Hemagglutinin Quantitative ELISA-based Potency Assay for Trivalent Seasonal Influenza Vaccine Using Group-Specific Universal Monoclonal Antibodies

Abstract

The assurance of vaccine potency is important for the timely release and distribution of influenza vaccines. As an alternative to Single Radial Immunodiffusion (SRID), we report a new quantitative enzyme-linked immunosorbent assay (ELISA) for seasonal trivalent influenza vaccine (TIV). The consensus hemagglutinin (cHA) stalks for group 1 influenza A virus (IAV), group 2 IAV, and influenza B virus (IBV) were designed and produced in bacterial recombinant host in a soluble form, and monoclonal antibodies (mAbs) were generated. The group-specific ‘universal’ mAbs (uAbs) bound to various subtypes of HAs in the same group from recombinant hosts, embryonated eggs, and commercial vaccine lots. The calibration curves were generated to assess the sensitivity, specificity, accuracy, and linear dynamic range. The quantitative ELISA was validated for the potency assay of individual components of TIV- H1, H3, and IBV- with good correlation with the SRID method. This new assay could be extended to pandemic or pre-pandemic mock-up vaccines of H5 of group 1 and H7 virus of group 2, and novel HA stalk-based universal vaccines.

Built‐in RNA‐mediated chaperone (chaperna) for antigen folding tailored to immunized hosts

“…Consequently, based on the antigenicity prediction, we selected the RBD (367–606 amino acids) and the HR2 domain of the S protein (1246–1295 amino acids) as target antigens. The cHA stalk of influenza HA were generated based on the consensus sequences of group 1 influenza A viruses (IAVs), as previously described (Figure 1a; Chae, Kim, Hwang, & Seong, 2019). The globular domain (gd) of HA was selected from influenza virus H5N1.…”

“…The HA stalk draws attention as potential target for universal influenza vaccine (Jang & Seong, 2019), and an ELISA‐based influenza vaccine potency assay (Chae, Kim, Kim, et al, 2019). Thus, mRID‐cHA stalk (IAV) was constructed based on the HA sequence of influenza A group1 viruses (H1, H2, H5 and H9), and mRID‐cHA stalk (IBV) from influenza B viruses including both Yamagata and Victoria lineages (Chae, Kim, Kim, et al, 2019). After immunization, positive hybridoma clones (ELISA O.D > 2.0) were selected by ELISA for mRID‐cHA stalk (IAV) and mRID‐cHA (IBV).…”

“…Likewise, mAb # 3 specifically bound to two different lineages of IBV with equal efficiency, without any cross‐reactivity to IAV (Figure 6c,d). The availability of group‐specific mAbs will be instrumental in establishing a novel ELISA‐based approach for the quantitation of trivalent seasonal influenza vaccines (Chae, Kim, Kim, et al, 2019; Gravel et al, 2015; Rajendran et al, 2018), pre‐pandemic vaccines, or HA stalk‐based cross‐protective universal vaccine (Erbelding et al, 2018; Jang & Seong, 2014). MAbs that bind to HA‐stalk have been reported to potentiate ADCC (Jegaskanda, Reading, & Kent, 2014).…”

“…MAbs against the conserved HA stalk are specific to influenza HAs within the same group (Figure 6c,d). They are used for the quantitation of individual components into trivalent seasonal vaccines that are independent of the annual supply of strain‐specific immunological reagents (Chae, Kim, Kim, et al, 2019; Kuck et al, 2017; Rajendran et al, 2018). The same approach is being extended to the HA of two lineages of IBVs for the quantitation of quadrivalent influenza vaccines.…”

Changing Cross-Reactivity for Different Immunoassays Using the Same Antibodies: Theoretical Description and Experimental Confirmation

“…A number of approaches have been presented in the literature to date that allow for a targeted influence on the selectivity of immunoassays. The most common is to design an antigenic preparation for the best presentation of either unique or common epitope(s) for antibody generation [14][15][16][17][18]. In a number of cases, the results of such a design have been successful and made it possible to obtain antibodies with the required spectrum of selectivity.…”