Taia T. Wang

Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain

ABSTRACT Although highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine. IMPORTANCE Current influenza vaccines are effective against only a narrow range of influenza virus strains. It is for this reason that new vaccines must be generated and administered each year. We now report progress toward the goal of an influenza virus vaccine which would protect against multiple strains. Our approach is based on presentation to the host immune system of a region of the influenza virus—called a “headless hemagglutinin” (headless HA)—which is similar among a multitude of diverse strains. We show that vaccination of mice with a headless HA confers protection to these animals against a lethal influenza virus challenge, thereby demonstrating the viability of the approach. Through further development and testing, we predict that a single immunization with a headless HA vaccine will offer effective protection through several influenza epidemics. Current influenza vaccines are effective against only a narrow range of influenza virus strains. It is for this reason that new vaccines must be generated and administered each year. We now report progress toward the goal of an influenza virus vaccine which would protect against multiple strains. Our approach is based on presentation to the host immune system of a region of the influenza virus—called a “headless hemagglutinin” (headless HA)—which is similar among a multitude of diverse strains. We show that vaccination of mice with a headless HA confers protection to these animals against a lethal influenza virus challenge, thereby demonstrating the viability of the approach. Through further development and testing, we predict that a single immunization with a headless HA vaccine will offer effective protection through several influenza epidemics.

Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins.

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.

Hemagglutinin stalk antibodies elicited by the 2009 pandemic influenza virus as a mechanism for the extinction of seasonal H1N1 viruses

After the emergence of pandemic influenza viruses in 1957, 1968, and 2009, existing seasonal viruses were observed to be replaced in the human population by the novel pandemic strains. We have previously hypothesized that the replacement of seasonal strains was mediated, in part, by a population-scale boost in antibodies specific for conserved regions of the hemagglutinin stalk and the viral neuraminidase. Numerous recent studies have shown the role of stalk-specific antibodies in neutralization of influenza viruses; the finding that stalk antibodies can effectively neutralize virus alters the existing dogma that influenza virus neutralization is mediated solely by antibodies that react with the globular head of the viral hemagglutinin. The present study explores the possibility that stalk-specific antibodies were boosted by infection with the 2009 H1N1 pandemic virus and that those antibodies could have contributed to the disappearance of existing seasonal H1N1 influenza virus strains. To study stalk-specific antibodies, we have developed chimeric hemagglutinin constructs that enable the measurement of antibodies that bind the hemagglutinin protein and neutralize virus but do not have hemagglutination inhibition activity. Using these chimeric hemagglutinin reagents, we show that infection with the 2009 pandemic H1N1 virus elicited a boost in titer of virus-neutralizing antibodies directed against the hemagglutinin stalk. In addition, we describe assays that can be used to measure influenza virus-neutralizing antibodies that are not detected in the traditional hemagglutination inhibition assay.

Type I and type II Fc receptors regulate innate and adaptive immunity

Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibodys Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.

PB1-F2 Expression by the 2009 Pandemic H1N1 Influenza Virus Has Minimal Impact on Virulence in Animal Models

ABSTRACT Unlike previous pandemic viruses, the 2009 H1N1 pandemic influenza virus does not code for the virulence factor PB1-F2. The genome of the 2009 H1N1 virus contains three stop codons preventing PB1-F2 expression; however, PB1-F2 production could occur following genetic mutation or reassortment. Thus, it is of great interest to understand the impact that expression of the PB1-F2 protein might have in the context of the 2009 pandemic influenza virus, A/California/04/2009 (Cal/09). We have addressed this question by generating two Cal/09 viruses with productive PB1-F2 open reading frames containing either an asparagine at position 66 of PB1-F2 (66N) or a serine at position 66 (66S): this N66S change has previously been shown to be associated with increased virulence in mice. We used these viruses to investigate the effect on virulence conferred by expression of the 66N or the 66S PB1-F2 protein in both in vitro and in vivo systems. Our results show enhanced replication of the 66S virus in A549 cells, while studies of BALB/c and DBA/2 mice and ferrets revealed no significant differences in symptoms of infection with wild-type Cal/09 versus the 66N or 66S virus variant. Also, coinfection of mice with Streptococcus pneumoniae and the different viruses (recombinant wild-type [rWT] Cal/09 and the 66N and 66S viruses) did not result in significant differences in mortality. Mice infected with either PB1-F2-expressing virus did demonstrate altered protein levels of proinflammatory cytokines; differences were observed to be greater in infection caused by the 66S virus. In summary, our study demonstrates that PB1-F2 expression by the Cal/09 virus modulates the immune response to infection while having a minimal effect on virus virulence in two mammalian models.

Seroevidence for H5N1 influenza infections in humans: meta-analysis.

One to two percent of 14,000 people tested in 20 studies showed evidence of prior H5N1 infection. The prevalence of avian H5N1 influenza A infections in humans has not been definitively determined. Cases of H5N1 infection in humans confirmed by the World Health Organization (WHO) are fewer than 600 in number, with an overall case fatality rate of >50%. We hypothesize that the stringent criteria for confirmation of a human case of H5N1 by WHO do not account for a majority of infections but rather the select few hospitalized cases that are more likely to be severe and result in poor clinical outcome. Meta-analysis shows that 1 to 2% of more than 12,500 study participants from 20 studies had seroevidence for prior H5N1 infection.

Unraveling the Mystery of Swine Influenza Virus

Influenza virus outbreaks occur with regularity, but the severity of outbreaks is not consistent. The recent flu epidemic caused by an H1N1 swine influenza virus presents an opportunity to examine what is known about virulence factors and the spread of infection to better prepare for major influenza outbreaks in the future.

Why Do Influenza Virus Subtypes Die Out? A Hypothesis

ABSTRACT Novel pandemic influenza viruses enter the human population with some regularity and can cause disease that is severe and widespread. The emergence of novel viruses, historically, has often been coupled with the disappearance of existing seasonal virus strains. Here, we propose that the elimination of seasonal strains during virus pandemics is a process mediated, at the population level, by humoral immunity. Specifically, we suggest that infection with a novel virus strain, in people previously exposed to influenza viruses, can elicit a memory B cell response against conserved hemagglutinin stalk epitopes and/or neuraminidase epitopes. The anti-stalk and/or anti-neuraminidase antibodies then act to diminish the clinical severity of disease caused by novel influenza viruses and to eliminate seasonal virus strains.

H5N1 influenza viruses: Facts, not fear

The ongoing controversy over publication of two studies involving the transmission in ferrets of H5N1 (H5) subtype influenza viruses and the recommendations of the National Science Advisory Board for Biosecurity to redact key details in the manuscripts call for an examination of relevant scientific facts. In addition, there are calls in the media to destroy the viruses, curtail future research in this area, and protect the public from such “frightening” research efforts. Fear needs to be put to rest with solid science and not speculation.

Universal epitopes of influenza virus hemagglutinins

The influenza virus has proved an elusive target in the development of broadly protective vaccines. A new study identifies an antibody with broad neutralizing activity against influenza viruses of different subtypes. The antibody recognizes a highly conserved region on the viral hemagglutinin that may be targeted to prevent infection.