expression-functional-recombinant-hemagglutinin-neuraminidase

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Icahn School of Medicine at Mount Sinai

The influenza virus hemagglutinin molecule possesses a globular head domain that mediates receptor binding and a stalk domain at the membrane-proximal region. We generated functional influenza viruses expressing chimeric hemagglutinins encompassing a variety of globular head and stalk combinations, not only from different hemagglutinin subtypes but also from different hemagglutinin phylogenetic groups. These chimeric recombinant viruses possess growth properties similar to those of wild-type influenza viruses and can be used as reagents to measure domain-specific antibodies in virological and immunological assays.

Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.

Immunological changes associated with age contribute to the high rates of influenza virus morbidity and mortality in the elderly. Compounding this problem, aged individuals do not respond to vaccination as well as younger, healthy adults. Efforts to increase protection to this demographic group are of utmost importance, as the proportion of the population above the age of 65 is projected to increase in the coming decade. Using a live influenza virus with a truncated nonstructural protein 1 (NS1), we are able to stimulate cellular and humoral immune responses of aged mice comparable to levels seen in young mice. Impressively, a single vaccination provided protection following stringent lethal challenge in aged mice.

NS1-truncated live attenuated virus vaccine provides robust protection to aged mice from viral challenge.

Recently, a new class of broadly neutralizing anti-influenza virus antibodies that target the stalk domain of the viral hemagglutinin was discovered. As such, induction, isolation, characterization, and quantification of these novel antibodies has become an area of intense research and great interest. Since most of these antibodies bind to conformational epitopes, the structural integrity of hemagglutinin substrates for the detection and quantification of these antibodies is of high importance. Here we evaluate the binding of these antibodies to soluble, secreted hemagglutinins with or without a carboxy-terminal trimerization domain based on the natural trimerization domain of T4 phage fibritin. The lack of such a domain completely abolishes binding to group 1 hemagglutinins and also affects binding to group 2 hemagglutinins. Additionally, the presence of a trimerization domain positively influences soluble hemagglutinin stability during expression and purification. Our findings suggest that a carboxy-terminal trimerization domain is a necessary requirement for the structural integrity of stalk epitopes on recombinant soluble influenza virus hemagglutinin.

A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates.

Influenza virus-like particles are currently evaluated in clinical trials as vaccine candidates for influenza viruses. Most commonly they are produced in baculovirus- or mammalian- expression systems. Here we used different vaccination schemes in order to systematically compare virus-like particle preparations generated in the two systems. Our work shows significant differences in immunogenicity between the two, and indicates superior and broader immune responses induced by the baculovirus-derived constructs. We demonstrate that these differences critically influence protection and survival in a mouse model of influenza virus infection. Finally, we show that the enhanced immunogenicity of the baculovirus-derived virus-like particles is caused by contamination with residual baculovirus which activates the innate immune response at the site of inoculation.

Residual baculovirus in insect cell-derived influenza virus-like particle preparations enhances immunogenicity.

Broadly neutralizing antibodies directed against the conserved stalk domain of the viral hemagglutinin have attracted increasing attention in recent years. However, only a limited number of stalk antibodies directed against group 2 influenza hemagglutinins have been isolated so far. Also, little is known about the general level of induction of these antibodies by influenza virus vaccination or infection. To characterize the anti-stalk humoral response in the mouse model as well as in humans, chimeric hemagglutinin constructs previously developed in our group were employed in serological assays. Whereas influenza virus infection induced high titers of stalk-reactive antibodies, immunization with inactivated influenza virus vaccines failed to do so in the mouse model. Analysis of serum samples collected from human individuals who were infected by influenza viruses also revealed the induction of stalk-reactive antibodies. Finally, we show that the hemagglutinin stalk-directed antibodies induced in mice and humans have broad reactivity and neutralizing activity in vitro and in vivo. The results of the study point toward the existence of highly conserved epitopes in the stalk domains of group 2 hemagglutinins, which can be targeted for the development of a universal influenza virus vaccine in humans.

H3N2 influenza virus infection induces broadly reactive hemagglutinin stalk antibodies in humans and mice.

Current influenza virus vaccine strategies stimulate immune responses toward the globular head domain of the hemagglutinin protein in order to inhibit key steps of the virus life cycle. Because this domain is highly variable across strains, new vaccine formulations are required in most years. Here we demonstrate a novel vaccine strategy that generates immunity to the highly conserved stalk domain by using chimeric hemagglutinin constructs that express unique head and stalk combinations. By repeatedly immunizing mice with constructs that expressed the same stalk but an irrelevant head, we specifically stimulated a stalk-directed response that provided broad-based heterologous and heterosubtypic immunity in mice. Notably, our vaccination scheme provides a universal vaccine approach that protects against challenge with an H5 subtype virus. Furthermore, through in vivo studies using passively transferred antibodies or depletion of CD8(+) T cells, we demonstrated the critical role that humoral mechanisms of immunity play in the protection observed. The present data suggest that a vaccine strategy based on the stalk domain of the hemagglutinin protein could be used in humans to broadly protect against a variety of influenza virus subtypes.

Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.

Severe human disease caused by the emerging H7N9 influenza virus in China warrants a rapid response. Here, we present a recombinant Newcastle disease virus expressing a North American lineage H7 influenza virus hemagglutinin. Sera from immunized mice were cross-reactive to a broad range of H7 subtype viruses and inhibited hemagglutination by the novel H7 hemagglutinin. Immunized mice were protected against a heterologous H7 subtype challenge, and genetic analysis suggested that cross-protective antibodies recognize conserved antigenic sites.

Induction of cross-reactive antibodies to novel H7N9 influenza virus by recombinant Newcastle disease virus expressing a North American lineage H7 subtype hemagglutinin.

Article	Year
Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes. Journal of virology\| PubMed ID: 22398287	2012
NS1-truncated live attenuated virus vaccine provides robust protection to aged mice from viral challenge. Journal of virology\| PubMed ID: 22787224	2012
A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates. PloS one\| PubMed ID: 22928001	2012
Residual baculovirus in insect cell-derived influenza virus-like particle preparations enhances immunogenicity. PloS one\| PubMed ID: 23236516	2012
H3N2 influenza virus infection induces broadly reactive hemagglutinin stalk antibodies in humans and mice. Journal of virology\| PubMed ID: 23408625	2013
Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. Journal of virology\| PubMed ID: 23576508	2013
Induction of cross-reactive antibodies to novel H7N9 influenza virus by recombinant Newcastle disease virus expressing a North American lineage H7 subtype hemagglutinin. Journal of virology\| PubMed ID: 23698299	2013

Irina Margine

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