Seasonal influenza vaccination is the strongest correlate of cross-reactive antibody responses in migratory bird handlers

Abstract

Avian species are reservoirs of influenza A viruses and could harbor viruses with significant pandemic potential. We examined the antibody and cellular immune responses to influenza A viruses in field or laboratory workers with a spectrum of occupational exposure to avian species for evidence of zoonotic infections. We measured the seroprevalence and T cell responses among 95 individuals with various types and degrees of prior field or laboratory occupational exposure to wild North American avian species using whole blood samples collected in 2010. Plasma samples were tested using endpoint enzyme-linked immunosorbent assay (ELISA) and hemagglutination (HA) inhibition (HAI) assays to subtypes H3, H4, H5, H6, H7, H8, and H12 proteins. Detectable antibodies were found against influenza HA antigens in 77% of individuals, while 65% of individuals tested had measurable T cell responses (gamma interferon [IFN-γ] enzyme-linked immunosorbent spot assay [ELISPOT]) to multiple HA antigens of avian origin. To begin defining the observed antibody specificities, Spearman rank correlation analysis showed that ELISA responses, which measure both head- and stalk-binding antibodies, do not predict HAI reactivities, which measure primarily head-binding antibodies. This result suggests that ELISA titers can report cross-reactivity based on the levels of non-head-binding responses. However, the strongest positive correlate of HA-specific ELISA antibody titers was receipt of seasonal influenza virus vaccination. Occupational exposure was largely uncorrelated with serological measures, with the exception of individuals exposed to poultry, who had higher levels of H7-specific antibodies than non-poultry-exposed individuals. While the cohort had antibody and T cell reactivity to a broad range of influenza viruses, only occupational exposure to poultry was associated with a significant difference in antibody levels to a specific subtype (H7). There was no evidence that T cell assays provided greater specificity for the detection of zoonotic infection. However, influenza vaccination appears to promote cross-reactive antibodies and may provide enhanced protection to novel influenza viruses.

Importance: Annual vaccinations are necessary to ameliorate influenza disease due to drifted viral variants that emerge in the population. Major shifts in the antigenicity of influenza viruses can result in immunologically distinct viruses that can cause more severe disease in humans. Historically, genetic reassortment between avian, swine, or human influenza viruses has caused influenza pandemics in humans several times in the last century. Therefore, it is important to design vaccines to elicit broad protective responses to influenza infections. Because avian influenza viruses have an important role in emerging infections, we tested whether occupational exposure to birds can elicit immune responses to avian influenza viruses in humans. Instead of a specific occupational exposure, the strongest association of enhanced cross-reactive antibody responses was receipt of seasonal influenza vaccination. Therefore, individuals with preexisting immune responses to seasonal human influenza viruses have substantial cross-reactive antibody and T cell responses that may lead to enhanced protection to novel influenza viruses.

FIG 1

Individuals exhibit cross-reactive antibody and cellular responses to multiple HA antigens of avian origin. (a) Endpoint ELISA titers were determined for group 1 avian H5, H6, H8, and H12, group 2 avian H4, and group 2 human H7 hemagglutinin antigens. (b) Heat map representing color-coded antibody titers. K-means clustering analysis revealed 7 distinct clusters of individuals based on the endpoint ELISA titers. Gray, samples not tested.

FIG 2

(a) The frequency distributions of participants with IFN-γ-producing cells as measured by ELISPOT assay to individual HA proteins are shown as spot-forming units (SFU) per million PBMCs. (b) Individual PBMC IFN-γ responses for each HA protein or total response to any HA protein are plotted by ELISA cluster. Frequencies have been corrected for background IFN-γ production in unstimulated control cultures.

FIG 3

Reported seasonal influenza virus vaccination is associated with enriched HA-specific antibody titers. Endpoint ELISA titers were determined for avian H4, H5, H6, H8, and H12 and human H7 hemagglutinin antigens. Seasonal vaccination status indicates whether participants had reported receipt of any seasonal (pre-2009) influenza vaccination or the H1N1pdm09 influenza vaccine. Whiskers of box plots represent 10th and 90th percentiles, and the horizontal line and plus sign in each box give the median and mean titers of individuals, respectively. Variability in titer is shown by plotting the first and third quartiles of the titers as the outer limits of the box. Points beyond the whiskers denote outliers. Groups were compared by two-tailed, unpaired t test, and false discovery rate-adjusted P values are indicated.

FIG 4

(a) Exposure to wild avian species is not associated with enriched HA-specific antibody titers. Endpoint ELISA titers were determined for avian H4, H5, H6, H8, and H12 and human H7 hemagglutinin antigens. (b) Contact with poultry is associated with increased levels of anti-H7 antibodies. Endpoint ELISA titers were determined for avian H4, H5, H6, H8, and H12 and human H7 hemagglutinin antigens. Whiskers of box plots represent 10th and 90th percentiles, and the horizontal line and plus sign in each box give the median and mean titers of individuals, respectively. Variability in titer is shown by plotting the first and third quartiles of the titers as the outer limits of the box. Points beyond the whiskers denote outliers. Box color represents the participant-reported avian species exposure (white, passerines; gray, waterfowl; red, shorebirds; blue, raptors). Groups were compared by two-tailed, unpaired t test, and false discovery rate-adjusted P values were determined.

FIG 5

ELISA responses do not predict HAI reactivity. A Spearman rank correlation was performed on each individual’s ELISA and HAI responses. Above the diagonal, significant associations are marked with a circle, while nonsignificant associations are blank. Similarly, below the diagonal, the correlation coefficient is listed in the square where significant associations were found. ELISA antigens are in green, while HAI antigens are listed in orange following the nomenclature of Table 4. A significance level of less than 0.1 was applied after FDR adjustment.