N-Glycosylation of Seasonal Influenza Vaccine Hemagglutinins: Implication for Potency Testing and Immune Processing.

Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a type A:H1N1 virus, a type A:H3N2 virus, and one or two type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza virus antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin-Darby canine kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccines. The reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012. Released glycan and HA-specific glycopeptide glycosylation patterns were examined. We also examined the sensitivity of the single radial immunodiffusion (SRID) potency test to differences in HA antigen glycosylation. Based on deglycosylation studies applied using standard assay procedures, the SRID assay was not sensitive to any HA antigen glycosylation status from any cell system. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions, and fusion domains, was performed and has implications for immune processing, immune responses, and antigenic shielding. Differences in glycosylation patterns, as dictated by the cell system used for expression, may impact these functions.IMPORTANCE In the present study, the glycosylation patterns of the 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed, and the sensitivity of the single radial immunodiffusion (SRID) potency test to glycosylation was tested. Differences in hemagglutinin glycosylation site composition and heterogeneity seen in antigens produced in different cell substrates suggest differences in processing and downstream immune responses. The SRID potency test used for vaccine release is not sensitive to differences in glycosylation under standard use conditions. This work reveals important differences in vaccine antigens and may point out areas where improvements may be made concerning vaccine antigen preparation, immune processing, and testing.

Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine.

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Avoiding risk information about breast cancer.

BACKGROUND: Learning about personal risk can provide numerous benefits yet people sometimes opt to remain ignorant. PURPOSE: Two studies examined the role of perceived control, coping resources, and anticipated regret in women's decision to avoid breast cancer risk information. METHODS: Women completed a health inventory and then read a brochure about either controllable or uncontrollable predictors of breast cancer, or received no brochure. Participants then received an opportunity to learn their lifetime risk for breast cancer based on their inventory responses. RESULTS: Reading about controllable predictors of breast cancer reduced avoidance of risk information compared with reading about uncontrollable predictors or receiving no information. In addition, fewer coping resources, anticipated greater regret over seeking breast cancer risk information, and less regret over avoiding breast cancer risk information predicted information avoidance. CONCLUSION: Reading about controllable predictors of breast cancer reduces avoidance of breast cancer risk information.

Why do I hate thee? Conflict misperceptions and intergroup mistrust.

Members of partisan social groups often exaggerate how much their own opinions differ from those of their rivals. In the present two studies, partisans estimated their own and their rivals' attitudes toward different issues related to the social conflict and also made a variety of evaluative judgments about their own and the rival group. The authors found that (a) partisans perceive more disagreement with their rivals about values that are central to their own sides' ideological position than those that are central to their rivals' position and (b) perceptions of disagreement about the partisans' own central values are what predicts partisans' global evaluations of members of the outgroup (e.g., disliking, trait stereotypes, perceived similarity). Furthermore, partisans believed their adversaries were motivated by an opposition to the partisans' own core values rather than by promotion of the adversaries' core values. Discussion concentrates on the theoretical and applied implications of these findings.

Heterogeneity of the MDCK cell line and its applicability for influenza virus research.

Single-cell clones have been established from the MDCK cell line, characterized for their morphology and evaluated for their suitability for influenza virus research. Three discrete cell morphotypes were identified using light microscopy. Besides morphological features, the cell types can be distinguished by the level of expression of surface glycans recognized by peanut agglutinin (PNA). All clones were susceptible to infection by influenza viruses of different subtypes of influenza A virus (H1N1, H1N1pdm09, H3N2, H5N1) and influenza B virus, and all possessed on their surface terminally sialylated glycans with both types of glycosidic linkage (α2-3 and α2-6). The Type-1 cell lines were able to support a multicycle replication of influenza A and B viruses without help of an exogenous trypsin. In contrast, cell lines exhibiting Type-2 morphology were unable to support multicycle replication of influenza A viruses without trypsin supplementation. Western blot analysis of the hemagglutinin of H1N1 strains demonstrated that Type-2 cells were deficient in production of proteolytically activated hemagglutinin (no cleavage between HA1/HA2 was observed). HA1/HA2 cleavage of influenza B viruses in the Type-2 cells was also significantly impaired, but not completely abrogated, producing sufficient amount of activated HA to support efficient virus replication without trypsin. In contrast, all clones of Type-1 cells were able to produce proteolytically activated hemagglutinin of influenza A and B viruses. However, the growth kinetics and plaque size of influenza A viruses varied significantly in different clones. Influenza B virus also showed different plaque size, with the biggest plaque formation in the Type-2 cells, although the growth kinetics and peak infectivity titers were similar in all clones. Taken together, the study demonstrates that the population of original MDCK cells is represented by various types of cells that differ in their capacities to support replication of influenza A and B viruses.