Abstract
For 50 years it has been known that antibodies to neuraminidase (NA) protect against infection during seasonal and pandemic influenza outbreaks. However, NA is largely ignored in the ...formulation and standardization of our current influenza vaccines. There are a number of factors that contributed to this antigen being forgotten, including the lack of an easily performed test to measure NA antibody. With the availability of that test, it has been possible to show its independent contribution to protection in various situations. The challenge now is to make it possible to include known amounts of NA in investigational vaccines or to routinely measure NA content in licensed vaccines. Vaccines containing optimal amounts of NA may be particularly useful when there are antigenic changes, either drift or shift, in the hemagglutinin because NA immunity offers broad protection. It is now time to remember the NA as we work toward improved influenza vaccines.
Neuraminidase is one of the two surface glycoproteins of influenza A and B viruses. It has enzymatic activity that cleaves terminal sialic acid from glycans, and that activity is essential at several ...points in the virus life cycle. While neuraminidase is a major target for influenza antivirals, it is largely ignored in vaccine development. Current inactivated influenza virus vaccines might contain neuraminidase, but the antigen quantity and quality are varied and not standardized. While there are data that show a protective role of anti-neuraminidase immunity, many questions remain unanswered. These questions, among others, concern the targeted epitopes or antigenic sites, the potential for antigenic drift, and, connected to that, the breadth of protection, differences in induction of immune responses by vaccination versus infection, mechanisms of protection, the role of mucosal antineuraminidase antibodies, stability, and the immunogenicity of neuraminidase in vaccine formulations. Reagents for analysis of neuraminidase-based immunity are scarce, and assays are not widely used for clinical studies evaluating vaccines. However, efforts to better understand neuraminidase-based immunity have been made recently. A neuraminidase focus group, NAction!, was formed at a Centers of Excellence for Influenza Research and Surveillance meeting at the National Institutes of Health in Bethesda, MD, to promote research that helps to understand neuraminidase-based immunity and how it can contribute to the design of better and broadly protective influenza virus vaccines. Here, we review open questions and knowledge gaps that have been identified by this group and discuss how the gaps can be addressed, with the ultimate goal of designing better influenza virus vaccines.
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•Neuraminidase (NA)-inhibiting antibodies are an independent correlate of vaccine efficacy.•Many NA-specific antibodies bind to conserved epitopes and protect against heterologous ...viruses.•Assays to measure NA content and immunogenicity of influenza vaccines have been developed.
Neuraminidase (NA) plays an essential role in influenza virus replication, facilitating multicycle infection predominantly by releasing virions from infected cells. NA-inhibiting antibodies provide resistance to disease and NA-specific antibodies contribute to vaccine efficacy. The primary reason NA vaccine content and immunogenicity was not routinely measured in the past, was the lack of suitable assays to quantify NA and NA-specific antibodies. These are now available and with recent appreciation of its contribution to immunity, NA content of seasonal and pandemic vaccines is being considered. An added benefit of NA as a vaccine antigen is that many NA-specific antibodies bind to domains that are well conserved within a subtype, protecting against heterologous viruses. This suggests NA may be a good choice for inclusion in universal influenza vaccines.
Pandemic 2009 H1N1 virus isolates containing the neuraminidase inhibitor resistance mutation H275Y have been reported. We describe rapid selection for the H275Y resistance mutation during therapy in ...2 immunocompromised individuals at 9 and 14 days of therapy, as well as the first described case of clinically significant resistance to peramivir.
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.
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•Flu virus infection induces many neuraminidase (NA)-reactive B cells and antibodies•NA antibodies have broad cross-reactivity and inhibit neuraminidase enzyme activity•Current flu vaccines poorly display key NA epitopes and do not produce NA antibodies•NA-reactive antibodies offer protection against lethal flu virus infection
Current influenza vaccines predominantly produce antibodies targeting the viral hemagglutinin (HA). However, during natural infection, the body also produces antibodies targeting the viral neuraminidase (NA). These NA antibodies can provide robust and broad protection and could potentially be elicited prophylactically or via new vaccine strategies or used therapeutically.
A(H1N1)pdm09 influenza A viruses predominated in the 2013-2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) ...inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses.
Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) ...antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.
The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) ...cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.
Seasonal epidemics caused by influenza virus are driven by antigenic changes (drift) in viral surface glycoproteins that allow evasion from preexisting humoral immunity. Antigenic drift is a feature ...of not only the hemagglutinin (HA), but also of neuraminidase (NA). We have evaluated the antigenic evolution of each protein in H1N1 and H3N2 viruses used in vaccine formulations during the last 15 y by analysis of HA and NA inhibition titers and antigenic cartography. As previously shown for HA, genetic changes in NA did not always lead to an antigenic change. The noncontinuous pattern of NA drift did not correspond closely with HA drift in either subtype. Although NA drift was demonstrated using ferret sera, we show that these changes also impact recognition by NA-inhibiting antibodies in human sera. Remarkably, a single point mutation in the NA of A/Brisbane/59/2007 was primarily responsible for the lack of inhibition by polyclonal antibodies specific for earlier strains. These data underscore the importance of NA inhibition testing to define antigenic drift when there are sequence changes in NA.
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