Infection with seasonal influenza virus elicits CD4 T cells specific for genetically conserved epitopes that can be rapidly mobilized for protective immunity to pandemic H1N1 influenza virus

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus.     

Seasonal FluMist vaccination induces cross-reactive T cell immunity against H1N1 (2009) influenza and secondary bacterial infections

T cell epitopes have been found to be shared by circulating, seasonal influenza virus strains and the novel pandemic H1N1 influenza infection, but the ability of these common epitopes to provide cross-protection is unknown. We have now directly tested this by examining the ability of live seasonal influenza vaccine (FluMist) to mediate protection against swine-origin H1N1 influenza virus infection. Naive mice demonstrated considerable susceptibility to H1N1 Cal/04/09 infection, whereas FluMist-vaccinated mice had markedly decreased morbidity and mortality. In vivo depletion of CD4(+) or CD8(+) immune cells after vaccination indicated that protective immunity was primarily dependent upon FluMist-induced CD4(+) cells but not CD8(+) T cells. Passive protection studies revealed little role for serum or mucosal Abs in cross-protection. Although H1N1 influenza infection of naive mice induced intensive phagocyte recruitment, pulmonary innate defense against secondary pneumococcal infection was severely suppressed. This increased susceptibility to bacterial infection was correlated with augmented IFN-γ production produced during the recovery stage of H1N1 influenza infection, which was completely suppressed in mice previously immunized with FluMist. Furthermore, susceptibility to secondary bacterial infection was decreased in the absence of type II, but not type I, IFN signaling. Thus, seasonal FluMist treatment not only promoted resistance to pandemic H1N1 influenza infection but also restored innate immunity against complicating secondary bacterial infections.     

Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.     

Human Cytotoxic T Lymphocytes Directed to Seasonal Influenza A Viruses Cross-React with the Newly Emerging H7N9 Virus

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8⁺ T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8⁺ T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8⁺ T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8⁺ T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8⁺ T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8⁺ T cells may afford some protection against infection with the new virus.     

Memory CD4 T Cells Direct Protective Responses to Influenza Virus in the Lungs through Helper-Independent Mechanisms

Memory CD4 T cells specific for influenza virus are generated from natural infection and vaccination, persist long-term, and recognize determinants in seasonal and pandemic influenza virus strains. However, the protective potential of these long-lived influenza virus-specific memory CD4 T cells is not clear, including whether CD4 T-cell helper or effector functions are important in secondary antiviral responses. Here we demonstrate that memory CD4 T cells specific for H1N1 influenza virus directed protective responses to influenza virus challenge through intrinsic effector mechanisms, resulting in enhanced viral clearance, recovery from sublethal infection, and full protection from lethal challenge. Mice with influenza virus hemagglutinin (HA)-specific memory CD4 T cells or polyclonal influenza virus-specific memory CD4 T cells exhibited protection from influenza virus challenge that occurred in the presence of CD8-depleting antibodies in B-cell-deficient mice and when CD4 T cells were transferred into lymphocyte-deficient RAG2^−/− mice. Moreover, the presence of memory CD4 T cells mobilized enhanced T-cell recruitment and immune responses in the lung. Neutralization of gamma interferon (IFN-γ) production in vivo abrogated memory CD4 T-cell-mediated protection from influenza virus challenge by HA-specific memory T cells and heterosubtypic protection by polyclonal memory CD4 T cells. Our results indicate that memory CD4 T cells can direct enhanced protection from influenza virus infection through mobilization of immune effectors in the lung, independent of their helper functions. These findings have important implications for the generation of universal influenza vaccines by promoting long-lived protective CD4 T-cell responses.Influenza virus poses substantial threats to world health due to the emergence of new pandemic strains through viral mutation and reassortment, including the 2009 H1N1 pandemic strain. Developing effective vaccines that can provide immune-mediated protection to multiple influenza virus strains remains a major challenge, as current vaccines generate neutralizing antibodies directed against the highly variable hemagglutinin (HA) and neuraminidase (NA) surface viral glycoproteins (18). These vaccines are only partially effective at protecting individuals from succumbing to seasonal strains and are largely ineffective at protecting individuals from new pandemics. In contrast, T lymphocytes have the potential to provide long-term cross-strain protection, through their recognition of invariant viral determinants (3, 9), generation of effector responses to coordinate both cellular and humoral immunity, and development of memory populations that persist for decades (34). In humans, influenza virus-specific CD4 and CD8 T cells recognize internal polymerase, matrix, and nucleoprotein components of influenza virus which are conserved in multiple strains (3). Influenza virus-specific memory T cells generated from virus exposure and vaccines can be detected readily in the peripheral blood of healthy older children and adults (16, 30). Elucidating the protective capacities of memory T cells in antiviral immunity and their underlying mechanisms is therefore crucial to understanding clinical responses to influenza and to developing strategies to boost T-cell-mediated immunity for the next emerging pandemic.The potent cytolytic responses of virus-specific CD8 T cells and their roles in antiviral primary and secondary responses have been well established (58); however, considerably less is known about the function of memory CD4 T cells in antiviral immunity. Memory CD4 T cells have the potential to play more diverse roles in coordinating secondary responses than those of memory CD8 T cells via their ability to “help” or promote cellular and humoral immunity, and also through direct effector functions. Compared to CD8 T-cell responses, memory CD4 T-cell responses in humans were found to recognize a more diverse array of influenza virus-specific epitopes (46-48) and to exhibit cross-reactivities with new pandemic strains, including avian H5N1 and 2009 H1N1 “swine flu” strains (23, 28, 36, 48). In addition, antiviral memory CD4 T cells generated as a result of influenza vaccination (22) were found to persist longer than CD8 T cells in vivo following smallpox vaccination (29). These findings suggest that memory CD4 T-cell responses could be potential targets for boosting long-term cellular immunity following vaccination, although their protective capacity remains undefined.The role of CD4 T cells in anti-influenza virus immunity has been elucidated mainly for primary responses, and less is known about the protective potential and mechanisms underlying memory CD4 T-cell-directed secondary responses. In primary influenza virus infection, CD4 T cells promote antibody production by B cells necessary for complete viral clearance (2, 17, 19, 39, 40, 57) and also promote the generation of memory CD8 T cells (4). Whether memory CD4 T cells have a similar helper-intensive role in promoting B cells and CD8 T cells in secondary influenza responses or whether effector responses predominate is not known. In this study, we investigated the mechanisms by which memory CD4 T cells mediate secondary responses and promote recovery from influenza virus infection in the clinically relevant scenario of a persisting CD4 T-cell response but no preexisting antibody response to a new influenza virus strain. We demonstrate that both influenza virus HA-specific and polyclonal influenza virus-specific memory CD4 T cells direct rapid lung viral clearance and protect from lethality via secondary antiviral responses in the absence of CD8 T cells, B cells, or any lymphocytes. Unlike primary responses to influenza virus, which can mediate protection independent of gamma interferon (IFN-γ), memory CD4 T-cell-mediated protection in the lung is dependent on secreted IFN-γ and is associated with localized interactions with lung airways and foci of T-cell-directed responses. Our findings reveal that memory CD4 T cells drive antiviral protection in the lung through a qualitatively distinct mechanism and have important implications for exploiting the protective role of persisting memory CD4 T cells in vaccines and immunotherapies.     

Cross-reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates

In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To "prime" cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 "primed" animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5-7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7-10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in "primed" animals, and reached peak frequencies in blood and lung 4-7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in "primed" animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, "primed" animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses.     

Assessment of Seasonal Influenza A Virus-Specific CD4 T-Cell Responses to 2009 Pandemic H1N1 Swine-Origin Influenza A Virus

Very limited evidence has been reported to show human adaptive immune responses to the 2009 pandemic H1N1 swine-origin influenza A virus (S-OIV). We studied 17 S-OIV peptides homologous to immunodominant CD4 T epitopes from hemagglutinin (HA), neuraminidase (NA), nuclear protein (NP), M1 matrix protein (MP), and PB1 of a seasonal H1N1 strain. We concluded that 15 of these 17 S-OIV peptides would induce responses of seasonal influenza virus-specific T cells. Of these, seven S-OIV sequences were identical to seasonal influenza virus sequences, while eight had at least one amino acid that was not conserved. T cells recognizing epitopes derived from these S-OIV antigens could be detected ex vivo. Most of these T cells expressed memory markers, although none of the donors had been exposed to S-OIV. Functional analysis revealed that specific amino acid differences in the sequences of these S-OIV peptides would not affect or partially affect memory T-cell responses. These findings suggest that without protective antibody responses, individuals vaccinated against seasonal influenza A may still benefit from preexisting cross-reactive memory CD4 T cells reducing their susceptibility to S-OIV infection.The outbreak of H1N1 swine-origin influenza A virus (S-OIV) in April 2009 has raised a new threat to public health (5, 6). This novel virus (with A/California/04/09 H1N1 as a prototypic strain) not only replicated more efficiently but also caused more severe pathological lesions in the lungs of infected mice, ferrets, and nonhuman primates than a currently circulating human H1N1 virus (9). Similarly, human patients with influenza-like illness who tested negative for S-OIV had a milder clinical course than those who tested positive (13). Another major concern is the lack of immune protection against S-OIV in the human population. Initial serum analysis indicated that cross-reactive antibodies to this novel viral strain were detected in only one-third of people over 60 years of age, while humoral immune responses in the population under 60 years of age were rarely detected (3, 8). In addition, vaccination with recent seasonal influenza vaccines induced little or no cross-reactive antibody responses to S-OIV in any age group (3, 8).Only a few studies address whether preexisting seasonal influenza A virus-specific memory T cells cross-react with antigenic peptides derived from S-OIV (7). In the absence of preexisting cross-reactive neutralizing antibodies, it is likely that T-cell-mediated cellular immunity contributes to viral clearance and reduces the severity of symptoms, although virus-specific T cells cannot directly prevent the establishment of infection (10). Greenbaum and colleagues recently compared published T-cell epitopes for seasonal influenza viruses with S-OIV antigens (Ags) using a computational approach (7). Several seasonal H1N1 epitopes were found to be identical to S-OIV sequences. This implies that seasonal flu-specific memory T cells circulating in the peripheral blood of vaccinated and/or previously infected individuals are able to recognize their S-OIV homologues.The first objective of this study was to determine the extent of cross-reactivity of seasonal H1N1 influenza A virus-specific CD4 T cells with S-OIV epitopes, especially those less conserved peptide sequences. We chose 17 immunodominant DR4-restricted T-cell epitopes derived from a seasonal H1N1 strain, compared the binding of these epitopes and their S-OIV homologous peptides to DR4, tested the ability of S-OIV peptides to drive seasonal influenza virus-specific T-cell proliferation in vitro, and estimated the frequency of S-OIV cross-reactive T cells in the periphery of noninfected donors. We found that most homologous S-OIV peptides were able to activate seasonal H1N1 virus-specific CD4 T cells. The second objective was to compare the antigen dosage requirement to activate those T cells. By assessing the alternations in the functional avidities (of T cells to the cognate peptide and S-OIV homologue) due to amino acid differences in S-OIV peptides, we showed how those cross-reactive CD4 T cells differentially responded to the antigenic peptides derived from seasonal H1N1 virus or S-OIV. This study leads to the conclusion that previous exposure to seasonal H1N1 viral antigens will generate considerable levels of memory CD4 T cells cross-reactive with S-OIV.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Healthy human subjects have CD4+ T cells directed against H5N1 influenza virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.     

Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus

Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.     

Cytotoxic T Lymphocytes Established by Seasonal Human Influenza Cross-React against 2009 Pandemic H1N1 Influenza Virus

While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Using influenza A virus matrix protein 1 (M1_58-66) epitope-specific CTLs isolated from healthy HLA-A2⁺ individuals, we further found that M1_58-66 epitope-specific CTLs efficiently killed both M1_58-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M1_58-66-specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M1_58-66 epitope-specific CTLs in 20% (4/20) of HLA-A2⁺ individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.Since its first identification in North America in April 2009, the novel pandemic H1N1 2009 (pdmH1N1) virus has been spreading in humans worldwide, giving rise to the first pandemic in the 21st century (13, 18). The pdmH1N1 virus contains a unique gene constellation, with its NA and M gene segments being derived from the Eurasian swine lineage while the other gene segments originated from the swine triple-reassortant H1N1 lineage. The triple-reassortant swine viruses have in turn derived the HA, NP, and NS gene segments from the classical swine lineage (20). The 1918 pandemic virus gave rise to both the seasonal influenza H1N1 and the classical swine H1N1 virus lineages (41). Evolution in different hosts during the subsequent 90 years has led to increasing antigenic differences between recent seasonal H1N1 viruses and swine H1 viruses (42). Thus, younger individuals have no antibodies that cross neutralize pdmH1N1, while those over 65 years of age are increasingly likely to have cross-neutralizing antibodies to pdmH1N1 (10, 25).Currently available seasonal influenza vaccines do not induce cross-reactive antibodies against this novel virus in any age group (10, 25). In animal models, it has been shown that pdmH1N1 replicated more efficiently and caused more severe pathological lesions than the current seasonal influenza virus (28). However, most patients with pdmH1N1 virus infection show a mild illness comparable to seasonal influenza (9, 42). The incidence of severe cases caused by pdmH1N1 was not significantly higher than that caused by human seasonal influenza viruses (43). These findings imply that seasonal influenza A virus-specific memory T cells preexisting in previously infected individuals may have cross-protection to this novel pdmH1N1.Cross-reactivity of influenza A virus-specific T-cell immunity against heterosubtypic strains which are serologically distinct has been demonstrated (5, 29, 33, 47). Humans who have not been exposed to avian influenza A (H5N1) virus do have cross-reactive memory CD4 and CD8 T cells to a wide range of H5N1 peptides (33, 47). More recently, one study also showed that some seasonal influenza A virus-specific memory T cells in individuals without exposure to prior pdmH1N1 infection can recognize pdmH1N1 (24). However, the results in most of these studies were determined by the gamma interferon (IFN-γ) responses to influenza virus peptides. Although the recalled IFN-γ response is commonly used to detect memory CD4 and CD8 T cells, the activated T cells that bind major histocompatibility complex (MHC)-presented peptide are not necessarily capable of lysing the target cells (6). In addition, the peptides, but not the whole virus, may not be able to fully represent the human cross-response against the virus as a whole. Therefore, in addition to cytokine production, the demonstration of direct antigen-specific cytotoxicity of cytotoxic T lymphocytes (CTLs) against both peptide-pulsed and virus-infected target cells is needed for better understanding of human CTL responses against pdmH1N1 virus.In this study, using bulk memory CTLs and epitope-specific CTLs established by seasonal influenza A viruses and epitope-specific peptide from healthy individuals, respectively, we evaluated their cross-cytotoxicity and cytokine responses to pdmH1N1. We also examined the expression of chemokine receptors CXCR3 and CCR5, which could help CTLs to migrate to the site of infection. In addition, to understand whether the seasonal influenza vaccines have benefit for people who have not been exposed to pdmH1N1, we further examined the ability of seasonal influenza vaccines to induce the conserved M1_58-66 epitope-specific CTLs in HLA-A2-seropositive healthy individuals.     

Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice

Previously, it has been shown that infection in humans with the pandemic swine influenza virus induces antibodies with specificity to the stalk domain of the viral hemagglutinin. Following the generation of these data, we sought to recapitulate these findings in the mouse model by sequential influenza virus infection. Mice that were inoculated with a seasonal influenza H1N1 virus followed by infection with a pandemic H1N1 strain produced higher antihemagglutinin stalk antibody titers than mice sequentially infected with drifted seasonal strains. In order to achieve antibody titers of comparable magnitude using sequential infection, mice had to be infected with 100- to 1,000-fold more of the drifted seasonal virus. The antistalk antibodies produced by these infections were influenza virus neutralizing, which illustrates the utility of the mouse model in which to study this interaction between virus and host.     

Type 1 responses of human Vγ9Vδ2 T cells to influenza A viruses

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.     

T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.     

Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally broad pattern of immunodominance in the primary HLA-DR1-restricted CD4 T-cell response to influenza virus hemagglutinin

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.     

Pandemic Influenza H1N1 2009, Innate Immunity,and the Impact of Immunosenescence on Influenza Vaccine

Seasonal and pandemic strains of influenza have widespread implications for the global economy and global health. This has been highlighted recently as the epidemiologic characteristics for hospitalization and mortality for pandemic influenza H1N1 2009 are now emerging. While treatment with neuraminidase inhibitors are effective for seasonal and pandemic influenza, prevention of morbidity and mortality through effective vaccines requires a rigorous process of research and development. Vulnerable populations such as older adults (i.e., > age 65 years) suffer the greatest impact from seasonal influenza yet do not have a consistent seroprotective response to seasonal influenza vaccines due to a combination of factors. This short narrative review will highlight the emerging epidemiologic characteristics of pandemic H1N1 2009 and focus on immunosenescence, innate immune system responses to influenza virus infection and vaccination, and influenza vaccine responsiveness as it relates to seasonal and H1N1 pandemic influenza vaccines.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02

We made an H1N1 vaccine candidate from a virus library consisting of 144 (?=?16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically na?ve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.