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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.     

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.     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.     

Humoral and cell-mediated immunity to pandemic H1N1 influenza in a Canadian cohort one year post-pandemic: implications for vaccination

We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity.     

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.     

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.     

Protective cross-reactive cellular immunity to lethal A/Goose/Guangdong/1/96-like H5N1 influenza virus is correlated with the proportion of pulmonary CD8(+) T cells expressing gamma interferon

A/Goose/Guangdong/1/96-like H5N1 influenza viruses now circulating in southeastern China differ genetically from the H5N1 viruses transmitted to humans in 1997 but were their precursors. Here we show that the currently circulating H9N2 influenza viruses provide chickens with cross-reactive protective immunity against the currently circulating H5N1 influenza viruses and that this protective immunity is closely related to the percentage of pulmonary CD8(+) T cells expressing gamma interferon (IFN-gamma). In vivo depletion of T-cell subsets showed that the cross-reactive immunity was mediated by T cells bearing CD8(+) and T-cell receptor (TCR) alpha/beta and that the Vbeta1 subset of TCR alpha/beta T cells had a dominant role in protective immunity. The protective immunity induced by infection with H9N2 virus declined with time, lasting as long as 100 days after immunization. Shedding of A/Goose/Guangdong/1/96-like H5N1 virus by immunized chickens also increased with the passage of time and thus may play a role in the perpetuation and spread of these highly pathogenic H5N1 influenza viruses. Our findings indicate that pulmonary cellular immunity may be very important in protecting na?ve natural hosts against lethal influenza viruses.     

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.     

Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.     

Standard Trivalent Influenza Virus Protein Vaccination Does Not Prime Antibody-Dependent Cellular Cytotoxicity in Macaques

Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011–2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.     

H5N1重组禽流感病毒样颗粒免疫原性研究

目的对构建的H5N1重组禽流感病毒样颗粒(VLPs)进行初步免疫原性探讨,并与H5N1全病毒灭活疫苗(WIV)进行体液免疫和细胞免疫的比较。方法在0周和3周分别以纯化H5N1重组禽流感病毒样颗粒、H5N1全病毒灭活疫苗及pH7.2 PBS腿部肌肉注射BALB/c小鼠,于不同时间收集血清,以血凝抑制试验(HI)和血清IgG抗体酶联免疫吸附试验(ELISA)评估体液免疫,CD4+、CD8+T细胞亚群及酶联免疫斑点试验(ELISPOT)评估细胞免疫,并以同型毒株滴鼻攻击,观察小鼠存活率。结果病毒样颗粒各组和全病毒灭活疫苗免疫后小鼠血清ELISA IgG效价均有升高;中和抗体效价除病毒样颗粒120 ng/只免疫剂量外其他免疫小鼠HI效价均达1︰40;小鼠脾CD4+T淋巴细胞亚群分类:全病毒灭活疫苗组(600μg/只)为36.56%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为26.58%,32.20%,29.25%;PBS组为26.65%;CD8+T淋巴细胞亚群分类:全病毒灭活疫苗组(600 ng/只)为10.78%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为1 3.53%,14.24%,1 3.35%;PBS组为10.69%。ELISPOT试验统计学数据显示,病毒样颗粒和全病毒灭活疫苗的小鼠脾单个核细胞分泌IFN-γ细胞与PBS组有显著性差异;小鼠保护性试验结果显示,除病毒样颗粒120 ng/只免疫剂量小鼠的存活率为87.5%外,其他病毒样颗粒实验组小鼠均为100%,PBS对照组为12.5%。结论 H5N1重组禽流感病毒样颗粒能诱导体液免疫和细胞免疫,并能抵御同型病毒株的攻击,可作为H5N1人用禽流感的候选疫苗。     

Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8–12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.