The problem: Influenza viruses compromise public health during seasonal outbreaks, infecting up to 1 billion people and claiming 400 000 lives every year according to estimates by the World Health Organization (WHO). Vaccination is an effective measure to alleviate the burden of the virus. However, due to the fast evolution of the virus, frequent vaccine updates are required to assure a matching, protective vaccine. To facilitate the production of protective vaccines the public and private sectors collaborate closely through an effective response system. Firstly, a global surveillance system (Global Influenza Surveillance and Response System, GISRS), organized by the WHO, continuously monitors the evolution of circulating and emerging viruses (4). This surveillance system consists of 144 national influenza centers and four international WHO collaborating centers, which collect patient samples worldwide and perform antigenic characterization of the collected viruses. Together they provide detailed information on the antigenic properties of circulating viruses in the human population and how efficiently antibodies raised by current vaccine strains can provide protection. Based on this information, the WHO recommends a suitable circulating strain to be used for vaccine production. Vaccine manufacturers are then responsible for producing high-growth vaccines that antigenically match with the recommendations of the WHO. In the last step, regulatory authorities request clinical studies to confirm the safety and immunogenicity of the newly produced vaccine. All these steps require antigenic characterization which is performed by the standard Hemagglutination Inhibition (HI) assay. The key requirement of this assay is the agglutination of red blood cells (RBC). Agglutination is caused by the binding of hemagglutinin, the viral receptor-binding protein, to 2,6-linked sialic acid moieties present on the surface of RBCs. These receptors are usually part of N-linked glycans, which are highly complex biomolecules composed of a core pentasaccharide modified by various numbers and patterns of glycan moieties. The HI assay is based on the ability of serum antibodies to block receptor binding to RBCs (“inhibition of agglutination”) and the extent of inhibition correlates with the protectiveness of antibodies. This inhibition with a binary output observed by the eye, has been a direct correlate of protection for the WHO for decades. The fast evolution of the virus, however, also affects the binding specificities of this hemagglutinin. This has resulted in the inability of contemporary influenza A/H3N2 viruses, a subgroup of influenza viruses associated with high mortality and morbidity, to agglutinate red blood cells . As a result, the standard HI assay cannot be used for antigenic characterization of A/H3N2 viruses anymore.
The solution: To solve the difficulties of antigenic characterization of contemporary A/H3N2 viruses and to reintroduce the standard HI assay, we have developed enzymatically engineered RBCs that express appropriate receptors for these viruses, this is not achievable by the WHO kit. It is the absence of appropriate elongated N-glycan receptors on wild-type RBCs that causes the failure of the standard HI assay.
The origin: As part of an ERC_STG Sugar-enable (#802780), Dr. de Vries and his coworkers studied the evolution of receptor specificity of human influenza A viruses using glycan microarray technology. It was demonstrated that the previous notion of a terminal sialic acid attached in an α2,6 manner on a single N acetyllactosamine as the prototypic human-type receptor does not apply for these viruses anymore. Instead, contemporary A/H3N2 viruses have evolved to recognize sialic acids presented on poly-N-acetyllactosamine (LacNAc) units on N-linked glycans . Applying this knowledge, functional receptors were installed on RBCs using recombinant glycosyltransferases to obtain RBCs that can efficiently be agglutinated by contemporary A/H3N2 viruses. These engineered RBCs facilitate the antigenic characterization of recent A/H3N2 viruses by the HI assay. It is the expectation that the application of the engineered RBCs allows WHO affiliated institutes (>140 institutes) and vaccine manufacturers to replace the current alternative (PRN assay) and reintroduce the standard HI assay. The HI assay is already established in all affected laboratories (applied for other virus subtypes), standardized, reproducible, and extremely straightforward.
