r = -0.56, p=0.004) (Figure 4E).

Neither total cytokine-secreting T-cells (Figure 4C) nor IFN-Y⁺IL-2^~T-cells (Figure 4F) specific for control CMV antigen correlated with total symptom score (r=-0.33; p=0.13 and r=-0.15; p=0.48, respectively) or decreased risk of a more severe illness (Figure 3 C,H, I ) suggesting that T-cell responses associated with limiting illness severity were influenza-specific.

Example 3 - Phenotype of cross-reactive CD8*IFN-y*IL-2 memory T-cells

To pinpoint the specific phenotype of the pre-existing influenza-specific protection- associated CD8⁺IFN-Y⁺IL-2^" T-cell population, we stratified it into its constituent memory subsets by multi-parameter flow-cytometry using surface markers CD45RA and CCR7 (Sallusto, F., Geginat, J. & Lanzavecchia, A. Annu Rev Immunol 22, 745- 763 (2004)).

The IFN-Y⁺IL-2~ T-cell response to live virus was dominated by CD8⁺ T-cells which comprised predominantly CD45RA CCR7^" effector-memory and CD45RA⁺CCR7~ late-effector T-cells (Figure 5A). The proportion of CD8⁺I FN-Y⁺I L-2^" T-cells in the CD45RA⁺CCR7~ late-effector subset was inversely correlated (r=-0.49, p=0.02) with total symptom score (Figure 5B). Functional characterisation of this protection- associated CD8⁺IFN-Y⁺IL-2-CD45RA⁺CCR7- T-cell population for lung-homing (CCR5), degranulation (CD107A/B) and cytokine secretion (TNF-a) revealed that 70% of these cells expressed CCR5 and 33% expressed CD107A B (Figure 5C) in response to live pH 1 1 virus, demonstrating capability for rapid cytotoxicity and lung- homing on virus exposure.

Although a recent report in an artificial experimental challenge model implicated CD4⁺ T-cells in limiting influenza illness severity, we found no association between the total symptom score and the proportion of virus-specific CD4⁺IFNg⁺IL-2~ (r = - 0.093; p = 0.68) or the late-effector CD45RA⁺CCR7~ subset of CD4⁺IFNg⁺IL-2^" T- cells (r = -0.012; p = 0.96) (Figure 12).

Our study was designed to test the role of CD8+ T-cells in heterosubtypic immunity and therefore longer peptides containing optimal CD4 epitopes were not used. Our experiments thus do not rule out a possible correlation of CD4 T cells with illness severity.

Example 4 - Heterosubtypic T cell responses and viral shedding As evidenced by the decrease in influenza incidence with school-closure during the 2009 pandemic (Cauchemez, S., et al. Lancet Infect Dis 9, 473-481 (2009)), limiting influenza transmission is critical for pandemic control. Frequencies of pre-existing live virus-specific I FN-Y⁺IL-2~ T-cells were significantly higher (p = 0.05) in individuals not shedding virus compared to those shedding virus, while there was no significant difference in total cytokine-secreting cells (Figure 10), consistent with results of experimental challenge studies associating T-cell frequencies with reduced viral shedding (Yewdell, J.W., Bennink, J.R., Smith, G.L. & Moss, B. Proc Natl Acad Sci U S A 82, 1785-1789 (1985); McMichael, A.J., Gotch, F. M., Noble, G.R. & Beare, P.A. N Engl J Med 309, 13-17 ( 983)).

Individuals might be misclassified as non-shedders if they collected nasal swabs later after onset of symptoms than non-shedders; however, there was no significant difference in the time from symptom onset to nasal swab collection between infected individuals shedding and not shedding virus.

Discussion of results of Examples 1-4

In influenza pandemics, where susceptible populations lack protective antibodies, the most favourable outcome of infection is symptom-free illness. We have identified, for the first time, a cellular immune correlate of protection against clinical illness following natural influenza infection by an antigenically-shifted reassortant virus.

In our cohort, a higher frequency of CD8⁺1FN-Y⁺1L-2^" cross-reactive T-cells was associated with decreased risk of fever, less I LI symptoms, reduced illness severity score and absence of viral shedding in individuals infected with pandemic virus. Within this functional CD8⁺ T-cell subset, we also identified CD45RA⁺CCR7^~ late- effector T-cells as the cellular immune correlate of protection against community- acquired pandemic influenza illness. These cells have direct antiviral cytotoxic potential, rapidly secreting IFN-γ and expressing the degranulation marker CD107 on recognition of live virus. Further, these cells express the lung homing marker CCR5 which is critical for directing CD8⁺ T-cells to the lungs during respiratory viral infection with genetic variants of the CCR5 gene potentially predisposing individuals to risk of severe pH1 N1 disease. Thus, the cytokine-secretion profile, capability for rapid cytotoxicity and lung-homing potential displayed by this T-cell population are all attributes compatible with mediating protection.

We found that the summed T-cell response to highly conserved CD8 epitopes from three immunodominant core proteins, PB1 , M1 and NP, was most strongly associated with limiting illness severity. This indicates that CD8⁺ HLA-class I- restricted T-cells specific for epitopes conserved across influenza A virus subtypes confer protection against illness for a broad range of influenza A viruses, in the absence of detectable cross-reactive neutralising antibodies. Despite the increasing interest in developing broadly protective universal influenza vaccines, the field is limited by the absence of an immunological surrogate of protection and identification of protective antigens. Our findings support the prioritisation of universal vaccines that induce durable CD8⁺ T-cell responses to these conserved epitopes.

In summary, our study in a natural infection setting identifies a cellular immunological correlate of protection against symptomatic influenza and demonstrates a role for cross-reactive CD8⁺ T-cells specific for highly conserved epitopes from core proteins in limiting illness severity during a pandemic.

Example 5 - Pharmaceutical formulations and administration

Also provided for are pharmaceutical formulations or compositions (such as vaccine formulations or compositions) comprising one or more compounds identified according to the third, fourth or fifth aspects of the invention (hereafter also referred to as "compounds of the invention") in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent or carrier. Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of the active ingredient.

The compounds of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. In human therapy, the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds of the invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecal^, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi- dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be from 1 mg/kg to 30 mg/kg. Thus, for example, the tablets or capsules of the compound of the invention may contain a dose of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage, which will be most suitable for any individual patient, and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention. The compounds of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 , 1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' delivers an appropriate dose of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The compounds of the invention may also be transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the compounds of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum. For application topically to the skin, the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Generally, in humans, oral or topical administration of the compounds of the invention is the preferred route, being the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the drug may be administered parenterally, e.g. sublingually or buccally.

For veterinary use, a compound of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration, which will be most appropriate for a particular animal.

Embodiments of the invention will now be described in the following two sets of numbered paragraphs:

Numbered paragraphs (set 1):

1. A method of determining prognosis of an influenza infection in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens; (iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-γ without secreting IL-2; and optionally

(iv) calculating the cells identified in (iii) as a percentage of those identified in

(i);

2. The method of paragraph 1 wherein the symptomatic disease is severe symptomatic disease.

3. The method of paragraphs 1 or 2 wherein the individual is at increased risk of developing symptomatic disease following an influenza infection if no cells are identified at step (iii).

4. The method of paragraphs 1 , 2 or 3 wherein the risk of the individual developing symptomatic disease following an influenza infection is inversely proportional to the percentage value calculated in step (iv) of the method.

5. A method of determining influenza immunity status in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

0);

wherein the identification of cells in step (iii) and/or the percentage of cells calculated in (iv) correlates to influenza immunity status of the individual. 6. Use of the method of paragraph 5 in a method of identifying one or more compounds that induces influenza immunity in an individual.

A method of identifying one or more compounds that induce influenza immunity in individual comprising:

(iv) calculating the cells identified in (iii) as a percentage of those identified in (i);

8. A method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more test compounds;

(0;

9. The method of paragraph 8 wherein a period of time is allowed to elapse between previous administration of the test compound and sample provision or the method of paragraph 9 wherein a period of time is allowed to elapse between steps (ii) and (iii). 10. The use of paragraph 6 or the method of any one of paragraphs 7 to 9 wherein the suitability of a test compound for inducing influenza immunity is indicated by the identification of one or more cells at step (iii).

11. The use of paragraph 6 or the method of any one of paragraphs 7 to 10 wherein the suitability of a test compound for inducing influenza immunity is proportional to the percentage value calculated in step (iv) of the method.

12. The use or method of any one of paragraphs 6 to 11 wherein the method comprises an additional step (v) of determining if the one or more candidate vaccine increases the percentage calculated at step (iv) in comparison to a control value. 13. The method of paragraph 12 wherein the control value is obtained by performing the method of paragraph 8 but using a sample of cells at step (i) obtained from the individual prior to their being administered with one or more test compounds. 14. The method of paragraph 12 wherein the control value is obtained by performing the method of paragraph 9 without including the one or more test compounds at step (ii) and/or including one or more influenza antigens in place of the one or more test compounds at step (ii). 15. The method of any one of paragraphs 1 to 5, 7 or 10 to 14 wherein the one or more influenza antigens in step (ii) comprise at least one influenza antigen that is conserved across multiple (preferably all known) influenza A strains.

16. The method of paragraph 15 wherein all of the one or more influenza antigens are conserved across multiple (or all known) influenza A strains.

17. The method of paragraphs 15 or 16 wherein the one or more influenza antigens comprise PB1 , NP, M1 , NS1 , NS2, PB1 F2, HA, NA, M2 and/or PB2 proteins. 18. The method of paragraph 17 wherein the one or more influenza antigens comprise one or more of the peptides described in Table 5.

19. The method of paragraph 18 wherein the one or more influenza antigens comprise all of the peptides described in Table 5.

20. The method or use of any previous paragraph wherein step (iii) is performed by multi-parameter flow cytometry.

21 . The method or use of any previous paragraph wherein the sample is a blood sample, preferably a PBMC sample.

22. The method or use of any previous paragraph wherein an additional step is first performed in order to determine that the sample of cells is either infected with influenza or is obtained from a subject infected with influenza. 23. The method of any one of paragraphs 1-4 or 15-22 wherein the symptomatic disease is associated with one or more of fever, cough, headache, myalgia, sore throat, diarrhoea and runny nose. 24. The method of any one of paragraphs 1-4 and 15-22 wherein the symptomatic disease is associated with viral shedding.

25. The method of any previous paragraph wherein the individual is a mammal, preferably a mouse, rat, rabbit, sheep, goat, dog or human.

26. The method or use of any previous paragraph wherein the T-cells are identified in step (ii) by identifying those cells that are CD3 positive.

27. The method or use of any previous paragraph wherein the T-cells identified in step (iii) are identified as live T-cells, preferably by use of a dead cell marker.

28. A vaccine composition comprising one or more compounds identified by the methods of paragraphs 7 to 27. 29. A composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-Y and IL-2. 30. The composition of paragraph 29 wherein the plurality of antibodies or antigen- binding fragments thereof is fixed to a solid support.

31. The composition of either of paragraphs 29 or 30 wherein the antibody or antigen-binding fragments thereof is selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments), single variable domains (e.g. VH and V_. domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]). 32. The composition of any one of paragraphs 29 to 31 wherein the antibodies or antigen-binding fragments thereof with a particular specificity are separately detectable to those with a different specificity. 33. The composition of any one of paragraphs 29 to 32 wherein the antibodies or antigen-binding fragments thereof are visually detectable. 34. The composition of any one of paragraphs 29 to 33 wherein the antibodies or antigen-binding fragments thereof are labelled (e.g. with a fluorescent label).

35. The composition of any one of paragraphs 29 to 34 for use in diagnosing individuals at risk of developing symptomatic disease following influenza infection.

36. A kit for determining influenza immunity status in an individual comprising:

(ii) instructions for use.

37. A kit as referred to in paragraph 36 comprising one or more influenza antigens, dead cell discriminators, negative and/or positive controls.

38. The kit as referred to in paragraph 37 wherein the one or more influenza antigens are as defined in any one of paragraphs 15 to 19.

39. The kit as referred to in any one of paragraphs 36 to 38 wherein the composition of (i) and/or (ii) as a whole is additionally able to bind to CD3 and/or dead cells and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3 and/or dead cells.

40. Use of a kit as referred to in any of paragraphs 36 to 39 in the method of any of paragraphs 1 -5 and 7 to 27. Numbered paragraphs (set 2):

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

(iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-γ without secreting IL-2;

(iv) identifying those cells of (iii) which are also CCR7 negative and CD45RA positive; and optionally

(v) calculating the cells identified in (iv) as a percentage of those identified in (iii);

wherein the identification of cells in step (iv) and/or the percentage of cells calculated in (v) correlates to the risk of the individual developing symptomatic disease following an influenza infection, and wherein steps (iii) and (iv) can be carried out either sequentially or simultaneously.

3. The method of paragraphs 1 or 2 wherein the individual is at increased risk of developing symptomatic disease following an influenza infection if no cells are identified at step (iv).

4. The method of paragraphs 1 , 2 or 3 wherein the risk of the individual developing symptomatic disease following an influenza infection is inversely proportional to the percentage value calculated in step (v) of the method.

5. The method of paragraph 4 wherein the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (v) is less than or equal to 30% to 75%.

6. A method of determining influenza immunity status in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

(iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-γ without secreting IL-2; (iv) identifying those cells of (iii) which are also CCR7 negative and CD45RA positive; and optionally

wherein the identification of cells in step (iv) and/or the percentage of cells calculated in (v) correlates to influenza immunity status of the individual, and wherein steps (iii) and (iv) can be carried out either sequentially or simultaneously.

7. Use of the method of paragraph 6 in a method of identifying one or more compounds that induces influenza immunity in an individual.

(ii) exposing the sample of (i) to one or more influenza antigens;

(iii) identifying T-cells in the sample that are CD8 positive and secrete IFN- Y without secreting IL-2;

(v) calculating the cells identified in (iv) as a percentage of those identified in (Ni);

wherein the identification of cells in step (iv) and/or the percentage of cells calculated in (v) correlates to the ability of the one or more test compounds to induce influenza immunity in an individual, and wherein steps (iii) and (iv) can be carried out either sequentially or simultaneously.

9. A method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more test compounds;

(v) calculating the cells identified in (iv) as a percentage of those identified in (iii); wherein the identification of cells in step (iv) and/or the percentage of cells calculated in (v) correlates to the ability of the one or more test compounds to induce influenza immunity in an individual, and wherein steps (iii) and (iv) can be carried out either sequentially or simultaneously.

10. The method of paragraph 8 wherein a period of time is allowed to elapse between previous administration of the test compound and sample provision or the method of paragraph 9 wherein a period of time is allowed to elapse between steps (ii) and (iii).

11. The use of paragraph 7 or the method of any one of paragraphs 8 to 10 wherein the suitability of a test compound for inducing influenza immunity is indicated by the identification of one or more cells at step (iv). 12. The use of paragraph 7 or the method of any one of paragraphs 8 to 11 wherein the suitability of a test compound for inducing influenza immunity is proportional to the percentage value calculated in step (v) of the method.

13. The use or method of paragraph 12 wherein a test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step

(v) is at least 30% to 75%.

14. The use or method of any one of paragraphs 8 to 13 wherein the method comprises an additional step (vi) of determining if the one or more candidate vaccine increases the percentage calculated at step (v) in comparison to a control value.

15. The method of paragraph 14 wherein the control value is obtained by performing the method of paragraph 8 but using a sample of cells at step (i) obtained from the individual prior to their being administered with one or more test compounds.

16. The method of paragraph 14 wherein the control value is obtained by performing the method of paragraph 9 without including the one or more test compounds at step (ii) and/or including one or more influenza antigens in place of the one or more test compounds at step (ii). 17 The method of any one of paragraphs 1 to 8 and 11 to 16 wherein the one or more influenza antigens in step (ii) comprise at least one influenza antigen that is conserved across multiple (preferably all known) influenza A strains. 18. The method of paragraph 17 wherein all of the one or more influenza antigens are conserved across multiple (or all known) influenza A strains.

19. The method of paragraphs 17 or 18 wherein the one or more influenza antigens comprise PB1 , NP, M1 , NS1 , NS2, PB1 F2, HA, NA, M2 and/or PB2 proteins.

20. The method of paragraph 19 wherein the one or more influenza antigens comprise one or more of the peptides described in Table 5.

21. The method of paragraph 20 wherein the one or more influenza antigens comprise all of the peptides described in Table 5.

22. The method of any previous paragraph wherein steps (iii) and/or (iv) are performed by multi-parameter flow cytometry. 23. The method of any previous paragraph wherein the sample is a blood sample, preferably a PBMC sample.

24. The method of any previous paragraph wherein an additional step is first performed in order to determine that the sample of cells is either infected with influenza or is obtained from a subject infected with influenza.

25. The method of any one of paragraphs 1-5 and 17-24 wherein the symptomatic disease is associated with one or more of fever, cough, headache, myalgia, sore throat, diarrhoea and runny nose.

26. The method of any one of paragraphs 1-5 and 17-24 wherein the symptomatic disease is associated with viral shedding.

27. The method of any previous paragraph wherein the individual is a mammal, preferably a mouse, rat, rabbit, sheep, goat, dog or human. 28. The method or use of any previous paragraph wherein the T-cells are identified in step (ii) by identifying those cells that are CD3 positive.

29. The method or use of any previous paragraph wherein the T-cells identified in step (iii) are identified as live T-cells, preferably by use of a dead cell marker.

30. A vaccine composition comprising one or more compounds identified by the methods of paragraphs 6 to 28. 31. The method or use of any previous paragraph wherein the T-cells are identified in step (ii) of the methods of the invention by identifying those cells that are CD3 positive.

32. A composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, CD45RA and CCR7.

33. The composition of paragraph 32 wherein the plurality of antibodies or antigen- binding fragments thereof additionally binds to each of IFN-γ and IL-2 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for each of IFN-γ and IL-2.

34. A composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-Y and IL-2.

35. The composition of any one of paragraphs 32 to 34 wherein the plurality of antibodies or antigen-binding fragments thereof is fixed to a solid support.

36. The composition of any one of paragraphs 32 to 35 wherein the antibody or antigen-binding fragments thereof is selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)₂ fragments), single variable domains (e.g. V_H and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]). 37. The composition of any one of paragraphs 32 to 36 wherein the antibodies or antigen-binding fragments thereof with a particular specificity are separately detectable to those with a different specificity.

38. The composition of any one of paragraphs 32 to 37 wherein the antibodies or antigen-binding fragments thereof are visually detectable.

39. The composition of any one of paragraphs 32 to 38 wherein the antibodies or antigen-binding fragments thereof are labelled (e.g. with a fluorescent label).

40. The composition of any one of paragraphs 22 to 39 for use in diagnosing individuals at risk of developing symptomatic disease following influenza infection.

A kit for determining influenza immunity status in an individual comprising:

(iii) instructions for use.

A kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2;

(ii) a composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD45RA and CCR7and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD45RA and CCR7; and

(iv) instructions for use. 43. A kit for determining influenza immunity status in an individual comprising:

(v) instructions for use.

44. A kit as referred to in any one of paragraphs 41 or 43 additionally comprising one or more influenza antigens, dead cell discriminators, negative and/or positive controls.

45. The kit as referred to in paragraph 44 wherein the one or more influenza antigens are as defined in any one of paragraphs 17 to 21.

46. The kit as referred to in any one of paragraphs 41 to 45 wherein the composition of (i) and/or (ii) as a whole is additionally able to bind to CD3 and/or dead cells and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3 and/or dead cells.

47. Use of a kit as referred to in any of paragraphs 41 to 43 in the method of any of paragraphs 1 to 29.

Claims

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

wherein the identification of cells in step (iii) and/or the percentage of cells calculated in (iv) correlates to the risk of the individual developing symptomatic disease following an influenza infection. 2. The method of claim 1 comprising the following additional steps:

3. The method of claims 1 or 2 wherein the symptomatic disease is severe symptomatic disease.

4. The method of claims 1 , 2 or 3 wherein the individual is at increased risk of developing symptomatic disease following an influenza infection if no cells are identified at step (iii) or (a).

5. The method of claims 1 , 2, 3 or 4 wherein the risk of the individual developing symptomatic disease following an influenza infection is inversely proportional to the percentage value calculated in step (iv) or (b) of the method.

6. The method of claim 5 wherein the individual is at increased risk of developing symptomatic disease following an influenza infection when the percentage calculated in step (iv) is less than or equal to 0.005% to 0.020% or, if step (b) is performed, the percentage calculated in step (b) is less than or equal to 30% to 75%.

7. A method of determining influenza immunity status in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more influenza antigens;

(0;

8. The method of claim 7 comprising the following additional steps:

9. Use of the method of claim 7 or 8 in a method of identifying one or more compounds that induces influenza immunity in an individual.

10. A method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(ii) exposing the sample of (i) to one or more influenza antigens;

(iii) identifying T-cells in the sample that are CD8 positive and secrete IFN-γ without secreting IL-2; and optionally (iv) calculating the cells identified in (iii) as a percentage of those provided in

CO;

11. A method of identifying one or more compounds that induce influenza immunity in an individual comprising:

(i) providing a sample comprising T-cells;

(ii) exposing the sample of (i) to one or more test compounds;

12. The method of claim 10 or 11 comprising the following additional steps:

b) calculating the cells identified in step (a) as a percentage of those identified in step (iii)

wherein the identification of cells in step (a) and/or the percentage of cells calculated in (b) correlates to the ability of the one or more test compounds to induce influenza immunity in an individual, and wherein steps (iii) and (a) can be carried out either sequentially or simultaneously. 13. The method of claim 10 or 12 wherein a period of time is allowed to elapse between previous administration of the test compound and sample provision or the method of claim 11 or 12 wherein a period of time is allowed to elapse between steps (ii) and (iii). 14. The use of claim 9 or the method of any one of claims 10 to 13 wherein the suitability of a test compound for inducing influenza immunity is indicated by the identification of one or more cells at step (iii) or (a).

15. The use of claim 9 or the method of any one of claims 10 to 13 wherein the suitability of a test compound for inducing influenza immunity is proportional to the percentage value calculated in step (iv) or (b) of the method.

16. The method of claim 15 wherein the test compound is identified as inducing influenza immunity in an individual when the percentage calculated in step (iv) is at least 0.005% to 0.020% or, if step (b) is performed, the percentage calculated in step (b) is at least 30% to 75%.

17. The use or method of any one of claims 9 to 16 wherein the method comprises an additional step (v) of determining if the one or more candidate vaccine increases the percentage calculated at step (iv) or (b) in comparison to a control value.

18. The method of claim 17 wherein the control value is obtained by performing the method of claim 10 or 12 but using a sample of cells at step (i) obtained from the individual prior to their being administered with one or more test compounds. 19. The method of claim 17 wherein the control value is obtained by performing the method of claim 11 or 12 without including the one or more test compounds at step (ii) and/or including one or more influenza antigens in place of the one or more test compounds at step (ii). 20. The method of any one of claims 1 to 8, 10, or 12 to 19 wherein the one or more influenza antigens in step (ii) comprise at least one influenza antigen that is conserved across multiple (preferably all known) influenza A strains.

21. The method of claim 20 wherein all of the one or more influenza antigens are conserved across multiple (or all known) influenza A strains.

22. The method of claims 20 or 21 wherein the one or more influenza antigens comprise PB1 , NP, M1 , NS1 , NS2, PB1 F2, HA, NA, M2 and/or PB2 proteins. 23. The method of claim 22 wherein the one or more influenza antigens comprise one or more of the peptides described in Table 5.

24. The method of claim 23 wherein the one or more influenza antigens comprise all of the peptides described in Table 5.

25. The method or use of any previous claim wherein step (iii) is performed by multi-parameter flow cytometry.

26. The method or use of any previous claim wherein the sample is a blood sample, preferably a PBMC sample. 27. The method or use of any previous claim wherein an additional step is first performed in order to determine that the sample of cells is either infected with influenza or is obtained from a subject infected with influenza.

28. The method of any one of claims 1-6 or 20-27 wherein the symptomatic disease is associated with one or more of fever, cough, headache, myalgia, sore throat, diarrhoea and runny nose.

29. The method of any one of claims 1-6 and 20-27 wherein the symptomatic disease is associated with viral shedding.

30. The method of any previous claim wherein the individual is a mammal, preferably a mouse, rat, rabbit, sheep, goat, dog or human.

31. The method or use of any previous claim wherein the T-cells are identified in step (iii) by identifying those cells that are CD3 positive.

32. The method or use of any previous claim wherein the T-cells identified in step (iii) are identified as live T-cells, preferably by use of a dead cell marker. 33. A vaccine composition comprising one or more compounds identified by the methods of claims 10 to 32.

34. A composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2.

35. The composition of claim 34 wherein the plurality of antibodies or antigen- binding fragments thereof additionally binds to each of CD45RA and CCR7 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD45RA and CCR7.

36. A composition comprising a plurality of antibodies or antigen-binding fragments thereof that binds to each of CD8, CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, CD45RA and CCR7.

37. The composition of any of claims 34 to 36 wherein the plurality of antibodies or antigen-binding fragments thereof is fixed to a solid support.

38. The composition of any of claims 34 to 37 wherein the antibody or antigen- binding fragments thereof is selected from the group consisting of Fv fragments

{e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)₂ fragments), single variable domains (e.g. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).

39. The composition of any one of claims 34 to 38 wherein the antibodies or antigen-binding fragments thereof with a particular specificity are separately detectable to those with a different specificity. 40. The composition of any one of claims 34 to 39 wherein the antibodies or antigen-binding fragments thereof are visually detectable.

41. The composition of any one of claims 34 to 40 wherein the antibodies or antigen-binding fragments thereof are labelled (e.g. with a fluorescent label).

42. The composition of any one of claims 34 to 41 for use in diagnosing individuals at risk of developing symptomatic disease following influenza infection.

43. A kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen- binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2; and

(ii) instructions for use. 44. The kit of claim 43, wherein the composition of (i) is as a whole additionally able to bind to each of CD45RA and CCR7 and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD45RA and CCR7.

A kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen- binding fragments thereof that binds to each of CD8, CD45RA and CCR7 and wherein the plurality comprises antibodies or antigen- binding fragments thereof that are individually specific for each of CD8, CD45RA and CCR7;

(ii) a composition comprising a plurality of antibodies or antigen- binding fragments thereof that binds to each of IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of IFN-γ and IL-2; and

(iii) instructions for use.

A kit for determining influenza immunity status in an individual comprising:

(i) a composition comprising a plurality of antibodies or antigen- binding fragments thereof that binds to each of CD8, IFN-γ and IL-2 and wherein the plurality comprises antibodies or antigen-binding fragments thereof that are individually specific for each of CD8, IFN-γ and IL-2;

(ii) a composition comprising a plurality of antibodies or antigen- binding fragments thereof that binds to each of CD45RA and CCR7and wherein the plurality comprises antibodies or antigen- binding fragments thereof that are individually specific for each of CD45RA and CCR7; and

(iii) instructions for use.

47. A kit as claimed in any of claims 43 to 46 comprising one or more influenza antigens, dead cell discriminators, negative and/or positive controls.

48. The kit as claimed in claim 47 wherein the one or more influenza antigens are as defined in any one of claims 20 to 24. 49. The kit as claimed in any one of claims 43 to 48 wherein the composition of (i) and/or (ii) as a whole is additionally able to bind to CD3 and/or dead cells and additionally comprises antibodies or antigen-binding fragments thereof that are individually specific for CD3 and/or dead cells. 50. Use of a kit as claimed in any of claims 43 to 49 in the method of any of claims 1 to 8 and 10 to 32.

51. A method substantially as described herein with reference to the Examples and Figures.

52. A composition substantially as described herein with reference to the Examples and Figures.

53. A kit substantially as described herein with reference to the Examples and Figures.

54. A use substantially as described herein with reference to the Examples and Figures.