Synthrocytes: Synthetic erythrocytes to substitute animal red blood cells in hemagglutination assays for global influenza surveillance

Abstract

Influenza viruses cause annually seasonal flu epidemics with an important morbidity and mortality, as well as a high economical cost worldwide. The severity of the annual influenza epidemic is proportional to the degree of immune protection of the population, which is related to the high change rate of the viral envelope of these viruses. Since annual vaccination is the main prevention measure, the high antigenic drift obliges to the annual reformulation of the vaccine composition. Accordingly, global virological surveillance of circulating influenza viruses is required in order to collect genetic and antigenic information necessary to determine the vaccine composition yearly, a task that is coordinated globally by the World Health Organization (WHO) and to which laboratories from all over the world contribute. The hemagglutination inhibition assay (HAI) is one of the methods recommended by the WHO for the antigenic characterization of circulating influenza viruses and for the titration of specific neutralizing antibodies (Ab) against them, two tasks that are crucial for the annual recommendation of the vaccine composition. HAI is based on the fact that influenza binds to red-blood cells producing their agglutination and changing their sedimentation pattern, a behaviour that is prevented in the presence of specific anti-influenza Ab. HAI is a relatively simple and economical assay, but it has to be carried out with fresh erythrocytes. This is a biological reagent that is variable, unstable and difficult to obtain in large quantities for worldwide distribution, and that is acquired independently by each laboratory, which introduces lots of intra- and inter-laboratory variability in the results produced. The main objective of this PhD Thesis was the development of a synthetic reagent that could substitute animal erythrocytes in the HAI assays. As it will be shown, a reagent called "synthrocyte" has been developed, which mimics the differential sedimentation displayed by animal RBCs in the absence and in the presence of agglutinating agents in <25 min. By the end of this Thesis work, synthrocytes could bind the 4 influenza seasonal subtypes (A(H1N1)pdm09, A(H3N2), B/Yamagata and B/Victoria), producing changes in sedimentation that resembled those generated by erythrocytes in 30-60 min, and entailing a protocol and level of handling similar to those in classical HAI assays. Moreover, synthrocytes were reproducible and more stable than their animal counterparts, could be lyophilized for long-term storage, and were mass-producible. This work laid the first stone for the creation of a new research line that has continued beyond this Thesis with the contribution of other Team Members. We envisage that the results summarized here, and those that followed and will follow, will pave the way to achieve the worldwide standardization of HAI tests with minimal equipment or training requirements and facilitate inter-laboratory result evaluation.

Date of Award	22 Feb 2024
Original language	English
Supervisor	Eva Baldrich Rubio (Director)