Contribution of murine innate serum inhibitors toward interference within influenza virus immune assays

Abstract

Background Prior to detection of an antibody response toward influenza viruses using the hemagglutination inhibition assay (HAI), sera are routinely treated to inactivate innate inhibitors using both heat-inactivation (56°C) and recombinant neuraminidase (receptor-destroying enzyme, RDE). Objectives We revisited the contributions of innate serum inhibitors toward interference with influenza viruses in immune assays, using murine sera, with emphasis on the interactions with influenza A viruses of the H3N2 subtype. Methods We used individual serum treatments: 56°C alone, RDE alone, or RDE + 56°C, to treat sera prior to evaluation within HAI, microneutralization, and macrophage uptake assays. Results Our data demonstrate that inhibitors present within untreated murine sera interfere with the HAI assay in a manner that is different than that seen for the microneutralization assay. Specifically, the γ class inhibitor α2-Macroglobulin (A2-M) can inhibit H3N2 viruses within the HAI assay, but not in the microneutralization assay. Based on these findings, we used a macrophage uptake assay to demonstrate that these inhibitors can increase uptake by macrophages when the influenza viruses express an HA from a 1968 H3N2 virus isolate, but not a 1997 H3N2 isolate. Conclusions The practice of treating sera to inactivate innate inhibitors of influenza viruses prior to evaluation within immune assays has allowed us to effectively detect influenza virus-specific antibodies for decades. However, this practice has yielded an under-appreciation for the contribution of innate serum inhibitors toward host immune responses against these viruses, including contributions toward neutralization and macrophage uptake.

Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction

“…Of note, heat-inactivated mouse serum retained most of its neutralizing effects against NpL ( Figure 8 ). This finding suggests that a significant contribution to Nipah virus resistance in mice may be heat-stable, innate immunity serum proteins, such as α2-macroglobulin, that are known to bind viruses 48 – 49 . As with any viral vector, delivery methods will need to be optimized before NpL will be useful in testing gene delivery in mouse models of disease.…”

A chimeric influenza hemagglutinin delivered by parainfluenza virus 5 vector induces broadly protective immunity against genetically divergent influenza a H1 viruses in swine

“…Hemagglutination inhibition (HAI) assays were performed, with receptor-destroying enzyme (RDE, Accurate Chemical, Westbury, NY) being used to treat serum samples prior to use in the HAI assay, as described previously ( 49 ). RDE-treated serum samples from pigs (1 mL volume) were incubated with chicken red blood cells (50 L) to remove nonspecific agglutinins in the serum, as described ( 50 , 51 ).…”

Influenza A Viruses in Peridomestic Mammals

“…These tests have the added advantage that they can identify specific subtypes, but they require reference viruses which is not problematic for experimental studies in which the test virus can be used, but for field studies reference viruses must be carefully selected to match study objectives or some exposures may be missed. Additionally, serum inhibitors can be a problem for HI testing of mammalian samples so samples must be treated (e.g., with receptor-destroying enzyme) to inactivate inhibitors [29]. A final consideration is erythrocyte source for these tests.…”