An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations

Abstract

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines.Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines.

A Live-Attenuated Prime, Inactivated Boost Vaccination Strategy with Chimeric Hemagglutinin-Based Universal Influenza Virus Vaccines Provides Protection in Ferrets: A Confirmatory Study

“…The allantoic fluid was harvested, purified by ultra-centrifugation through a sucrose cushion, and incubated with 0.03% of formaldehyde at 4 °C for 72 h to inactivate the viruses. The HA content of the virus vaccine preparations was measured as previously described and each dose was adjusted to contain 5 µg of HA [ 26 ]. The B-cH9/1 virus used for priming of the ferrets was based on a recombinant B/Yamagata/16/88 backbone with an HA that features the HA stalk domain from an H1N1 strain PR8 and the head domain from an H9N2 strain A/guinea fowl/Hong Kong/WF10/99 [ 8 , 27 ].…”

A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies

“…The LAIV was administered in a 2 mL volume via the intranasal route. For the second boost, a dose of inactivated chimeric influenza A virus expressing cH5/1 (H5 head domain from A/Vietnam/1203/04 on top of an H1 stalk domain from A/California/04/09, N1 from A/California/04/09 and a A/Puerto Rico/8/34 backbone) equivalent to 15 μg of hemagglutinin [ 38 ] was administered in a 2 mL volume intramuscularly with (groups G1 Emul and G2 Emul) or without (groups G1 and G2) adjuvant. UV-inactivated H1N2 virus (32 HA units) with adjuvant and human seasonal vaccine (TIV) containing 15 μg of inactivated pH1N1 with adjuvant were given in a 2 mL volume intramuscularly as a prime and booster 4 weeks apart for group G3 and G4, respectively (see Table 1 ).…”

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

“…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.…”