| Abstract: | The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.The emergence of novel influenza virus strains poses a continuous public health threat (1, 2). The World Health Organization estimates that influenza viruses infect one-billion people annually, with three- to five-million cases of severe illness, and up to 500,000 deaths worldwide (3). Following influenza virus infection, humoral immune responses against the viral hemagglutinin (HA) protein may persist for decades in humans (4). These anti-HA responses correlate strongly with protection against influenza infection (5). Serological memory is maintained by antibody-secreting long-lived plasma cells and reinforced by memory B cells, which can rapidly differentiate into antibody-secreting cells upon antigen reexposure (6).Influenza vaccine efficacy is constantly undermined by antigenic variation in the circulating viral strains, particularly in the HA and neuraminidase (NA) proteins. Current influenza vaccination strategies rely on changing the HA and NA components of the annual human vaccine to ensure that they antigenically match circulating influenza strains (7, 8). Developing an influenza vaccine that is capable of providing broad and long-lasting protective antibody responses remains the central challenge for influenza virus research.HA is a trimer, with each monomer comprised of two subunits: HA1, which includes the HA globular head, and HA2, whose ectodomain together with the N- and C-terminal parts of HA1 constitute the HA stem region (9). Phylogenetically, the 18 HA subtypes characterized so far are divided into two groups. Among strains that have recently caused disease in humans, H1 and H5 HAs belong to group 1, whereas H3 and H7 HAs belong to group 2 (10). Conventional anti-HA neutralizing antibodies primarily target a few immunodominant epitopes located in proximity to the receptor-binding domain within the globular head region of the molecule (11, 12). Although these antibodies are potentially protective, they are strain-specific because of the high variability of such epitopes, and thus lack, in general, the much-desired broad neutralizing activity. Recently, broadly neutralizing human (13–18) and murine (19) monoclonal antibodies (mAbs) directed against distinct epitopes within the HA stem region have been extensively characterized. These mAbs were shown to interfere with the influenza viruses’ life cycle in different ways (20). By generating monoclonal antibodies from plasmablasts isolated ex vivo, we demonstrated that these broadly neutralizing antibodies could be retrieved from patients infected with or vaccinated against the pandemic H1N1 2009 influenza virus (18, 21). Recent observations that HA stem epitopes are accessible on the majority of HA trimers on intact virions (22), and that a stable HA stem protein that is immunologically intact could be produced (23), provided further hope for the feasibility of a stem-based universal influenza vaccine (24).Notably, HA stem-specific mAbs isolated from humans showed a high degree of affinity maturation, suggesting a memory B-cell origin. These results raised two important questions that we address in the current study. First, what are the baseline levels of broadly cross-reactive stem-binding antibodies and memory B cells? Second, using current influenza vaccines, to what extent can HA stem-specific responses be boosted in comparison with those directed against the HA globular head?Structural studies have clearly demonstrated that the main neutralizing antibody epitopes within the HA stem region are conformation-dependent, and that the integrity of these epitopes requires the presence of the HA1 subunit in addition to the HA2 subunit, which constitute the bulk of the HA stem (16, 17). To be able to directly measure HA stem-reactive antibodies and memory B cells, we used a chimeric HA molecule that expresses the globular head of H9 HA on H1 backbone (25). Our data demonstrate that post-2009 trivalent inactivated vaccines (TIV) induced minimal stem-specific responses in comparison with head-specific responses. On the other hand, immunization with H5N1 generated relatively strong anti-HA stem responses, demonstrating that it is feasible to elicit broadly neutralizing responses in humans given the right immunogen design. |
