Aims and Methods To facilitate antigenic characterization of the influenza A 2009 pandemic H1N1 [A(H1N1)pdm09] hemagglutinin (HA), we generated a panel of murine monoclonal antibodies (mAbs) using as the immunogen mammalian‐derived virus‐like particles containing the HA of the A/California/04/2009 virus. The antibodies were specific for the A/California/04/2009 HA, and individual mAbs suitable for use in several practical applications including ELISA, immunofluorescence, and Western blot analysis were identified. Results and Conclusions As the panel of mAbs included antibodies with hemagglutination inhibition (HI) and virus neutralizing activities, this allowed identification and characterization of potentially important antigenic and neutralizing epitopes of the A/California/04/2009 HA and comparison of those epitopes with the HAs of other influenza viruses including seasonal H1N1 viruses as well as the A/South Carolina/1918 and A/New Jersey/1976 H1N1 viruses. Three mAbs with the highest HI and neutralizing titers were able to provide passive protection against virus challenge. Two other mAbs without HI or neutralizing activities were able to provide partial protection against challenge. HA epitopes recognized by the strongest neutralizing mAbs in the panel were identified by isolation and selection of virus escape mutants in the presence of individual mAbs. Cloned viruses resistant to HI and antibody neutralization were sequenced to identify mutations, and two unique mutations (D127E and G155E) were identified, both near the antigenic site Sa. Using human post‐vaccination sera, however, there were no differences in HI titer between A/California/04/2009 and either escape mutant, suggesting that these single mutations were not sufficient to abrogate a protective antibody response to the vaccine.
Co-circulation of two antigenically and genetically distinct lineages of influenza B virus, represented by prototype viruses B/Victoria/2/1987 and B/Yamagata/16/1988, has led to the development of quadrivalent influenza vaccines that contain two influenza B antigens. The inclusion of two influenza B antigens presents challenges for the production and regulation of inactivated quadrivalent vaccines, including the potential for cross-reactivity of the reagents used in identity and potency assays because of the relative close relatedness of the hemagglutinin (HA) from the two virus lineages. Monoclonal antibodies (mAbs) specific for the two lineages of influenza B HA were generated and characterized and used to set-up simple identity tests that distinguish the influenza B antigens in inactivated trivalent and quadrivalent vaccines. The lineage-specific mAbs bound well to the HA of influenza B strains included in influenza vaccines over a period of more than 10 years, suggesting that identity tests using such lineage-specific mAbs would not necessarily have to be updated with every influenza B vaccine strain change. These lineage-specific mAbs were also used in an antibody capture ELISA format to quantify HA in vaccine samples, including monovalent, trivalent, and quadrivalent vaccine samples from various manufacturers. The results demonstrated correlation with HA values determined by the traditional single radial immunodiffusion (SRID) assay. Further, the antibody-capture ELISA was able to distinguish heat-stressed vaccine from unstressed vaccine, and was similar to the SRID in quantifying the resultant loss of potency. These mAb reagents should be useful for further development of antibody-based alternative influenza B identity and potency assays.
BackgroundThe potency of inactivated influenza vaccines is determined using a single radial immunodiffusion (SRID) assay. This assay is relatively easy to standardize, it is not technically demanding, and it is capable of measuring the potency of several vaccine strain subtypes in a multivalent vaccine. Nevertheless, alternative methods that retain the major advantages of the SRID, but with a greater dynamic range of measurement and with reduced reagent requirements, are needed.ObjectivesThe feasibility of an ELISA-based assay format was explored as an alternative potency assay for inactivated influenza vaccines.MethodsSeveral murine monoclonal antibodies (mAbs), specific for the 2009 pandemic H1N1 influenza virus hemagglutinin (HA), were evaluated for their potential to capture and quantify HA antigen. Vaccine samples, obtained from four licensed influenza vaccine manufacturers, included monovalent bulk vaccine, monovalent vaccine, and trivalent vaccine. Traditional SRID potency assays were run in parallel with the mAb–ELISA potency assay using the reference antigen standard appropriate for the vaccine samples being tested.ResultsThe results indicated that the ELISA potency assay can quantify HA over a wide range of concentrations, including vaccine at subpotent doses, and the ELISA and SRID potency values correlated well for most vaccine samples. Importantly, the assay was capable of quantifying A/California HA in a trivalent formulation.ConclusionsThis study demonstrates the general feasibility of the mAb approach and strongly suggests that such ELISAs have potential for continued development as an alternative method to assay the potency of inactivated influenza vaccines.
Influenza subtypes such as H7 have pandemic potential since they are able to infect humans with severe consequences, as evidenced by the ongoing H7N9 infections in China that began in 2013. The diversity of H7 viruses calls for a broadly cross-protective vaccine for protection. We describe the construction of recombinant modified vaccinia virus Ankara (MVA) vectors expressing the hemagglutinin (HA) or neuraminidase (NA) from three H7 viruses representing both Eurasian and North American H7 lineages – A/mallard/Netherlands/12/2000 (H7N3), A/Canada/rv444/2004 (H7N3), and A/Shanghai/02/2013 (H7N9). These vectors were evaluated for immunogenicity and protective efficacy against H7N3 virus in a murine model of intranasal challenge. High levels of H7-, N3-, and N9-specific antibodies, including neutralizing antibodies, were induced by the MVA-HA and MVA-NA vectors. Mice vaccinated with MVA vectors expressing any of the H7 antigens were protected, suggesting cross-protection among H7 viruses. In addition, MVA vectors expressing N3 but not N9 elicited protection against H7N3 virus challenge. Similar outcomes were obtained when immune sera from MVA vector-immunized mice were passively transferred to naïve mice prior to challenge with the H7N3 virus. The results support the further development of an MVA vector platform as a candidate vaccine for influenza strains with pandemic potential.
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