Background
In April 2009, an outbreak of H1N1 influenza initially hit Mexico and rapidly spread to other countries and regions of the world. On June 11, 2009, World Health Organization (WHO) declared the first state of emergency about the influenza pandemic in 21
st century [
1]. Historically, there were four worldwide pandemics of influenza which had caused millions of death. The first influenza pandemic in 1918 which caused by H1N1 strain killed 40 to 50 million people [
2]. In 1957, the prevalent strain of influenza virus H1N1 in human abruptly disappeared and replaced by a new reassortant of influenza virus H2N2, which contained three new segments from the avian source and maintained the other five segments from the H1N1 strain of 1918 lineage [
3]. In 1968, the circulating influenza virus H2N2 subtype transformed to H3N2 subtype by reassortment of the novel hemagglutinin (HA) and polymerase PB1 segments [
4]. In January 1976, a novel virus subtype, A/New Jersey/76 H1N1, was identified in an outbreak of respiratory disease occurred among soldiers returning to an Army base in Fort Dix, New Jersey. However, this virus subtype did not escape from the base [
5]. In 1977, separate emergence of another H1N1 virus successfully propagated [
6], and then co-circulated with 1968 H3N2 subtype in human population globally. The 2009 pandemic H1N1 virus (H1N1pdm) was antigenically similar to pre-1950 influenza strains [
7] and A/New Jersey/76 H1N1 strain [
8].
The prevalence of pre-existing memory B cell against 2009 H1N1pdm in human population has been rarely evaluated. The ELISpot assay had been developed to count antigen-specific memory B cells in human blood by Crotty et al. [
9]. These detected antigen specific memory B cells satisfied the canonical surface phenotype of human memory B cells: CD19
+CD20
+Ig
+CD27
+ [
9]. This method had been widely used to assess the immunological memory of B cells response to various infectious diseases [
10].
As estimated in China and other countries, overall age-standardized H1N1pdm cumulative incidence varied significantly by age with the highest in children 5–19 and 0–4 years old [
11]. This age distribution was different from the seasonal influenza which mostly infected the elderly population [
12]. In the present study, we evaluated neutralization antibodies against H1N1pdm as well as recent circulating seasonal H1N1 viruses in serum samples collected before and after the pandemic. The samples were further resorted by birth year to estimate the age-specific H1N1pdm infection. In addition, we examined H1N1pdm and seasonal H1N1 specific memory B and IFN-γ
+ T cells frequencies using ELISpot assay in healthy individuals, whose blood had been collected in 2006, and aimed to evaluate the potential connections of pre-existing cellular immunities and age-dependent H1N1pdm influenza infections.
Discussion
Compared with other countries [
2,
18‐
21], elderly people in China had a relatively low baseline of antibody production responding to the 2009 H1N1pdm [
22,
23]. In a serosurvey of large samples (n = 2379), only 2.0% elder Chinese people had pre-existing antibodies responding to H1N1pdm virus [
22]. In the present study, our data further demonstrated a relatively low frequency of pre-existing H1N1pdm virus-specific MBCs in the elderly population in China. It was not fully understood why Chinese elder people had a lower infection rate than the youth during the 2009 H1N1pdm.
The pre-existing CD8
+ T lymphocytes (CTL) memory can mediate heterologous immunity among different subtype influenza A viruses [
24]. Although recent seasonal influenza induced little cross-reactive antibody production against H1N1pdm virus [
21], cellular responses might provide immune protection by targeting invariable or cross-reactive epitopes. A total of 49% of the epitopes in recent seasonal H1N1 were found totally conserved in H1N1pdm [
25], furthermore, CD8
+ T cells specific for conserved epitopes could lyse pandemic influenza infected cells in vitro [
26]. One research indicated that H1N1pdm virus shared immunogenic peptides with the catastrophic 1918 H1N1 strain as well as viruses circulating prior to 1945 [
27]. Cross-reactive CTL immunity between the H1N1pdm strain and the 1918 Spanish H1N1 strain might be related with the lower susceptibility to 2009 H1N1pdm in people over 65 years of age [
27]. Another recent study suggested that high susceptibility of children to the pandemic H2N2 in 1957 might be more closely linked to the number of influenza exposures [
28].
Our study indicated that live influenza virus (both pandemic and seasonal influenza) stimulated poor responses of IFN-γ
+ T cells in the older population, consisting with the other studies using peptide pools stimulation [
29]. We supposed the reduced T cell responses in the older group might be due to aging of the immune system and reduced responsiveness. Aging has a significant impact on CTL responses in murine model [
30]. Not only naïve epitope-specific CD8
+ T cells decline with age [
31], but also TCR repertoire diversity decreases with age [
32]. All these reduced T cell function in the older individuals might lead to underestimated response of magnitude in IFN-γ
+ ELISpot assay. It seemed that early priming of CTL response prior to aging was the key for establishment of long-lasting and protective immunity.
In our IFN-γ
+ ELISpot assay, we used the live influenza virus. It was important to understand the responds of IFN-γ
+ T cells to live virus, because these cells were presumably reacting to processed virus from within infected antigen presenting cells. With the use of live virus, adequate epitopes through the natural infection of APCs for the activation of virus specific IFN-γ
+ T cells could be generated [
29,
33,
34]. However, it was difficult to directly assess the HA specific humoral and cellular immunity responses when using live virus to stimulate APCs. Virus activated IFN-γ
+ T cells could produce kinds of pro-inflammatory cytokines or directly clear virus from the infected cells, thus benefit to the rapid recovery from influenza infection [
35]. The influenza virus specific IFN-γ
+ T cells responses were mainly initiated by conserved antigens (such as nucleoprotein and M protein) and therefore the ELISpot assay could detect the influenza virus with highly diverse subtypes [
36].
Pre-existing sero-antibodies and memory B cells against H1N1pdm virus were low across all studied population. Although seropositives of H1N1pdm virus was higher in people over 60 years than in ones under 60 years, the difference is too small (7.8 vs 4.9%) to deduce one solid conclusion. Moreover, the GMT titers were not significantly different between two age groups. In contrast, sero-antibodies against seasonal influenza H1N1 were higher in the elder than in the younger people. The high seroantibody responses in the elderly might be the results of high infection rate in this susceptible population during the seasonal influenza season in Northern China [
37]. However, seasonal HA specific MBC was not high in the older people. We proposed that an impaired memory B cell response in the elderly as observed in vaccination subjects might be one of the plausibility [
38]. However the real mechanism might be much more complex. We observed that even with low numbers of seasonal H1N1-specific MBCs elderly individuals maintained higher serum antibody levels than younger people and theses were boosted after the pandemic wave. But this was not the case for pdmH1N1 where there were low padmH1N1-specific MBCs and low serum antibody levels. The pattern of repeated exposure to seasonal strains in population might be expected as one of the explanations. However, it could not fully explain the discrepant memory responses between seasonal and pandemic influenza H1N1.
There were several limitations in the present study. Firstly, the serum samples of pre and post-pandemic were not well paired, and the vaccination history and health status of participants were not identified. These might yield biased prevalence of H1N1pdm in different age groups. Secondly, the blood samples used for the serological assays and cell immunity assays were not well paired, and the number of samples used for cell immunity assay was not enough to get the statistical power. Thirdly, the lack of additional experiments to better characterized the memory T or B cell responses (such as CD45RA, CD45RO, CD4+IFN-γ+, CD8+ IFN-γ+), cytokine profile under stimulation with influenza peptides, and so on, due to the limited accessible samples, made further evaluation of immune response impossible.
Acknowledgements
The authors thank Dr. Yue Long Shu (Chinese National Influenza Center), Prof. Shane Crotty (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and Dr. Adam Meijer (National Institute for Public Health and the Environment (RIVM), the Netherlands) for providing experimental reagents. We thank Chris Li (University of Oxford) for technical support.