Soluble trimeric hemagglutinins to study receptor binding and immunogenic properties

Abstract

Influenza A viruses (IAVs) are important pathogens of animals and man. In humans, IAV is the cause of seasonal epidemics and occasional pandemics. The pandemics are caused by suddenly appearing IAVs from animal reservoirs, to which most humans have no immunity. The hemagglutinin (HA) protein plays crucial roles in the early stages of virus infection as it binds to sialic acid receptors, which results in endocytic uptake of the virions, and is responsible for the low pH-induced fusion of viral and cellular membranes. Because of its role in sialic acid receptor binding, it is the most important determinant of virus tropism. In addition, the HA protein is also the major immunogen. Antibody levels against HA correlate with protection against infection and disease. This thesis focuses on these two key features of the HA protein. The HA-receptor interactions were studied by using recombinant soluble stable HA trimers, which are secreted from cells, by replacing the transmembrane anchor of HA with a GCN4 trimerization motif. First we studied to what extent the expression system used, and thus the glycosylation state of the recombinant proteins, interfered with HA-receptor interactions. The results show that this is indeed the case. Differences in receptor specificity between different HA proteins could be demonstrated using recombinant proteins produced in mammalian cells, but not with the HA proteins made in insect cells. This technology was subsequently applied to compare the receptor-binding properties of the HA protein from the swine origin new pandemic H1N1 virus with a closely related H1 protein derived from a swine virus. While the new pandemic H1 protein exhibited hardly any binding to different substrates, the swine protein efficiently bound to a number of sialylated glycans. The two amino acid residues responsible for this difference were identified, which provided new insight into HA-receptor interactions. Also the vaccine potential of the recombinant soluble trimeric HA proteins was evaluated. Immunization of chickens, mice ferrets or swine with the HA trimer preparations was shown to protect against a homologous (lethal) challenge with IAV, including the highly pathogenic avian influenza virus H5N1. Vaccination with the trimers induced strong antibody responses and diminished virus replication and excretion upon challenge and decreased clinical effects of infection. In addition, we investigated to what extent the glycosylation state of the HA preparations would affect their immunogenicity. The results consistently demonstrate that HA proteins carrying terminal mannose residues, such as insect-cell produced HA proteins, induce significantly lower antibody titers against HA when compared to fully glycosylated HA preparations. In summary, the research described in this thesis demonstrates the potential of recombinant trimeric soluble HA for the study of HA-receptor interactions and for vaccination purposes.