A Complex Dance: Measuring the Multidimensional Worlds of Influenza Virus Evolution and Anti-Influenza Immune Responses

Abstract

The human antibody response to influenza virus infection or vaccination is as complicated as it is essential for protection against flu. The constant antigenic changes of the virus to escape human herd immunity hinder the yearly selection of vaccine strains since it is hard to predict which virus strains will circulate for the coming flu season. A "universal" influenza vaccine that could induce broad cross-influenza subtype protection would help to address this issue. However, the human antibody response is intricate and often obscure, with factors such as antigenic seniority or original antigenic sin (OAS), and back-boosting ensuring that each person mounts a unique immune response to infection or vaccination with any new influenza virus strain. Notably, the effects of existing antibodies on cross-protective immunity after repeated vaccinations are unclear. More research is needed to characterize the mechanisms at play, but traditional assays such as hemagglutinin inhibition (HAI) and microneutralization (MN) are excessively limited in scope and too resource-intensive to effectively meet this challenge. In the past ten years, new multiple dimensional assays (MDAs) have been developed to help overcome these problems by simultaneously measuring antibodies against a large panel of influenza hemagglutinin (HA) proteins with a minimal amount of sample in a high throughput way. MDAs will likely be a powerful tool for accelerating the study of the humoral immune response to influenza vaccination and the development of a universal influenza vaccine.

Keywords: broad neutralizing antibody(bnAb); hemagglutinin (HA) of influenza virus; heterosubtypic immunity of influenza; humoral response; influenza virus; mPLEX-Flu assay; multiple dimensional assay (MDA); original antigenic sin “OAS”; protein microarray assay; “universal” influenza vaccine.

Figure 1

The principle of a multiple dimensional assay (MDA). The schematic diagram demonstrates the principle of the mPLEX-Flu assay, an example of a MDA, which is a Luminex based assay. Each color-coded xMAP bead is coupled with the purified recombinant hemagglutinin (rHAs) of one influenza virus strain. The different colors of influenza strain HA-specific beads are mixed and incubated with the serum sample. Bound anti-HA antibodies are subsequently detected using detection antibodies specific to each antibody isotope. The magnetic beads are read on a dual-laser flow-based Luminex reader. One laser classifies the bead and identifies the analyte being detected. The second laser determines the magnitude of the PE-derived signal, which is in direct proportion to the amount of analyte bound. Median fluorescence intensity (MFI) is converted to absolute concentration using a standard curve, at which point multiple dimensional data analysis can begin.

Figure 2

The current applications of multiple dimensional assays (MDAs). Four major influenza research applications of MDAs are listed, along with the multidimensional data set that each can generate.