Heterosubtype neutralizing responses to influenza A (H5N1) viruses are mediated by antibodies to virus haemagglutinin

Abstract

Background: It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA) or haemagglutinin (HA). We use pseudoviral particles (H5pp) coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin.

Methodology/principal findings: We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN) and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer > or = 20) H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients.

Conclusions/significance: Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.

Figure 1. Comparison of H5 neutralizing antibodies tested by MN and H5pp tests for different age groups.

Titers were measured from paired sera of 98 children (A, B), 20 adults (C, D) and 118 elderly (E, F) given seasonal influenza vaccination; (G, H) 42 pairs sera from adults recipient of H5N1 clinical candidate vaccines. Dashed line marks the positivity criteria (titer≥20). Each line represents a paired serum from one individual. Where more than one person has identical pre and post antibody titers, the number of persons with these overlapping results is denoted as n. The MN titers were determined at HKU using the MN-HKU method (see methods).

Figure 2. Comparison of titers obtained with H5pp versus MN assays for two panels of paired sera.

Titers (reciprocal dilution of serum that gives 50% neutralization) were determined for sera from elderly (>65 yr) recipient of H5N1 vaccine selected to include 16 with pre-vaccination MN H5N1 titer ≥20 and 10 randomly selected seronegative persons with MN titer <20 and HI <8. The post vaccination was sampled 21 days post vaccination. The graph (A) shows the correlation of antibody titers in pre- (circles) and post (square) vaccination sera obtained with the H5 MN (sanofi pasteur method) and H5pp tests for those who were MN seropositive (black markers) and seronegative (open markers) pre-vaccination. Graph (B) shows the pre- and post H5N1 vaccination H5pp antibody titers in those with (“seropositive”) and without (“seronegative”) pre-vaccine antibody. For computation, the sera with undetectable antibody titers were given the value of 10. Positivity criterion (titer≥20) is represented by dashed lines.