1976 and 2009 H1N1 influenza virus vaccines boost anti-hemagglutinin stalk antibodies in humans

Abstract

Background: Infection with pandemic H1N1 influenza A viruses (IAVs) containing hemagglutinin (HA) proteins with globular heads that differ substantially from seasonal strains results in a boost in broadly cross-reactive antibodies that bind to the HA stalk. Boosting these antibodies has become an attractive strategy for creating a universal IAV vaccine. Therefore, it was essential to determine whether vaccines containing H1N1 viruses whose head domains differ substantially compared to seasonal strains could also achieve this boost.

Methods: Prospective samples of subjects who had received the A/New Jersey/1976 (NJ/76) vaccine and healthy, age-matched controls were assessed for the presence of anti-HA stalk antibodies before and after receiving the A/California/04/2009 (Cal/09) vaccine between October 2009 and January 2010.

Results: Individuals who received either the NJ/76 vaccine or the Cal/09 vaccine experienced a robust boost in HA stalk-reactive, neutralizing antibodies similar to what has been observed in individuals infected with Cal/09.

Conclusions: These results demonstrate that vaccines containing viruses whose HA head domains that differ substantially from seasonal strains are capable of boosting titers of HA stalk antibodies. Furthermore, anti-HA stalk antibodies elicited by vaccination appear to be long-lived and therefore could be targeted for the generation of a universal IAV vaccine.

Figure 1.

NJ/76 vaccine recipients had elevated anti-hemagglutinin (HA) stalk antibodies prior to Cal/09 vaccination. Serial dilutions of serum from NJ/76 vaccinees (n = 20) or age-matched control subjects (n = 15) were tested for their reactivity to (A) cH6/1 HA or (B) NC/99 HA by ELISA and immunoglobulin G (IgG) endpoint titers where calculated. Due to limited quantities of available serum, pre-Cal/09 vaccination IgG endpoint titers were also determined for pooled NJ/76 vaccinees (N = 5) and control subjects (n = 7) against (C) cH6/1 and (D) NC/99. Each pool consisted of all individuals from each group for whom both pre- and post-Cal/09 vaccination samples were available. Horizontal lines indicate the geometic mean titer. Endpoint titer limit of detection = 100. Unpaired Student t-tests were performed and 2-tailed P values < .05 were considered statistically significant. Abbreviations: ND, not detected; NS, not significant. *statistically significant.

Figure 2.

NJ/76 vaccine recipients had elevated hemagglutination inhibition (HAI) titers against Cal/09 prior to Cal/09 vaccination. (A) HAI titers were determined for pre-Cal/09 vaccination pooled serum samples from NJ/76 vaccinees (n = 5) and control subjects (n = 7) against Cal/09 and France/76 using chicken red blood cells. (B) HAI assays against Cal/09 were also performed using serum samples corresponding to the individual subjects from within each pool in order to ensure that the pooled results were representative of the group as a whole. (C) Pre-Cal/09 vaccination serum was pooled from all control subjects and NJ/76 vaccines and was used to perform an HAI assay against NJ/76. Horizontal lines indicate the geometic mean titer. HAI limit of detection = 20. Unpaired Student t-tests were performed, and 2-tailed P values < .05 were considered statistically significant. Abbreviation: ND, not detected. *statistically significant.

Figure 3.

NJ/76 and Cal/09 vaccines boosted broadly neutralizing antibodies. Microneutralization assays were performed on MDCK against (A) cH5/1 N3 and (B) Cal/09 virus using TPCK-trypsin-treated, pooled serum samples collected from NJ/76 vaccinees (n = 5) and control subjects (n = 7) before and after Cal/09 vaccination. Following infection, cells were stained with an anti-NP antibody and an HRP-conjugated secondary antibody. Neutralization titers were defined as the lowest serum dilution that resulted in at least 50% reduction in specific signal. Microneutralization assay limit of detection = 30.