Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

Abstract

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.

Identification of novel influenza A virus exposures by an improved high‐throughput multiplex MAGPIX platform and serum adsorption

“…Seroconversion with a ≥ 4‐fold rise in HI or MN antibody titers is indicative of recent influenza virus infection or vaccination . In recent years, magnetic multiplex fluorescence microsphere immunoassays (MAGPIX) have been developed to investigate antibody profiles following influenza vaccination/infection and aid in the diagnosis of influenza . Previously, we developed a high‐throughput MAGPIX assay with serum adsorption using two ectodomain recombinant HAs (rHAs) from A(H1N1)pdm09 (A/California/7/2009, CA/09) and A/Perth/16/2009 (Perth/09, A(H3N2)) to identify novel influenza exposures when well‐paired human serum samples were available .…”

“…1,2 In recent years, magnetic multiplex fluorescence microsphere immunoassays (MAGPIX) have been developed to investigate antibody profiles following influenza vaccination/ infection and aid in the diagnosis of influenza. [3][4][5] Previously, we developed a high-throughput MAGPIX assay with serum adsorption using two ectodomain recombinant HAs (rHAs) from A(H1N1) pdm09 (A/California/7/2009, CA/09) and A/Perth/ 16/2009 (Perth/09, A(H3N2)) to identify novel influenza exposures when well-paired human serum samples were available. 4 However, during novel influenza virus outbreak investigations, paired serum collection may not always be feasible, posing challenges to determine influenza virus infections through serology.…”

Application of Volumetric Absorptive Micro Sampling to Measure Multidimensional Anti-Influenza Hemagglutinin Igg Antibodies by MPlex-Flu Assay

“…The mPlex-Flu assay was performed as described previously [7,8,12] [7,12], and the standard curve for each influenza virus strain was generated by 1:4 serially diluting STD01 for each batch of samples. 50µL of the diluted sample was added into each reaction well.…”

“…The influenza HA bead panel used in this study is shown in Table ??, comprising 33 separate rHAs. 50µL of bead mix was added to each well of the plate as previously described [7,8,12]. Plates were then incubated with gentle shaking for 2 hours at room temperature, and then washed (PBS + 1% Bris + 0.1% BSA).…”

“…Thus, developing a solution would require both a simple method for in-the-field collection of small amounts of peripheral blood or serum, coupled with an assay that uses very small sample sizes (10-20 µL). Here we describe and validate such a system, using a combination of volumetric absorptive microsampling (VAMS) [6] coupled with a Luminex-based assay (mPLEX-Flu) [7,8,9,10,11,12] to quantitatively measure IgG antibodies against 33 strains of influenza hemagglutinin.…”