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.     

Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.     

Induction of Cross-Reactive Antibodies to Novel H7N9 Influenza Virus by Recombinant Newcastle Disease Virus Expressing a North American Lineage H7 Subtype Hemagglutinin

Severe human disease caused by the emerging H7N9 influenza virus in China warrants a rapid response. Here, we present a recombinant Newcastle disease virus expressing a North American lineage H7 influenza virus hemagglutinin. Sera from immunized mice were cross-reactive to a broad range of H7 subtype viruses and inhibited hemagglutination by the novel H7 hemagglutinin. Immunized mice were protected against a heterologous H7 subtype challenge, and genetic analysis suggested that cross-protective antibodies recognize conserved antigenic sites.     

Broadly Protective Adenovirus-Based Multivalent Vaccines against Highly Pathogenic Avian Influenza Viruses for Pandemic Preparedness

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.     

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.     

Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8(+) T-cell memory

The recall of CD8(+) T-cell memory established by infecting H-2(b) mice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8(+) effector and memory T cells specific for the shared, immunodominant D(b)NP(366) epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8(+) memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2-->H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8(+) D(b)NP(366)(+) T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8(+) D(b)NP(366)(+) set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8(+) T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8(+) memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.     

Vaccine-Induced Boosting of Influenza Virus-Specific CD4 T Cells in Younger and Aged Humans

Current yearly influenza virus vaccines induce strain-specific neutralizing antibody (NAb) responses providing protective immunity to closely matched viruses. However, these vaccines are often poorly effective in high-risk groups such as the elderly and challenges exist in predicting yearly or emerging pandemic influenza virus strains to include in the vaccines. Thus, there has been considerable emphasis on understanding broadly protective immunological mechanisms for influenza virus. Recent studies have implicated memory CD4 T cells in heterotypic immunity in animal models and in human challenge studies. Here we examined how influenza virus vaccination boosted CD4 T cell responses in younger versus aged humans. Our results demonstrate that while the magnitude of the vaccine-induced CD4 T cell response and number of subjects responding on day 7 did not differ between younger and aged subjects, fewer aged subjects had peak responses on day 14. While CD4 T cell responses were inefficiently boosted against NA, both HA and especially nucleocaspid protein- and matrix-(NP+M) specific responses were robustly boosted. Pre-existing CD4 T cell responses were associated with more robust responses to influenza virus NP+M, but not H1 or H3. Finally pre-existing strain-specific NAb decreased the boosting of CD4 T cell responses. Thus, accumulation of pre-existing influenza virus-specific immunity in the form of NAb and cross-reactive T cells to conserved virus proteins (e.g. NP and M) over a lifetime of exposure to infection and vaccination may influence vaccine-induced CD4 T cell responses in the aged.     

Protective Role of Gamma Interferon during the Recall Response to Influenza Virus

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4⁺ and CD8⁺ T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.     

Protection against a Lethal Avian Influenza A Virus in a Mammalian System

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig^+/+) and Ig^−/− mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8⁺ T-cell memory in the Ig^−/− mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig^+/+ mice, generating secondary CD8⁺ and CD4⁺ T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.     

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.     

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

Immunity to influenza A H9N2 viruses induced by infection and vaccination

Avian influenza A H9N2 viruses are widespread among domestic poultry and were recently isolated from humans with respiratory illness in China. Two antigenically and genetically distinct groups of H9N2 viruses (G1 and G9) are prevalent in China. To evaluate a strategy for vaccination, we compared G1 and G9 viruses for their relative immunogenicity and cross-protective efficacy. Infection of BALB/c mice with representative viruses of either group protected against subsequent challenge with the homologous or heterologous H9N2 virus in the absence of detectable cross-reactive serum hemagglutination inhibition antibody. Mice injected intramuscularly with inactivated G1 whole virus vaccine were completely protected from challenge with either H9N2 virus. In contrast, mice administered inactivated G9 vaccine were only partially protected against heterologous challenge with the G1 virus. These results have implications for the development of human vaccines against H9N2 viruses, a priority for pandemic preparedness.     

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.     

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A (H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses (compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.     

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.     

Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

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.     

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HA_m) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HA_m constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.