Project description:A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase-antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days.
Project description:Neuraminidase (NA) is an important surface protein on influenza virions, playing an essential role in the viral life cycle and being a key target of the immune system. Despite the importance of NA-based immunity, current vaccines are focused on the hemagglutinin (HA) protein as the target for protective antibodies, and the amount of NA is not standardized in virion-based vaccines. Antibodies targeting NA are predominantly protective, reducing infection severity and viral shedding. Recently, NA-specific monoclonal antibodies have been characterized, and their target epitopes have been identified. This review summarizes the characteristics of NA, NA-specific antibodies, the mechanism of NA inhibition, and the recent efforts towards developing NA-based and NA-incorporating influenza vaccines.
Project description:Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among?>?50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.
Project description:Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
Project description:Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators of protection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, in vivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48 hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.
Project description:Better vaccines against influenza virus are urgently needed to provide broader protection against diverse strains, subtypes, and types. Such efforts are assisted by the identification of novel broadly neutralizing epitopes targeted by protective antibodies. Influenza vaccine development has largely focused on the hemagglutinin, but the other major surface antigen, the neuraminidase, has reemerged as a potential target for universal vaccines. We describe three human monoclonal antibodies isolated from an H3N2-infected donor that bind with exceptional breadth to multiple different influenza A and B virus neuraminidases. These antibodies neutralize the virus, mediate effector functions, are broadly protective in vivo, and inhibit neuraminidase activity by directly binding to the active site. Structural and functional characterization of these antibodies will inform the development of neuraminidase-based universal vaccines against influenza virus.
Project description:Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site (S364N, N369T, and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D, and Q313K/R). This work increases our understanding of how human antibody responses target the N1 protein. IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses.
Project description:The H7N9 influenza virus has been circulating in China for more than six years. The neuraminidase (NA) has gained great concern for the development of antiviral drugs, therapeutic antibodies, and new vaccines. In this study, we screened seven mouse monoclonal antibodies (mAbs) and compared their protective effects against H7N9 influenza virus. The epitope mapping from escape mutants showed that all the seven mAbs could bind to the head region of the N9 NA close to the enzyme activity sites, and four key sites of N9 NA were reported for the first time. The mAbs D3 and 7H2 could simultaneously inhibit the cleavage of the sialic acid of fetuin protein with large molecular weight and NA-XTD with small molecule weight in the NA inhibition experiment, prevent the formation of virus plaque at a low concentration, and effectively protect the mice from the challenge of the lethal dose of H7N9 virus.
Project description:As targets of adaptive immunity, influenza viruses are characterized by the fluidity with which they respond to the selective pressure applied by neutralizing antibodies. This mutability of structural determinants of protective immunity is the obstacle in developing universal influenza vaccines. Towards the development of such vaccines and other immune therapies, our studies are designed to identify regions of influenza viruses that are conserved and that mediate virus neutralization. We have specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the pandemic of 1968. Three monoclonal antibodies have been identified that are broadly-neutralizing against H3 influenza viruses spanning 40 years. The antibodies react with the hemagglutinin glycoprotein and appear to bind in regions that are refractory to the structural variation required for viral escape from neutralization. The antibodies demonstrate therapeutic efficacy in mice against H3N2 virus infection and have potential for use in the treatment of human influenza disease. By mapping the binding region of one antibody, 12D1, we have identified a continuous region of the hemagglutinin that may act as an immunogen to elicit broadly protective immunity to H3 viruses. The anti-H3 monoclonal antibodies were identified after immunization of mice with the hemagglutinin of four different viruses (A/Hong Kong/1/1968, A/Alabama/1/1981, A/Beijing/47/1992, A/Wyoming/3/2003). This immunization schedule was designed to boost B cells specific for conserved regions of the hemagglutinin from distinct antigenic clusters. Importantly, our antibodies are of naturally occurring specificity rather than selected from cloned libraries, demonstrating that broad-spectrum humoral immunity to influenza viruses can be elicited in vivo.
Project description:Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.