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. 2017 Sep 19;8(5):e01463-17.
doi: 10.1128/mBio.01463-17.

Influenza Virus Hemagglutinin Stalk-Specific Antibodies in Human Serum are a Surrogate Marker for In Vivo Protection in a Serum Transfer Mouse Challenge Model

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Influenza Virus Hemagglutinin Stalk-Specific Antibodies in Human Serum are a Surrogate Marker for In Vivo Protection in a Serum Transfer Mouse Challenge Model

Henning Jacobsen et al. mBio. .

Abstract

The immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines.IMPORTANCE Influenza viruses are a serious concern for public health and cause a large number of deaths worldwide every year. Current influenza virus vaccines can confer protection from disease, but they often show low efficacy due to the ever-changing nature of the viruses. Novel vaccination approaches target conserved epitopes of the virus, including the hemagglutinin stalk domain, to elicit universally protective antibodies that also bind to mutated viruses or new subtypes of viruses. Importantly, the hemagglutination inhibition assay-the only assay that has been accepted as a correlate of protection by regulatory authorities-cannot measure antibodies against the hemagglutinin stalk domain. Therefore, novel correlates of protection and assays to measure vaccine immunogenicity need to be developed. In this study, we correlated the results from multiple assays with protection in mice after transfer of human serum and a lethal virus challenge to investigate potential novel serological surrogate markers for protection.

Keywords: ADCC; ELISA; correlate of protection; hemagglutinin; hemagglutinin stalk; influenza; influenza vaccines; surrogate marker; universal influenza virus vaccine.

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Figures

FIG 1
FIG 1
Hemagglutination inhibition assay and ELISA results. Serum samples from individuals pre- and postvaccination were measured individually. Serum samples are shown as individual symbols. Bars represent geometric mean titers. (A) Hemagglutination inhibition was measured against H1N1pdm09 virus. The gray dashed line indicates the limit of detection. (B) Hemagglutinin stalk-specific antibodies were measured by ELISA using a recombinantly produced cH6/1 hemagglutinin. The construct contains an exotic hemagglutinin head domain to which humans are generally not exposed and the H1N1pdm09 hemagglutinin stalk domain. (C) Antibodies against H1N1pdm09 hemagglutinin were measured against recombinantly produced protein. (D) Antibodies against H1N1pdm09 neuraminidase were measured against recombinantly produced protein.
FIG 2
FIG 2
Serum transfer and H1N1 mouse challenge setup and results. (A) Serum samples for each group and time point were pooled, and 150 µl was intraperitoneally injected into mice. Five hours later, animals were sedated, retroorbitally bled to confirm successful serum transfer, and intranasally infected with H1N1pdm09 virus. A negative-control group that received PBS was included and is shown in all weight loss graphs. Mice were monitored daily for weight loss for 14 days. Points indicate group means, and error bars show the standard errors of the means. (B) Weight loss curves for mice that received pooled sera from pre- or post-seasonal influenza vaccination are shown. Percent survival is indicated for each group in the figure legend. (C) Weight loss curves for mice that received pooled sera from prevaccinated, post-1×, or post-2× nonadjuvanted H5N1 vaccination are shown in blue. Percent survival is indicated for each group in the figure legend. (D) Weight loss curves for mice that received pooled sera from prevaccination or post-1× or post-2× adjuvanted H5N1 vaccination are shown. Percent survival is indicated for each group in the figure legend. (E) The maximum weight loss throughout the 14-day observation period was calculated for all animals. Mice that died were assigned the maximum weight loss percentage (25%). Each dot represents a single animal, and white bars show the group geometric mean titers. All groups were compared in a one-way ANOVA Dunnett’s multiple-comparisons test for the post-seasonal influenza vaccination group (standard of care). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 3
FIG 3
Microneutralization, neuraminidase inhibition, and ADCC reporter assay results. Serum samples from individuals pre- and postvaccination were measured individually. Results for serum samples are shown as individual symbols. Bars represent geometric mean titers. (A) Microneutralization was measured against H1N1pdm09 virus. The gray dashed line indicates the limit of detection. (B) Neuraminidase inhibition was measured against H1N1pdm09 virus in an ELLA. (C) ADCC was measured in an FcγRIIIa-expressing Jurkat cell line-based luciferase reporter assay.
FIG 4
FIG 4
Correlation of assay results and maximum weight loss. The mean maximum weight loss and standard error of the mean are plotted on the y axis. The geometric mean titers are plotted on the x axis for each group. Each symbol represents one time point of one group. Semilog lines (y axis is linear, x axis is logarithmic) were plotted for each graph and are shown as black solid lines. Pearson r and P values for the correlation analysis are shown for each graph. To adjust for multiple comparisons, the P values were analyzed with the two-stage step-up method of Benjamini, Krieger, and Yekutieli with a false-discovery rate of 5%. H1 stalk ELISA (q = 0.0008), H1pdm09 ELISA (q = 0.0003), N1pdm09 ELISA (q = 0.0017), and ADCC (q = 0.0114) were confirmed as discoveries.

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