Heterosubtypic T cell–mediated immunity to influenza A viruses in humans has been widely reported (24
). While cross-reactive T cells cannot prevent establishment of infection, they can enhance recovery by promoting virus clearance and reduce the severity of illness (15
). It has also been demonstrated that in vitro T cell cultures derived from healthy individuals can lyse target cells infected with swine or avian influenza viruses (28
). Together with a large number of studies in mice that demonstrated cross-protection conferred by influenza A–specific T cells during subsequent challenges (reviewed in ref. 11
), these studies suggest that vaccines stimulating cross-reactive T cell populations, potentially used in conjunction with the current antibody-based strategy, may confer broader and more effective protection against a potential pandemic strain. However, most studies using T cell cultures expanded in vitro were unable to assess accurately the overall influenza-specific memory T cell response and the extent of cross-reactivity to heterologous strains, particularly regarding the breadth, magnitude, and immunodominance hierarchy of the responses, at the population level. If a novel vaccine formula designed to stimulate cross-protective T cells is to be developed, it is crucial to address these questions first.
In the present study, we provide for what we believe to be the first time, a comprehensive ex vivo assessment of virus-specific CD4+ and CD8+ memory T cell responses against the full proteome of the human influenza A (H3N2) virus together with measurement of the extent of cross-reactivity to avian influenza A (H5N1) using freshly isolated PBMCs from 48 healthy individual from the UK and 42 healthy individuals from Viet Nam. Of 90 healthy donors with no prior exposure to H5N1, 81% and 93% of healthy donors living in the UK and Viet Nam, respectively, displayed detectable cross-reactive CD4+ and/or CD8+ memory T cell responses to the H5N1 internal proteins. In contrast, cross-recognition of the H5N1 HA and NA was exhibited by only 9 and 7, respectively, of 20 Vietnamese participants tested, and the T cell pools were of considerably lower frequencies compared with those specific for the H3N2 HA and NA. It is worth noting that HA and NA, predominantly targeted by CD4+ T cell populations, appear less immunogenic for T cell recognition than most internal proteins, regardless of the subtypes tested. Despite relatively high amino acid sequence identity (approximately 80%) between the NA of the H5N1 and human H1N1 strains, ex vivo cross-reactive T cell recognition of the NA was unremarkable. Thus, it would seem reasonable to suggest that internal protein regions, highly conserved and more frequently recognized by both CD4+ and CD8+ cross-reactive memory T cells, may serve as effective targets for vaccines designed to boost potentially cross-protective memory T cell populations.
Previous reports have identified M1 and NP as major targets of influenza-specific CTL recognition in humans (20
). Our ex vivo study verified M1 and NP as the most immunogenic protein targets of H5N1 cross-reactive CD4+
T cell responses. Moreover, CD4+
T cell clones specific for M1 or NP epitopes displayed cross-reactive effector functions against the target cells expressing the H5N1 M1 or NP, respectively. Transfer of NP-specific CTL clone protects mice from otherwise lethal challenges with homologous and heterologous influenza A viruses (22
). Although no such direct evidence exists in humans, a good correlation has been documented between pre-existing influenza A virus–specific CTL activities and enhanced viral clearance upon nasal challenge (15
). Thus, it is likely that our observations on cross-reactive influenza-specific memory T cells in healthy individuals may correlate with enhanced antiviral protection upon exposure to H5N1 influenza A viruses. Interestingly, the dominance of M1- or NP-specific responses observed at the population level during peptide pool screening was evident at the individual level when positive pool responses were confirmed at the single peptide level: M1- or NP-specific epitope regions were consistently immunodominant in a number of participants of diverse HLA backgrounds (data not shown). This held true for both CD4+
H5N1 cross-reactive and H3N2 strain–specific T cell epitope regions. It appears that M1 and/or NP may serve as potential universal influenza vaccine targets to provide broad protection to populations of diverse genetic backgrounds. Notably, vaccination of mice with DNA encoding NP elicited NP-specific CD4+
effector T cells and provided measurable protection against otherwise lethal heterosubtypic challenges (38
Influenza A–specific T cells detectable by ex vivo IFN-γ ELISpot have been previously characterized as a subset of circulating memory T cell populations capable of immediate effector function upon antigen encounter without a need to differentiate or proliferate (39
). IFN-γ secretion, an important component of antiviral protection in influenza infection (40
), was detectable within 6–12 hours of stimulation with peptides. In view of the diverse tissue tropism associated with highly pathogenic H5N1 viruses (41
), it is possible that circulating H5N1 cross-reactive memory T cell populations capable of immediate effector function could confer partial protection against human infection with H5N1 viruses.
Highly cross-reactive CD4+
effector functions against target cells infected with rVACVs expressing the H5N1 M1 or NP were detected both at the single-cell and polyclonal levels. In addition to exhibiting good cytolytic activities (CD8+
clones), cross-reactive T cell clones also displayed a high degree of polyfunctionality, simultaneously secreting IFN-γ, TNF-α, and IL-2 (CD4+
clones), together with upregulation of CD107a in recognition of H5N1 antigens. The induction of such multifunctional T cell populations in mouse and man has been associated with superior antiviral protection and vaccine efficacy (42
). Further characterization of such broadly cross-reactive and highly functional CD4+
T cell responses is in progress. This includes epitope optimization and determination of HLA restrictions, not included in the comprehensive analysis reported here.
Population differences in H5N1 cross-reactive T cell responses were observed between the cohorts from the UK and Viet Nam. A higher percentage of Vietnamese participants displayed detectable H5N1 cross-reactive memory T cell responses ex vivo. In addition, although the difference did not reach statistical significance, we detected ex vivo IFN-γ T cell responses of higher magnitude to the H5N1 internal proteins from Vietnamese participants (mean overall magnitudes: 390 (Viet Nam) and 287 (UK) SFU/106
= 0.058). Potential explanations for these differences include host genetic differences, exposure to region-specific, unrelated pathogens (an example is discussed in ref. 45
), and variations in the circulating influenza virus strains in the 2 regions. Moreover, there is a clearly defined influenza season in the UK, whereas in southern Viet Nam, acute influenza cases are reported throughout the year.
Considerable variation in virus-specific T cell recognition patterns was observed between different individuals in aspects such as the magnitude and breadth of the responses, CD4 and CD8 dependence, cross-reactivity, and dominant protein targets. McMichael et al. demonstrated a correlation between pre-existing influenza A–specific CTL activity and enhanced virus clearance upon subsequent intranasal challenge with live virus even in the absence of cross-protective antibodies (15
). This suggests that pre-existing influenza-specific memory T cells played a clear role in protection against disease in subsequent infection with heterologous strains. Thus, it is likely that individual variations in circulating memory T cell pools, particularly in the memory pools that are cross-reactive between subtypes, would at least partially contribute to the determination of clinical outcome of subsequent influenza infections together with other variables such as HLA haplotypes (46
), age (47
), antigen doses (48
), and exposure history to influenza viruses and other pathogens (45
). It may even be possible that such variations have contributed to differential outcomes in human infection cases with highly pathogenic H5N1. We are currently investigating this possibility in Viet Nam.
In a recent retrospective epidemiological analysis, Epstein provided a good example of heterosubtypic protection against influenza A infection conferred by immunological memory established from previous natural infection: adults who had experienced natural infection with the H1N1 strain in 1957 were significantly better protected from the H2N2 pandemic strain later that year (50
). In view of the absence of evidence suggesting the existence of neutralizing antibodies that are cross-reactive between the H1N1 and H2N2 strains, this observation further suggests that pre-existing cross-reactive memory T cells may be beneficial in subsequent heterosubtypic infection. However, it is worth noting that T cell memory to influenza in humans, particularly CD8+
CTL activities, is short-lived, with a half-life of 2–3 years (51
), and needs regular boosting by natural infection or appropriately designed vaccines.
There is growing interest in targeting conserved regions of internal proteins, such as M1 and NP, in combination with the existing vaccine formula in preparation for a potential pandemic. In view of the 80%–90% amino acid sequence identity of these proteins between the avian and human type A strains, our data pertaining to the immunodominant protein regions recognized by healthy individuals of diverse HLA backgrounds could serve as a potent catalyst for such endeavors. For instance, the epitope regions we identified that frequently elicited highly cross-reactive CD4+ and CD8+ T cell responses merit special attention for the potential development of a universal vaccine that would generate or boost T cell responses against seasonal human influenza as well as avian influenza viruses.
Despite extensive HLA polymorphism, the majority of HLA-A and -B allelic variants can be classified into 9 supertypes based on their shared peptide-binding specificity (52
). Epitope-based vaccines containing conserved peptides recognized by various MHC molecules may therefore confer broad and potent protection against influenza. However, developing such vaccines that can provide broad coverage across the diverse populations remains a challenge, as different HLA types are expressed at varying frequencies in different ethnic groups. Thus, the analysis of cross-reactivity of and population coverage by (54
) epitopes of interest should parallel comprehensive epitope mapping efforts (55
). Such endeavors may be facilitated by a centralized database, such as the Immune Epitope Database and Analysis Resource (http://www.immuneepitope.org), which provides comprehensive T and B cell epitope listing of various organisms as well as relevant analysis tools and resources (30
Currently available inactivated subunit vaccines are inefficient at boosting influenza-specific CTLs, as antigens cannot reach the cytosol and thus cannot be processed and presented as MHC-peptide complexes for T cell recognition. Stimulation of cross-reactive memory T cells could be potentially achieved by cold adapted live attenuated influenza vaccines (LAIVs) that closely mimic natural infection. Several trials have reported that LAIVs can boost virus-specific CTLs as well as mucosal and serum antibodies and provide broad cross-protection against heterologous human influenza A viruses (58
). While the safety and efficacy of the reassortant viruses bearing modified H5 HAs (60
) in humans remains to be determined, it would be worth evaluating the extent of cross-protection against H5N1 potentially conferred by currently available seasonal human LAIVs. Memory T cell populations boosted by these vaccines may in theory cross-react and provide partial protection against H5N1 by targeting highly conserved internal virus proteins.
The aim of such T cell–based approaches would be to provide broader partial protection against overwhelming infection and help lower morbidity and mortality rather than to provide complete protection against establishment of infection. This would be a highly relevant and perhaps more realistic public health goal in a pandemic situation.
In summary, the data presented here form an important basis from which to evaluate the role of virus-specific cross-reactive T cells in broad partial protection from human infection with avian (H5N1) and human influenza A viruses.