Inactivated influenza vaccine stress can affect in vitro potency assay relationship to immunogenicity

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. The capability of hemagglutinin (HA), the main antigen in inactivated influenza vaccines (IIVs), to elicit functional neutralizing antibodies determines IIV effectiveness. When HA is subjected to environmental stress during manufacturing or while stored prior to administration, such as low pH and temperature excursions, the HA immunological activity can be affected. Single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, is believed to specifically detect immunologically active HA and has been applied to evaluate HA stability against stress. Here we report that transient low pH treatment and freeze/thaw cycles with HA in PBS abolish SRID-quantified in vitro potency for all HAs of multiple influenza strains. Raised temperature substantially decreases in vitro potency with more extensive HA structural changes. Chemical stress and mechanical stress moderately change SRID in vitro potency values in a strain-dependent manner. Trypsin digestion, which selectively degrades stressed HA, followed by RP-HPLC quantification as a candidate alternative in vitro potency assay yields results comparable to SRID. Mouse immunogenicity studies confirm that HA stressed by transient low pH treatment does not elicit functional antibodies in vivo, nor does it have a measureable SRID value. However, HA stressed by raised temperature elicits high titers of functional antibodies in vivo despite substantial loss of SRID in vitro potency. This discrepancy between SRID in vitro potency and vaccine immunogenicity suggests that SRID may not reliably indicate IIV potency under all conditions. Further efforts to develop alternate potency assays that can better predict in vivo immunogenicity should continue along with additional studies exploring HA conformation, SRID values and consequent immunogenicity.