Heterologous viral protein interactions within licensed seasonal influenza virus vaccines

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.

Characterization of Inactivated Influenza Vaccines Used in the Russian National Immunization Program

“…The densitometry results obtained for the gels shown in Figure S1A,B are represented in Tables S2 and S3. The correlation between HA concentrations measured by the SDS-PAGE assay for the U3, U4, and VG vaccine samples and normative HA concentrations (recovery, %) varied from 69.23% to 75.71% (Table 2), which approximately corresponded to the previously described degree of proximity of SRID and SDS-PAGE results [11,15,26]. Notes: For U3, U4, and VG, the image in Figure 2B was used for analysis; total protein concentration-our protein measurements ( Figure 1); HA content based on SDS-PAGE measured as the HA proportion using the weight of total protein concentration; and recovery (%)-ratio of HA concentration measured by SDS-PAGE to normative HA concentration expressed as a percentage.…”

“…This finding is consistent with results of non-HA protein quantitation in commercially available vaccines using Western blot analysis. Differences in the non-HA protein concentration correlate with the intensities of cellular and humoral immune responses [15]. Thus, our study indicated the potential variability of commercially available IIVs licensed in Russia by their ability to elicit an immune response to non-HA proteins of the influenza virus, and, therefore, potentially stimulate development of cross-protective immunity against various types and subtypes of the influenza virus.…”

“…Since the influenza virus HA is an intensely N-glycosylated glycoprotein, the peptide N-glycosidase F (PNGase F) treatment of vaccine samples before application on gels leads to carbohydrate cleavage from this protein and consequently to increased electrophoretic mobility of HA1 and HA2, and to decreased fuzziness of the respective protein bands [15]. After deglycosylation, HA1 and HA2 bands may be reliably separated from NP and M1, respectively.…”

“…From this point of view, abundance of non-HA (or other) viral proteins, in comparison with the abundance of HA, reflects the success of a strategy for vaccine enrichment with hemagglutinin. However, much of the currently available experimental evidence was on the role of non-HA proteins in protective immunity against influenza [15]. Considering this, one of the aims of our study was to quantify non-HA viral proteins in different IIVs.…”

“…Thus, the current WHO guidelines for controlling the final bulk-protein composition (i.e., the step immediately preceding the filling process) of IIVs include three tests: determining the HA, total protein, and ovalbumin contents of each strain of the influenza virus [14]. Depending on the manufacturing year and the manufacturer, relative amounts of NA, matrix protein (M1), and nuclear protein (NP) (as well as the immune responses induced by them) in different commercially available inactivated split vaccines may vary significantly, from several up to dozens of times [15]. Thus, new sophisticated platforms for quick and efficient characterization of the manufactured vaccine products should be launched.…”