Genetic and Antigenic Evolution of Influenza A (H3N2) Virus Neuraminidase

There is still much uncertainty about the underlying mechanisms that govern antigenic drift of influenza viruses, and several theories have been proposed. These theories consider hemagglutinin (HA) to be the primary driving force, while the second key surface glycoprotein (neuraminidase, NA) and the other viral proteins have largely been ignored. This thesis focuses on all influenza A (H3N2) virus proteins, with special emphasis on NA, to better understand—and ultimately predict—the complex evolution of A(H3N2) viruses. The genetic evolution, with respect to antigenic change, was analyzed by comparing HA and NA (chapter 2) and by studying the whole-genome (chapter 3). We next optimized the enzyme-linked lectin assay (ELLA) as an NA inhibition (NI) assay for human serology (chapter 5) and rapid antigenic characterization (chapter 6). With the optimized NI ELLA, we analyzed NA from 1968 till the 2010-2011 season and subsequently compared it to HA (chapter 7). Our work clearly shows that there is antigenic evolution for NA, thus warranting the inclusion of NAs representing emerging influenza A strains in vaccines. Increasing knowledge on which mutations cause changes in the phenotype of NA can help to perform more targeted influenza surveillance. It would then be advisable to integrate genetic and antigenic NA data with sequence and antigenic data of HA, epidemiological data, and geographical data during influenza surveillance. This will facilitate consideration of NA content, and improve next generation influenza vaccines

Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses

Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. BLAST analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa

Genomewide analysis of reassortment and evolution of human influenza A(H3N2) viruses circulating between 1968 and 2011

Influenza A(H3N2) viruses became widespread in humans during the 1968 H3N2 virus pandemic and have been a major cause of influenza epidemics ever since. These viruses evolve continuously by reassortment and genomic evolution. Antigenic drift is the cause for the need to update influenza vaccines frequently. Using two data sets that span the entire period of circulation of human influenza A(H3N2) viruses, it was shown that influenza A(H3N2) virus evolution can be mapped to 13 antigenic clusters. Here we analyzed the full genomes of 286 influenza A(H3N2) viruses from these two data sets to investigate the genomic evolution and reassortment patterns. Numerous reassortment events were found, scattered over the entire period of virus circu