Heterologous viral protein interactions within licensed seasonal influenza virus vaccines

Marina Koroleva,Katherine Richards,Frances Batarse,Andrea J Sant,Savannah Moritzky,Francisco Chaves,Florian Krammer,Patrick Wilson,Carole Henry

doi:10.1038/s41541-019-0153-1

Abstract

Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells.

Highlights

CD4 T cell help is essential for production of high affinity antibodies in response to vaccination
Licensed influenza vaccines are highly complex with regard to viral protein composition We first performed a survey of four different split virion licensed influenza virus vaccines (IIVs) (Fluzone 2014–2015, 2015–2016 and FluLaval 2013–2014, 2015–2016) prepared from embryonated chicken eggs in order to characterize their viral protein composition
To compare the abundance of the influenza virus proteins within these vaccines, as well as an estimate of reproducibility across independent experiments, densitometry analyses were performed (Supplementary Fig. 1). Examination of these data suggest that the assays are reproducible and that while HA content can differ by twofold among vaccines as estimated by western blotting, NA, NP, and M1 content can vary as much as 3–4-fold, depending on the year of production and the manufacturer. These data argue that the composition of viral proteins in split vaccines is complex, and in addition to the HA proteins derived from each virus strain, the administered vaccine contains NA, NP, and M1, present in variable quantities per dose, depending on the year and vaccine manufacturer

Summary

Introduction

CD4 T cell help is essential for production of high affinity antibodies in response to vaccination (reviewed in refs 1–3). Some biochemical studies have suggested that beyond HA, IIV can contain additional influenza viral proteins such as neuraminidase (NA) (reviewed in refs 4–6). Recent biochemistry studies have shown that NA in licensed influenza vaccines can be quantified by stable isotope dimethyl labeling in conjunction with mass spectrometry.[7] Published studies have shown that inactivated vaccines can contain other viral components such as matrix protein (M1) and nucleoprotein (NP) and can elicit both CD4 T cell and antibody responses to inactivated influenza vaccination.[8,9,10,11,12] These studies have suggested the potential of these induced specificities to provide broadly protective immunity to influenza. Our own studies of CD4 T cell responses to licensed vaccines in mice and humans, quantifying virus-specific CD4 T cell reactivity, indicate that in addition to HA and NA, expansion of CD4 T cells specific for NP and M1 occurs.[11,13,14] The impact of these additional influenza virus proteins in IIV and the responses to them on human immunity to the virus are unknown at this time but can be envisioned to have functional consequences in both the B cell and CD4 T cell compartments

Methods

Results

Conclusion