Determination of influenza B identity and potency in quadrivalent inactivated influenza vaccines using lineage-specific monoclonal antibodies

Abstract

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.

Fig 1. Location of HA amino acid changes in influenza B escape mutants.

(A) Antigenic structure of the B/Victoria lineage HA trimer (B/Brisbane/60/2008 PDB ID: 4FQM) and location of the escape mutants to mAbs BR5A1 (blue), BR8E12 (magenta), BR7B7 (yellow), and CR2F11 (green). (B) Antigenic structure of the B/Yamagata lineage HA trimer (B/Yamanashi/166/1998 PDB ID: 4M40) and location of the escape mutants to mAbs MA1H4 (orange), WI3E8 (red), MA3B2 (cyan), and CR2F11 (green). Escape mutants to the cross-reactive mAb CR2F11 were derived from both B/Victoria and B/Yamagata viruses.

Fig 2. SRID analysis of quadrivalent influenza vaccines using sheep polyclonal antiserum produced to B/Brisbane/60/2008.

Dilutions of quadrivalent influenza vaccine containing B/Brisbane/60/2008 and B/Massachusetts/2/2012 (A) or B/Brisbane/60/2008 and B/Texas/02/2013 (B) were loaded onto agarose gels (rows 2) along with dilutions of the two corresponding reference antigens (rows 1 and 3) and analyzed by standard SRID using B/Brisbane/60/2008 reference antiserum (Lot B-Ab-1108).

Fig 3. ELISA identity analysis of trivalent and quadrivalent influenza vaccines using lineage-specific mAbs.

ELISA plates were coated with the indicated purified mAbs at 2 μg/ml and used to capture Reference Antigens B/Brisbane/60/2008 (60 μg/ml) and B/Massachusetts/2/2012 (58 μg/ml), a quadrivalent vaccine containing B/Brisbane and B/Mass antigens at 24 and 40 μg/ml, respectively, a trivalent vaccine containing B/Florida/4/2006 (25 μg/ml), and a trivalent vaccine containing B/Brisbane (27 μg/ml). The starting dilution for all reference antigens and vaccines was 1:300.

Fig 4. Lineage specificity of mAb-capture ELISA.

ELISA plates were coated with the indicated purified mAbs at 2 μg/ml and used to capture Reference Antigens B/Brisbane/60/2008 (60 μg/ml), B/Malaysia/2506/2004 (76 μg/ml), B/Hong Kong/330/2001 (69 μg/ml), B/Massachusetts/2/2012 (58 μg/ml), B/Florida/4/2006 (79 μg/ml), B/Texas/6/2011 (80 μg/ml), and B/Phuket/3073/2013 (78 μg/ml). The starting dilution for all reference antigens was 1:300.

Fig 5. Correlation between SRID potency values and ELISA potency values determined for vaccines containing B/Victoria antigens.

(A) Eight vaccine samples, including 3 monovalent vaccines (■), 2 trivalent vaccines (▲), and 3 quadrivalent vaccines (●) were analyzed for HA content by standard SRID and mAb-capture ELISA using B/Victoria mAbs BR5A1 (closed symbols) and BR8E12 (open symbols). (B) SRID potency values for each vaccine plotted against the mAb-capture ELISA potency value using the combined (mean) BR5A1 and BR8E12 ELISA potency values.

Fig 6. Correlation between SRID potency values and ELISA potency values determined for vaccines containing B/Yamagata antigens.

(A) Six vaccine samples, including 3 trivalent vaccines (▲) and 3 quadrivalent vaccines (●) were analyzed for HA content by standard SRID and mAb-capture ELISA using B/Yamagata mAbs MA1H4 (closed symbols) and WI3E8 (open symbols). (B) SRID potency values for each vaccine plotted against the mAb-capture ELISA potency value using the combined (mean) MA1H4 and WI3E8 ELISA potency values.