Abstract
Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches.
Highlights
Influenza continues to have a significant impact on public health and is still responsible for high morbidity and mortality in humans, with annual attack rates estimated to be up to 10% in adults and 30% in children [1]
As a proof-of-concept to evaluate the performance of enzyme-linked immunosorbent assays (ELISAs) in distinguishing between head- and stalk-specific differences, a total of 16 pairs of serum samples from adult subjects and eight pairs of serum samples from children were selected on the basis of their
Titers (5/40); three subjects with very high boost (5/1280) of hemagglutination inhibition (HI) titers after vaccination; and one subject with a pre-existing HI titer of 160 which only marginally increased to 320 after vaccination. These selected samples were titrated by serological assays that are generally used in order to evaluate the immunogenicity of an influenza vaccine (MN, single radial hemolysis (SRH) and enzyme-linked lectin assay (ELLA)), along with the competitive head/stalk-specific ELISA described here (Table 1 and Figure 2A)
Summary
Influenza continues to have a significant impact on public health and is still responsible for high morbidity and mortality in humans, with annual attack rates estimated to be up to 10% in adults and 30% in children [1]. Influenza A and B viruses, which are responsible for annual epidemics in humans, undergo antigenic changes within the antibody-binding sites of the hemagglutinin (HA) and neuraminidase (NA) antigens; these changes are able to render the new strains different enough to at least partially avoid the immunity. Despite the efforts of the WHO Collaborating Centers and the new mathematical modelling approach [6,9] to monitor antigenic drift, an intrinsic uncertainty concerning the match between the circulating viruses and the vaccine strains remains [10]
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