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The stalk of the influenza virus hemagglutinin (HA) is an attractive target for antibody-based universal influenza virus vaccine development. While antibodies that target this part of the virus can be neutralizing, it has been shown in recent years that Fc receptor-mediated effector functions are of significant importance for the protective effect of anti-stalk antibodies. Several assays to measure Fc-Fc receptor interaction-based effector functions like antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis exist, but they suffer from limitations such as low throughput and high run-to-run variability. Reporter assays for antibody-dependent cellular cytotoxicity based on reporter cells that express luciferase upon engagement of human FcγRIIIa with the Fc of antigen-bound antibodies have been developed as well. These reporter assays can be used in a higher throughput setting with limited run-to-run assay variability but since they express only one Fc receptor, their biological relevance is unclear. Here we optimized an antibody-dependent cellular cytotoxicity reporter assay to measure the activity of antibodies to the conserved stalk domain of H1 hemagglutinin. The assay was then correlated to a CD107a-based degranulation assay, and a strong and significant correlation could be observed. This data suggests that the FcγRIIIa-based reporter assay is a good substitute for functional assays, especially in settings where larger sample numbers need to be analyzed.

Influenza viruses remain a severe threat to human health, causing up to 650,000 deaths annually1,2. Seasonal influenza virus vaccines can prevent infection, but are rendered ineffective by antigenic drift. To provide improved protection from infection, novel influenza virus vaccines that target the conserved epitopes of influenza viruses, specifically those in the hemagglutinin stalk and neuraminidase, are currently being developed3. Antibodies against the hemagglutinin stalk confer protection in animal studies4–6. However, no data exist on natural infections in humans, and these antibodies do not show activity in the hemagglutination inhibition assay, the hemagglutination inhibition titer being the current correlate of protection against influenza virus infection7–9. While previous studies have investigated the protective effect of cellular immune responses and neuraminidase-inhibiting antibodies, additional serological correlates of protection from infection could aid the development of broadly protective or universal influenza virus vaccines10–13. To address this gap, we performed a household transmission study to identify alternative correlates of protection from infection and disease in naturally exposed individuals. Using this study, we determined 50% protective titers and levels for hemagglutination inhibition, full-length hemagglutinin, neuraminidase and hemagglutinin stalk-specific antibodies. Further, we found that hemagglutinin stalk antibodies independently correlated with protection from influenza virus infection. Study of influenza virus transmission in humans provides evidence that hemagglutinin stalk-specific antibodies correlate with protection from infection.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

We investigated humoral immune response to influenza A(H1N1)pdm infection and found 32 (22%) of the infected individuals identified by PCR failed to produce a ≥ 4-fold hemagglutinin inhibition assay (HAI) response; a subset of 18 (56%) produced an alternate antibody response (against full-length HA, HA stalk, or neuraminidase). These individuals had lower pre-existing HAI antibody titers and showed a pattern of milder illness. An additional subset of 14 (44%) did not produce an alternate antibody response, had higher pre-existing antibody titers against full-length & stalk HA, and were less sick. These findings demonstrate that some individuals mount an alternate antibody response to influenza infection. In order to design more broadly protective influenza vaccines it may be useful to target these alternate sites. These findings support that there are influenza cases currently being missed by solely implementing HAI assays, resulting in an underestimation of the global burden of influenza infection.

The high variation of the influenza virus hemagglutinin (HA), particularly of its immunodominant head epitopes, makes it necessary to reformulate seasonal influenza virus vaccines every year. Novel influenza virus vaccines that redirect the immune response toward conserved epitopes of the HA stalk domain should afford broad and durable protection. Sequential immunization with chimeric HAs (cHAs) that express the same conserved HA stalk and distinct exotic HA heads has been shown to elicit high levels of broadly cross-reactive Abs. In the current mouse immunization studies, we tested this strategy using inactivated split virion cHA influenza virus vaccines (IIV) without adjuvant or adjuvanted with AS01 or AS03 to measure the impact of adjuvant on the Ab response. The vaccines elicited high levels of cross-reactive Abs that showed activity in an Ab-dependent, cell-mediated cytotoxicity reporter assay and were protective in a mouse viral challenge model after serum transfer. In addition, T cell responses to adjuvanted IIV were compared with responses to a cHA-expressing live attenuated influenza virus vaccine (LAIV). A strong but transient induction of Ag-specific T cells was observed in the spleens of mice vaccinated with LAIV. Interestingly, IIV also induced T cells, which were successfully recalled upon viral challenge. Groups that received AS01-adjuvanted IIV or LAIV 4 wk before the challenge showed the lowest level of viral replication (i.e., the highest level of protection). These studies provide evidence that broadly cross-reactive Abs elicited by cHA vaccination demonstrate Fc-mediated activity. In addition, cHA vaccination induced Ag-specific cellular responses that can contribute to protection upon infection.

Background Influenza causes a substantial burden worldwide, and current seasonal influenza vaccine has suboptimal effectiveness. To develop better, more broadly protective vaccines, a more thorough understanding is needed of how antibodies that target the influenza virus surface antigens, hemagglutinin (HA) (including head and stalk regions) and neuraminidase (NA), impact influenza illness and virus transmission. Methods We used a case-ascertained, community-based study of household influenza virus transmission set in Managua, Nicaragua. Using data from 170 reverse transcriptase–polymerase chain reaction (RT-PCR)–confirmed influenza virus A(H1N1)pdm infections and 45 household members with serologically confirmed infection, we examined the association of pre-existing NA, hemagglutination inhibiting, and HA stalk antibody levels and influenza viral shedding and disease duration using accelerated failure time models. Results Among RT-PCR–confirmed infections in adults, pre-existing anti-NA antibody levels ≥40 were associated with a 69% (95% confidence interval [CI], 34–85%) shortened shedding duration (mean, 1.0 vs 3.2 days). Neuraminidase antibody levels ≥80 were associated with further shortened shedding and significantly shortened symptom duration (influenza-like illness, 82%; 95% CI, 39–95%). Among RT-PCR–confirmed infections in children, hemagglutination inhibition titers ≥1:20 were associated with a 32% (95% CI, 13–47%) shortened shedding duration (mean, 3.9 vs 6.0 days). Conclusions Our results suggest that anti-NA antibodies play a large role in reducing influenza illness duration in adults and may impact transmission, most clearly among adults. Neuraminidase should be considered as an additional target in next-generation influenza virus vaccine development. We found that antibodies against neuraminidase were associated with significantly shortened viral shedding, and among adults they were also associated with shortened symptom duration. These results support neuraminidase as a potential target of next-generation influenza virus vaccines.