Induction of Cross-Clade Antibody and T-Cell Responses by a Modified Vaccinia Virus Ankara-Based Influenza A(H5N1) Vaccine in a Randomized Phase 1/2a Clinical Trial.

Background: High-pathogenicity avian influenza viruses continue to circulate in poultry and wild birds and occasionally infect humans, sometimes with fatal outcomes. Development of vaccines is a priority to prepare for potential pandemics but is complicated by antigenic variation of the surface glycoprotein hemagglutinin. We report the immunological profile induced by human immunization with modified vaccinia virus Ankara (MVA) expressing the hemagglutinin gene of influenza A(H5N1) virus A/Vietnam/1194/04 (rMVA-H5). Methods: In a double-blinded phase 1/2a clinical trial, 79 individuals received 1 or 2 injections of rMVA-H5 or vector control. Twenty-seven study subjects received a booster immunization after 1 year. The breadth, magnitude, and properties of vaccine-induced antibody and T-cell responses were characterized. Results: rMVA-H5 induced broadly reactive antibody responses, demonstrated by protein microarray, hemagglutination inhibition, virus neutralization, and antibody-dependent cellular cytotoxicity assays. Antibodies cross-reacted with antigenically distinct H5 viruses, including the recently emerged subtypes H5N6 and H5N8 and the currently circulating subtype H5N1. In addition, the induction of T cells specific for H5 viruses of 2 different clades was demonstrated. Conclusions: rMVA-H5 induced immune responses that cross-reacted with H5 viruses of various clades. These findings validate rMVA-H5 as vaccine candidate against antigenically distinct H5 viruses. Clinical Trials Registration: NTR3401.

Effects of pre-existing orthopoxvirus-specific immunity on the performance of Modified Vaccinia virus Ankara-based influenza vaccines.

The replication-deficient orthopoxvirus modified vaccinia virus Ankara (MVA) is a promising vaccine vector against various pathogens and has an excellent safety record. However, pre-existing vector-specific immunity is frequently suggested to be a drawback of MVA-based vaccines. To address this issue, mice were vaccinated with MVA-based influenza vaccines in the presence or absence of orthopoxvirus-specific immunity. Importantly, protective efficacy of an MVA-based influenza vaccine against a homologous challenge was not impaired in the presence of orthopoxvirus-specific pre-existing immunity. Nonetheless, orthopoxvirus-specific pre-existing immunity reduced the induction of antigen-specific antibodies under specific conditions and completely prevented induction of antigen-specific T cell responses by rMVA-based vaccination. Notably, antibodies induced by vaccinia virus vaccination, both in mice and humans, were not capable of neutralizing MVA. Thus, when using rMVA-based vaccines it is important to consider the main correlate of protection induced by the vaccine, the vaccine dose and the orthopoxvirus immune status of vaccine recipients.

Increased Protein Degradation Improves Influenza Virus Nucleoprotein-Specific CD8+ T Cell Activation In Vitro but Not in C57BL/6 Mice.

Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in the case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long-lasting immunity are preferred. Because influenza virus-specific CD8+ T cells are directed mainly against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8+ T cells. To optimize the induction of CD8+ T cell responses, we made several modifications to NP, aiming at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve the induction of CD8+ T cell responses. We showed that NP with increased degradation rates improved CD8+ T cell activation in vitro if the amount of antigen was limited or if CD8+ T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and wild-type NP (NPwt), since NPwt already induced optimal CD8+ T cell responses. IMPORTANCE: Due to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8+ T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8+ T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the modified vaccinia virus Ankara (MVA) vaccine vector. These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on major histocompatibility complex (MHC) class I molecules to CD8+ T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less-immunogenic proteins.

Heterosubtypic immunity to H7N9 influenza virus in isogenic guinea pigs after infection with pandemic H1N1 virus.

Heterosubtypic immunity is defined as immune-mediated (partial) protection against an influenza virus induced by an influenza virus of another subtype to which the host has not previously been exposed. This cross-protective effect has not yet been demonstrated to the newly emerging avian influenza A viruses of the H7N9 subtype. Here, we assessed the induction of protective immunity to these viruses by infection with A(H1N1)pdm09 virus in a newly developed guinea pig model. To this end, ten female 12-16 week old strain 2 guinea pigs were inoculated intratracheally with either A(H1N1)pdm09 influenza virus or PBS (unprimed controls) followed 4 weeks later with an A/H7N9 influenza virus challenge. Nasal swabs were taken daily and animals from both groups were sacrificed on days 2 and 7 post inoculation (p.i.) with A/H7N9 virus and full necropsies were performed. Nasal virus excretion persisted until day 7 in unprimed control animals, whereas only two out of seven H1N1pdm09-primed animals excreted virus via the nose. Infectious virus was recovered from nasal turbinates, trachea and lung of all animals at day 2 p.i., but titers were lower for H1N1pdm09-primed animals, especially in the nasal turbinates. By day 7 p.i., relatively high virus titers were found in the nasal turbinates of all unprimed control animals but infectious virus was isolated from the nose of only one of four H1N1pdm09-primed animals. Animals of both groups developed inflammation of variable severity in the entire respiratory tract. Viral antigen positive cells were demonstrated in the nasal epithelium of both groups at day 2. The bronchi(oli) and alveoli of unprimed animals showed a moderate to strong positive signal at day 2, whereas H1N1pdm09-primed animals showed only minimal positivity. By day 7, only viral antigen positive cells were found after H7N9 virus infection in the nasal turbinates and the lungs of unprimed controls. Thus infection with H1N1pdm09 virus induced partially protective heterosubtypic immunity to H7N9 virus in (isogenic) guinea pigs that could not be attributed to cross-reactive virus neutralizing antibodies.

Virus replication kinetics and pathogenesis of infection with H7N9 influenza virus in isogenic guinea pigs upon intratracheal inoculation.

Since 2013, avian influenza viruses of subtype H7N9 have been transmitted from poultry to humans in China and caused severe disease. Concerns persist over the pandemic potential of this virus and further understanding of immunity and transmission is required. The isogenic guinea pig model uniquely would allow for investigation into both. Eighteen female isogenic guinea pigs 12-16 weeks were inoculated intratracheally with either A/H7N9 virus (n=12) or PBS (n=6) and sacrificed on days 2 and 7 post-inoculation. Nasal and pharyngeal swabs were taken daily to assess viral replication kinetics and necropsies were performed to study pathogenesis. All animals showed peak virus titers in nasal secretions at day 2 post-inoculation and by day 7 post-inoculation infectious virus titers had decreased to just above detectable levels. At day 2, high virus titers were found in nasal turbinates and lungs and moderate titers in trachea and cerebrum. At day 7, infectious virus was detected in the nasal turbinates only. Histology showed moderate to severe inflammation in the entire respiratory tract and immunohistochemistry (IHC) demonstrated large numbers of viral antigen positive cells in the nasal epithelium at day 2 and fewer at day 7 post-inoculation. A moderate number of IHC positive cells was observed in the bronchi(oli) and alveoli at day 2 only. This study indicates that isogenic guinea pigs are a promising model to further study immunity to and transmission of H7N9 influenza virus.

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage.

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

Pathogenesis of infection with 2009 pandemic H1N1 influenza virus in isogenic guinea pigs after intranasal or intratracheal inoculation.

To elucidate the pathogenesis and transmission of influenza virus, the ferret model is typically used. To investigate protective immune responses, the use of inbred mouse strains has proven invaluable. Here, we describe a study with isogenic guinea pigs, which would uniquely combine the advantages of the mouse and ferret models for influenza virus infection. Strain 2 isogenic guinea pigs were inoculated with H1N1pdm09 influenza virus A/Netherlands/602/09 by the intranasal or intratracheal route. Viral replication kinetics were assessed by determining virus titers in nasal swabs and respiratory tissues, which were also used to assess histopathologic changes and the number of infected cells. In all guinea pigs, virus titers peaked in nasal secretions at day 2 after inoculation. Intranasal inoculation resulted in higher virus excretion via the nose and higher virus titers in the nasal turbinates than intratracheal inoculation. After intranasal inoculation, infectious virus was recovered only from nasal epithelium; after intratracheal inoculation, it was recovered also from trachea, lung, and cerebrum. Histopathologic changes corresponded with virus antigen distribution, being largely limited to nasal epithelium for intranasally infected guinea pigs and more widespread in the respiratory tract for intratracheally infected guinea pigs. In summary, isogenic guinea pigs show promise as a model to investigate the role of humoral and cell-mediated immunities to influenza and their effect on virus transmission.

A single immunization with modified vaccinia virus Ankara-based influenza virus H7 vaccine affords protection in the influenza A(H7N9) pneumonia ferret model.

Since the first reports in early 2013, >440 human cases of infection with avian influenza A(H7N9) have been reported including 122 fatalities. After the isolation of the first A(H7N9) viruses, the nucleotide sequences became publically available. Based on the coding sequence of the influenza virus A/Shanghai/2/2013 hemagglutinin gene, a codon-optimized gene was synthesized and cloned into a recombinant modified vaccinia virus Ankara (MVA). This MVA-H7-Sh2 viral vector was used to immunize ferrets and proved to be immunogenic, even after a single immunization. Subsequently, ferrets were challenged with influenza virus A/Anhui/1/2013 via the intratracheal route. Unprotected animals that were mock vaccinated or received empty vector developed interstitial pneumonia characterized by a marked alveolitis, accompanied by loss of appetite, weight loss, and heavy breathing. In contrast, animals vaccinated with MVA-H7-Sh2 were protected from severe disease.

Assessment of the antiviral properties of recombinant surfactant protein D against influenza B virus in vitro.

The armamentarium of antiviral drugs against influenza viruses is limited. Furthermore, influenza viruses emerge that are resistant to existing antiviral drugs like the M2 and NA inhibitors. Therefore, there is an urgent need for the development of novel classes of antiviral drugs. Here we investigated the antiviral properties of recombinant porcine surfactant protein D (RpSP-D), an innate defense molecule with lectin properties, against influenza B viruses. We have previously shown that porcine SP-D has more potent neutralizing activity against influenza A viruses than human SP-D. Here we show that RpSP-D neutralizes influenza B viruses efficiently and inhibited the binding of these viruses to epithelial cells of the human trachea.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

Novel G3/DT adjuvant promotes the induction of protective T cells responses after vaccination with a seasonal trivalent inactivated split-virion influenza vaccine.

Vaccines used against seasonal influenza are poorly effective against influenza A viruses of novel subtypes that may have pandemic potential. Furthermore, pre(pandemic) influenza vaccines are poorly immunogenic, which can be overcome by the use of adjuvants. A limited number of adjuvants has been approved for use in humans, however there is a need for alternative safe and effective adjuvants that can enhance the immunogenicity of influenza vaccines and that promote the induction of broad-protective T cell responses. Here we evaluated a novel nanoparticle, G3, as an adjuvant for a seasonal trivalent inactivated influenza vaccine in a mouse model. The G3 adjuvant was formulated with or without steviol glycosides (DT, for diterpenoid). The use of both formulations enhanced the virus-specific antibody response to all three vaccine strains considerably. The adjuvants were well tolerated without any signs of discomfort. To assess the protective potential of the vaccine-induced immune responses, an antigenically distinct influenza virus strain, A/Puerto Rico/8/34 (A/PR/8/34), was used for challenge infection. The vaccine-induced antibodies did not cross-react with strain A/PR/8/34 in HI and VN assays. However, mice immunized with the G3/DT-adjuvanted vaccine were partially protected against A/PR/8/34 infection, which correlated with the induction of anamnestic virus-specific CD8(+) T cell responses that were not observed with the use of G3 without DT. Both formulations induced maturation of human dendritic cells and promoted antigen presentation to a similar extent. In conclusion, G3/DT is a promising adjuvant formulation that not only potentiates the antibody response induced by influenza vaccines, but also induces T cell immunity which could afford broader protection against antigenically distinct influenza viruses.

Recombinant porcine surfactant protein D inhibits influenza A virus replication ex vivo.

Influenza is a major burden to public health. Due to high mutation rates and selection pressure, mutant viruses emerge which are resistant to currently used antiviral drugs. Therefore, there is a need for the development of novel classes of antiviral drugs that suffer less from the emergence of resistant viruses. Antiviral drugs based on collectin-like surfactant protein D (SP-D) may fulfil these requirements. Especially porcine SP-D displays strong antiviral activity to influenza A viruses. In the present study the antiviral activity of recombinant porcine SP-D was investigated in ex vivo cultures of respiratory tract tissue infected with human influenza A virus of the H3N2 subtype. Porcine SP-D has antiviral activity in these test systems. It is suggested that porcine SP-D may be used as a venue to develop a novel class of antiviral drugs.

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.

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis.

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza.

Infection of the upper respiratory tract with seasonal influenza A(H3N2) virus induces protective immunity in ferrets against infection with A(H1N1)pdm09 virus after intranasal, but not intratracheal, inoculation.

The clinical symptoms caused by infection with influenza A virus vary widely and depend on the strain causing the infection, the dose and route of inoculation, and the presence of preexisting immunity. In most cases, seasonal influenza A viruses cause relatively mild upper respiratory tract disease, while sometimes patients develop an acute severe pneumonia. Heterosubtypic immunity induced by previous infections with influenza A viruses may dampen the development of clinical symptoms caused by infection with influenza A viruses of another subtype, as is the case during influenza pandemics. Here we show that ferrets acquire protective immunity after infection of the upper respiratory tract with a seasonal influenza A(H3N2) virus against subsequent infection with influenza A(H1N1)pdm09 virus inoculated by the intranasal route. However, protective heterosubtypic immunity was afforded locally, since the prior infection with the A(H3N2) virus did not provide protection against the development of pneumonia induced after intratracheal inoculation with the A(H1N1)pdm09 virus. Interestingly, some of these animals developed more severe disease than that observed in naïve control animals. These findings are of interest in light of the development of so-called universal influenza vaccines that aim at the induction of cross-reactive T cell responses.

Binding of DC-SIGN to the hemagglutinin of influenza A viruses supports virus replication in DC-SIGN expressing cells.

Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.

Human T-cells directed to seasonal influenza A virus cross-react with 2009 pandemic influenza A (H1N1) and swine-origin triple-reassortant H3N2 influenza viruses.

Virus-specific CD8(+) T-cells contribute to protective immunity against influenza A virus (IAV) infections. As the majority of these cells are directed to conserved viral proteins, they may afford protection against IAVs of various subtypes. The present study assessed the cross-reactivity of human CD8(+) T-lymphocytes, induced by infection with seasonal A (H1N1) or A (H3N2) influenza virus, with 2009 pandemic influenza A (H1N1) virus [A(H1N1)pdm09] and swine-origin triple-reassortant A (H3N2) [A(H3N2)v] viruses that are currently causing an increasing number of human cases in the USA. It was demonstrated that CD8(+) T-cells induced after seasonal IAV infections exerted lytic activity and produced gamma interferon upon in vitro restimulation with A(H1N1)pdm09 and A(H3N2)v influenza A viruses. Furthermore, CD8(+) T-cells directed to A(H1N1)pdm09 virus displayed a high degree of cross-reactivity with A(H3N2)v viruses. It was concluded that cross-reacting T-cells had the potential to afford protective immunity against A(H1N1)pdm09 viruses during the pandemic and offer some degree of protection against infection with A(H3N2)v viruses.

The number and position of N-linked glycosylation sites in the hemagglutinin determine differential recognition of seasonal and 2009 pandemic H1N1 influenza virus by porcine surfactant protein D.

C-type lectins are important molecules of the innate immune system. These molecules, like surfactant protein D (SP-D) can recognize glycans on pathogens and neutralize these. Also influenza viruses are recognized by SP-D and their susceptibility to neutralization by SP-D is dependent on the number of N-linked glycosylation sites in the hemagglutinin in particular. Porcine SP-D displayed stronger neutralizing activity to human influenza A viruses than to swine influenza A viruses. Although viruses from these species differ with regard to the number of glycosylation sites in the hemagglutinin, the mechanism underlying the differential recognition by porcine SP-D is poorly understood. Here we investigated the molecular basis for the differential recognition of a seasonal H1N1 and a 2009 pandemic H1N1 virus by porcine SP-D. We demonstrated that the number and position of glycosylation sites determine viral susceptibility to the neutralizing activity of porcine SP-D. However, predicting the effect remains difficult as it was shown to be dependent on the strain and the position of the glycosylation sites.

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.

Cross-protective immunity against influenza pH1N1 2009 viruses induced by seasonal influenza A (H3N2) virus is mediated by virus-specific T-cells.

Influenza A (H1N1) viruses of swine origin were introduced into the human population in 2009 and caused a pandemic. The disease burden in the elderly was relatively low, which was attributed to the presence of cross-reacting serum antibodies in this age group, which were raised against seasonal influenza A (H1N1) viruses that circulated before 1957. It has also been described how infection with heterosubtypic influenza viruses can induce some degree of protection against infection by a novel strain of influenza virus. Here, we assess the extent of protective immunity against infection with the 2009 influenza A (H1N1) pandemic influenza virus that is afforded by infection with a seasonal influenza A (H3N2) virus in mice. Mice that experienced a primary A (H3N2) influenza virus infection displayed reduced weight loss after challenge infection and cleared the 2009 influenza A (H1N1) virus infection more rapidly. To elucidate the correlates of protection of this heterosubtypic immunity to pandemic H1N1 virus infection, adoptive transfer experiments were carried out by using selected post-infection lymphocyte populations. Virus-specific CD8(+) T-cells in concert with CD4(+) T-cells were responsible for the observed protection. These findings may not only provide an explanation for epidemiological differences in the incidence of severe pandemic H1N1 infections, they also indicate that the induction of cross-reactive virus-specific CD8(+) and CD4(+) T-cell responses may be a suitable approach for the development of universal influenza vaccines.