CD8+ T cell immunity to 2009 pandemic and seasonal H1N1 influenza viruses
Introduction
Since April 2009, pandemic H1N1 (pH1N1) virus has caused over 59 million infections worldwide and led to 0.4% of the hospitalizations during that period (www.cdc.gov/flu/weeklyarchives). Of the hospitalized cases reported, up to 46% of patients required intensive care unit-admittance. The estimated mortality rate was up to 46% in intensive care unit-admitted patients [1]. The virus attack rate was substantially higher than that of seasonal flu, based on morbidity and mortality estimators [2], [3], [4], but the pH1N1 mortality rate considering both hospitalized and non-hospitalized was similar to seasonal influenza at <0.5% [5].
The patterns of susceptibility and morbidity seen with pH1N1 infections observed early in the pandemic suggested that subjects previously exposed to antigenically similar influenza strains or vaccine had some immunity [1], [6]. Hancock et al. [6] measured the cross-protective antibody titers in subjects and found titers of >1:80 in 34% of subjects born prior to 1950. In contrast, fewer than 5% of those born after 1980 had protective antibody titers, which was reflected in their greater susceptibility to infection. Although young subjects were more susceptible, additional factors are thought to have contributed to disease attenuation in this population.
Epidemiologic studies clearly indicate that anti-influenza serum antibody levels provide important cross-protection from similar influenza virus clades and the prevention of secondary influenza infections from identical virus [6], [7], [8], [9], [10], [11], [12]. Humans have a natural library of highly specific protective antibodies formed in response to natural infection and vaccination. Pandemic influenza outbreaks can occur when genetic shifts in the virus lead to changes in hemagglutinin (HA) and neuraminidase (NA) on the virus surface; under these conditions, patients have no or poor cross-protective anti-influenza antibodies. A rapid response of the immune system may depend on conserved viral T cell epitopes recognized by cross-reactive T cells.
The majority of known influenza epitopes recognized through Class I MHC complexes by CD8+ T cells are derived from internal virus proteins [13], [14]. In the case of pH1N1, there is >80% amino acid conservation of internal proteins compared to seasonal influenza, but only 30–50% amino acid conservation of HA and NA [15]. A robust circulating memory CD8+ T cell response is expected to occur with prior antigen exposure. The H1N1 pandemic provides an excellent opportunity to study the role of memory CD8+ T cells in healthy adults to cross-react to a strain of influenza that is essentially novel to the individual in terms of serum neutralizing antibodies. Furthermore, it is vital to assess not only cross-reactivity, but also functional capacity of circulating CD8+ T cells. Subjects who have not been infected with influenza within the last three years might be expected to harbor T cells that are more quiescent and exhibit a limited array of cytokines when initially re-activated in vitro.
Based on the high degree of internal epitope conservation in pH1N1, as compared to seasonal influenza [14], [16], [17], [18], we hypothesized that subjects presumed naïve to pH1N1 would nonetheless have a measurable CD8+ T cell response as a result of previous priming with seasonal influenza strains. Furthermore, we hypothesized that the quality of CD8+ T cell antigen-specific cytokine response in subjects not recently infected with influenza would exhibit very few multiple-cytokine secreting (polyfunctional) cells. To test these hypotheses, we chose an experimental approach utilizing whole virus for in vitro stimulation of peripheral blood mononuclear cells (PBMCs) drawn from seronegative subjects, or collected prior to emergence of pH1N1. Our results show evidence of existing CD8+ T cell immunity to pH1N1 that is characterized by predominantly single and dual cytokine producing cells.
Section snippets
Samples
Normal healthy donors had unit bags of blood collected in the New York Influenza Center of Excellence (NYICE) Vaccine Research Unit from October 2008 to October 2009 (Table 1). Approval for human sample collection was obtained from The University of Rochester Institutional Review Board. All donors were consented for sample donation with a brief questionnaire, and procedures were consistent with the NYICE Healthy Donor Protocol #07-0090. PBMCs were isolated by ficoll-paque density gradient
Study cohort
Subject samples were collected from NYICE healthy donors between October 2008 and August 2009. Subject ages ranged from 19 to 49 years. Since pandemic influenza activity did not become locally widespread until after July 2009, samples collected from October 2008 and March 2009 were presumed to be naïve to the pH1N1 virus. Subjects reported no pH1N1 vaccination or influenza-like illness during the 2008–2009 season. A subset of samples collected between April and August of 2009 were tested by
Discussion
We set out to test the prediction that adults not known to have been exposed to the pH1N1 influenza virus would have memory CD8+ T cells that cross-react to the novel strain as well as to recent and prototypic influenza strains. Our results support the hypothesis that CD8+ T cells previously primed with multiple strains of seasonal influenza virus are activated when stimulated with pandemic influenza virus. Single cytokine producing cells were by far the predominant type of CD8+ T cells
Conclusion
Our study found evidence for existing CD8+ T cell immunity to pandemic strain H1N1 influenza virus in presumed naïve subjects, which was characterized by predominantly single-cytokine producers, then dual IFNγ+ TNFα+ cells, and very low frequencies of triple-cytokine secreting cells. The most active IFNγ-secreting populations were found in association with IL2 production, as measured by mean fluorescence intensity. These cross-reactive CD8+ T cells may have contributed to disease attenuation in
Acknowledgements
We would like to thank the healthy donor volunteers for their generous donation of time and cells, and the University of Rochester Vaccine Research Unit and the University of Rochester Retrovirology and Processing Lab for specimen processing. We are grateful to the NIH Biodefense and Emerging Infections Research Resources Repository for supplying the seed stock for A/Ca/04/2009 virus, and to Andrea Sant for providing A/New Caledonia/20/99 virus. We also thank Dr. Gloria Pryhuber for her
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