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.     

Different Arms of the Adaptive Immune System Induced by a Combination Vaccine Work in Concert to Provide Enhanced Clearance of Influenza

Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8⁺ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8⁺ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.     

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-Allele Cytotoxic T Lymphocyte Responses against 2009 Pandemic H1N1 Influenza A Virus among HLA-A24 and HLA-A3 Supertype-Positive Individuals

Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24⁺ population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11⁺ and HLA-A24⁺ donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11⁺ population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.     

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.     

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.     

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.     

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.     

Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines

Background

Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.

Methodology/Principal Findings

During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-γ⁺ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-γ⁺ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56^dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56^dim NK and DC.

Significance

These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.     

Cutting edge: CD4 T cells generated from encounter with seasonal influenza viruses and vaccines have broad protein specificity and can directly recognize naturally generated epitopes derived from the live pandemic H1N1 virus

The unexpected emergence of pandemic H1N1 influenza has generated significant interest in understanding immunological memory to influenza and how previous encounters with seasonal strains influence our ability to respond to novel strains. In this study, we evaluate the memory T cell repertoire in healthy adults to determine the abundance and protein specificity of influenza-reactive CD4 T cells, using an unbiased and empirical approach, and assess the ability of CD4 T cells to recognize epitopes naturally generated by infection with pandemic H1N1 virus. Our studies revealed that most individuals have abundant circulating CD4 T cells that recognize influenza-encoded proteins and that a strikingly large number of CD4 T cells can recognize autologous cells infected with live H1N1 virus. Collectively, our results indicate that a significant fraction of CD4 T cells generated from priming with seasonal virus and vaccines can be immediately mobilized upon infection with pandemic influenza strains derived from antigenic shift.     

Cooperativity Between CD8+ T Cells,Non-Neutralizing Antibodies,and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.     

Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

CD8⁺ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8⁺ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8⁺ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8⁺ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M2_82-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8⁺ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8⁺ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8⁺ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8⁺ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8⁺ T cells. These memory CD8⁺ T cells had lower cytokine expression than effector CD8⁺ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8⁺ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8⁺ T cell cytokine expression.     

The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4<Superscript>+</Superscript> T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8⁺ T cells are particularly promising because of the cytotoxicity and specificity of CD8⁺ T cells for tumor cells. Optimal CD8⁺ T cell activity requires the co-activation of CD4⁺ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4⁺ T cells. MHC II vaccines are MHC class I⁺ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii⁺ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4⁺ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii⁺ APC for priming and boosting Type 1 CD4⁺ T cells. MHC II vaccines consistently induce greater expansion of CD4⁺ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4⁺ T cells as measured by T cell receptor Vβ usage, compared to Ii⁺ APC. Therefore, the absence of Ii facilitates a robust CD4⁺ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii⁻ vaccine cells.     

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.     

Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice

During many infections, large numbers of effector CD8⁺ T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8⁺ T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8⁺ T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8⁺ T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4⁺ T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.     

TLR7 Recognition Is Dispensable for Influenza Virus A Infection but Important for the Induction of Hemagglutinin-Specific Antibodies in Response to the 2009 Pandemic Split Vaccine in Mice

Recognition of pathogen-associated molecular patterns by pattern recognition receptors of the innate immune system is crucial for the initiation of innate and adaptive responses and for immunological memory. We investigated the role of TLR7 in the induction of adaptive immunity and long-term memory following influenza virus infection and vaccination in C57BL/6 mice. During infection with influenza A/PR8/34 virus, the absence of either TLR7 or MyD88 leads to reduced virus-specific antibodies in the serum and antibody-secreting cells in their secondary lymphoid organs, particularly in bone marrow. In spite of this, the absence of TLR7/MyD88 signaling did not impair the production of protective antibodies. Following immunization with the 2009 pandemic inactivated split vaccine, TLR7(-/-) mice had significantly lower levels of germinal center formation, antibody-secreting cells, and circulating influenza virus-specific antibodies than control animals. Consequently, TLR7(-/-) mice failed to develop protective immunological memory upon challenge. Furthermore, the immunogenicity of the split vaccine was likely due to TLR7 recognition of virion RNA, as its removal from the split vaccine significantly reduced the levels of influenza virus-specific antibodies and compromised the vaccine protective efficacy in mice. Taken together, our data demonstrate that TLR7 plays an important role in vaccine-induced humoral immune responses to influenza virus through the interaction with viral RNA present in the split vaccine.     

Influenza Vaccine Induces Intracellular Immune Memory of Human NK Cells

Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)⁺CD56^dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.     

Agonistic Anti-CD40 Enhances the CD8+ T Cell Response during Vesicular Stomatitis Virus Infection

Intracellular pathogens are capable of inducing vigorous CD8⁺ T cell responses. However, we do not entirely understand the factors driving the generation of large pools of highly protective memory CD8⁺ T cells. Here, we studied the generation of endogenous ovalbumin-specific memory CD8⁺ T cells following infection with recombinant vesicular stomatitis virus (VSV) and Listeria monocytogenes (LM). VSV infection resulted in the generation of a large ovalbumin-specific memory CD8⁺ T cell population, which provided minimal protective immunity that waned with time. In contrast, the CD8⁺ T cell population of LM-ova provided protective immunity and remained stable with time. Agonistic CD40 stimulation during CD8⁺ T cell priming in response to VSV infection enabled the resultant memory CD8⁺ T cell population to provide strong protective immunity against secondary infection. Enhanced protective immunity by agonistic anti-CD40 was dependent on CD70. Agonistic anti-CD40 not only enhanced the size of the resultant memory CD8⁺ T cell population, but enhanced their polyfunctionality and sensitivity to antigen. Our data suggest that immunomodulation of CD40 signaling may be a key adjuvant to enhance CD8⁺ T cell response during development of VSV vaccine strategies.     

Annual vaccination against influenza virus hampers development of virus-specific CD8⁺ T cell immunity in children

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8(+) T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8(+) T cell immunity in children is currently unknown. Here we compared the virus-specific CD8(+) T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4(+) T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8(+) T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8(+) T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.     

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.