Our findings provide insight into the human B cell responses to a pandemic influenza virus strain. The unique genetic composition of the pandemic H1N1 influenza virus meant that our relatively young cohort probably had little or no preexisting specific antibody-mediated immunity to this virus before infection (Brockwell-Staats et al., 2009
; Dawood et al., 2009
; Garten et al., 2009
; Hancock et al., 2009
). Thus, two sources of B cells could have contributed to this response: newly recruited naive B cells and preexisting memory B cells that bound to epitopes conserved between past seasonal strains and the pandemic H1N1 strain. We theorize that predominant activation of the latter, preexisting memory cells can account for the observed high frequency of neutralizing antibodies (11/15 HA-binding antibodies), the majority (9/11) of which are cross-reactive with seasonal H1N1 strains () and other group 1 influenza strains, including H5 HA. Several observations support this conjecture.
Most convincingly, there was a particularly high frequency of cross-reactive antibodies overall, with a high level of somatic mutations found particularly among the variable genes of cross-reacting cells ( and Fig. S3). In fact, by ELISA most antibodies were cross-reactive and one third of the antibodies bound to past annual viral antigens at lower concentrations, suggesting higher avidity to past influenza strains than to the current pandemic H1N1 virus. Further, cross-reacting cells that bind with higher affinity to the pandemic H1N1 strain also have the highest frequency of variable-gene mutations (Fig. S3 B). Antibodies that were broadly cross-reactive were among the more highly mutated clones (Fig. S3 B). We propose that many of these cells were specific for cross-reactive epitopes present in annual influenza strains that then underwent further affinity maturation and adaptation to the infecting pandemic H1N1 virus. Supporting this conjecture, Corti et al. (2010)
first demonstrated that naturally occurring HA stalk–reactive memory B cells could be isolated from the blood of people recently immunized with the annual vaccine, before the outbreak of pandemic H1N1. The nature of that study was to screen EBV-transformed memory cell lines, thus precluding the determination of precise frequencies of these stalk-reactive B cells. However, these antibodies were estimated to be quite rare; occurring at one in thousands to one in hundreds of influenza-binding B cells, varying by individual. In contrast, we show that plasmablasts activated by infection with the highly novel pandemic H1N1 influenza strain have substantially increased targeting to the HA stalk region epitopes, totaling 10% of all influenza-specific antibodies and half of the neutralizing antibodies (). In fact, most specific antibodies isolated in this study were cross-reactive to past influenza strains. Collectively, the data described supports a model in which divergent viruses that are conserved only at the most critical regions for function will elicit a higher proportion of cross-reactive and neutralizing antibodies. Thus, although the activated plasmablasts of relatively few patients could be analyzed in detail at the monoclonal antibody level, we proffer that with the proper immunogen, the long-sought development of a pan-influenza vaccine might be possible.
Interestingly, the highly specific antibody EM-4C04 was derived from a patient that had a very severe disease course, with persistent viral shedding over several weeks. In addition, the variable genes from the plasmablasts of this patient had the lowest average number of somatic mutations (, outlier, and Fig. S3 B). Collectively, the unique specificity against pandemic H1N1, the low levels of somatic mutation, and the unusually severe disease in the absence of predisposing conditions suggest that this patient may have mounted a primary immune response to the pandemic H1N1 influenza infection. The complete lack of preexisting immunity may have contributed to the more severe disease observed in this patient. In contrast, the activation of broadly cross-neutralizing memory B cells in those with immune experience to annual strains might have contributed to the less severe disease of most infected patients during the pandemic.
It is notable that there is a discrepancy between patients for serum MN titers, the severity of disease, and the frequency of plasmablasts expressing neutralizing antibodies ( and ). For example, patient EM, despite having the worst disease course, had the greatest HAI and MN serum titers. This may be caused by the time from infection (day 31), allowing full seroconversion, or by the presence of highly potent antibodies, such as EM-4C04, whose activities were less likely to titer out. The highly specific nature of the response from this patient may have contributed to this advantage, ultimately better targeting the epitopes of the pandemic H1N1 strain. In contrast, patient 1009 had relatively low HAI and MN serum titers but the highest frequency of broadly neutralizing antibodies and a less severe disease course. One possibility is that our sampling from this patient was done before peak serological responses. Another possibility is that the high frequency of these potent antibodies in the memory B cell compartment may have resulted in rapid resolution of infection, precluding the development of a high serological response. A third possibility is that despite broader protection, the stalk-reactive antibodies are on the whole less potent and more rapidly titrated out than the highly specific antibodies to the HA globular head. These various possibilities will be of significant interest to study in the future.
Finally, we report the development of a large panel of human mAbs induced by pandemic H1N1 infection. Prophylactic therapy with polyclonal or mAbs has successfully been used for RSV, rabies, Hepatitis A and B, and varicella. In the case of influenza, mAbs have been shown to provide prophylactic or therapeutic protection in mice and other animal models (Reuman et al., 1983
; Sweet et al., 1987
; Palladino et al., 1995
; Renegar et al., 2004
). Passive transfer of maternal antibodies in humans has also been shown to confer protection (Puck et al., 1980
). Several of the antibodies we isolated have broad neutralization capacity in vitro against divergent influenza strains and show potent prophylactic and therapeutic activity when used to treat mice that were lethally infected with influenza. These antibodies could provide much needed pandemic therapeutics to treat severe cases of influenza and to protect high-risk populations.
In conclusion, analyses of the 46 mAbs induced by pandemic H1N1 infection indicated frequent activation of broadly reactive B cells. We propose that these cells had a memory cell origin caused by cross-reactivity to conserved and functionally important epitopes. If true, it will be important to characterize the efficacy of the pandemic H1N1 vaccine to induce a similarly cross-protective response.