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Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

ABSTRACT: We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.

SUBMITTER: Fleishman SJ

PROVIDER: S-EPMC3164876 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

Fleishman Sarel J SJ Whitehead Timothy A TA Ekiert Damian C DC Dreyfus Cyrille C Corn Jacob E JE Strauch Eva-Maria EM Wilson Ian A IA Baker David D

Science (New York, N.Y.) 20110501 6031

We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 ...[more]

PMID: 21566186

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Project description:Influenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.N-linked glycosylation sites in the stem regions of influenza virus hemagglutinin (HA) proteins are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Our studies indicate that unmasking the HA2 stem N-glycans of recombinant HA proteins from H5N1 and pH1N1 viruses induced more potent neutralizing antibody titers against homologous and heterosubtypic viruses. However, only immunization with the H5N1 HA2 stem mutant protein can refocus B antibody responses to the helix A epitope for inducing more CR6261-like/FI6v3-like and fusion inhibition antibodies in antisera, resulting in a significant improvement for the protection against lethal H5N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

Project description:Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Dataset Information

Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

Publications

Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

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