Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines

Lu, Yuan; Welsh, John P.; Swartz, James R.

doi:10.1073/pnas.1308701110

Cited by 191 publications

(153 citation statements)

References 30 publications

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“…HA-immunized mouse mortality was as high as that of the control groups, possibly because HA did not acquire its native structure. The so-called foldon element from the T4 bacteriophage is absent in our constructions but is necessary for correct HA folding and trimer assembly (49).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

Pascual

Mata

Gómez-Blanco

et al. 2015

J Virol

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Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ϳ70-nmdiameter T‫31؍‬ IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic ␣ helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this ␣ helix assemble into genuine VLPs only when expressed with an N-terminal His 6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ϳ78,000 nm 3 ) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (ϳ240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA-and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. IMPORTANCEVirus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.

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Section: Discussionmentioning

confidence: 99%

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

Pascual

Mata

Gómez-Blanco

et al. 2015

J Virol

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“…It is known that the HA 2 trimer without HA 1 forms the postfusion structure (22,39) and that HA 1 undergoes no conformational change at low pH (7). The tendency for HA 1 to dissociate at low pH can be easily understood from the high isoelectric point of the HA 1 (pI∼8) compared with HA 2 (pI∼5) (40). Therefore, for simplicity, we removed HA 1 from the model.…”

Section: Methodsmentioning

confidence: 99%

Order and disorder control the functional rearrangement of influenza hemagglutinin

Lin

Eddy

Noel

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Influenza hemagglutinin (HA), a homotrimeric glycoprotein crucial for membrane fusion, undergoes a large-scale structural rearrangement during viral invasion. X-ray crystallography has shown that the pre-and postfusion configurations of HA 2 , the membranefusion subunit of HA, have disparate secondary, tertiary, and quaternary structures, where some regions are displaced by more than 100 Å. To explore structural dynamics during the conformational transition, we studied simulations of a minimally frustrated model based on energy landscape theory. The model combines structural information from both the pre-and postfusion crystallographic configurations of HA 2 . Rather than a downhill drive toward formation of the central coiled-coil, we discovered an order-disorder transition early in the conformational change as the mechanism for the release of the fusion peptides from their burial sites in the prefusion crystal structure. This disorder quickly leads to a metastable intermediate with a broken threefold symmetry. Finally, kinetic competition between the formation of the extended coiled-coil and C-terminal melting results in two routes from this intermediate to the postfusion structure. Our study reiterates the roles that cracking and disorder can play in functional molecular motions, in contrast to the downhill mechanical interpretations of the "springloaded" model proposed for the HA 2 conformational transition.protein folding | structure-based model H emagglutinin (HA) is a viral receptor-binding and membrane-fusion glycoprotein involved in the invasion of influenza virions into host cells (1). Structural rearrangements of HA during membrane fusion are crucial for the delivery of the viral genome. The postfusion conformation of HA shows considerable similarity to other viral fusion proteins and eukaryotic membrane receptors involved in intracellular vesicle trafficking (2), suggesting there may be common mechanisms in the function of these proteins. Therefore, HA may serve as a model system, allowing characterization of the molecular and energetic details that underlie its conformational transition to provide insights into general principles of membrane fusion (3).HA is a homotrimer consisting of two domains connected by disulfide bonds (4): a globular receptor binding domain (HA 1 ), and a coiled-coil membrane-fusion domain anchored to the viral membrane (HA 2 ). Recognized by the sialic acid receptor of a host cell, the intact virus enters the cell via endocytosis. Low pH in a late endosome then induces the dissociation of HA 1 from HA 2 (1) and an irreversible conformational transition of HA 2 . Experimentally, this conformational change can be triggered by either low pH, high temperature, or urea denaturation (5).Structures of HA in pre-and postfusion pH conformations have been solved by X-ray crystallography. The structure of the prefusion ectodomain contains both HA 1 and HA 2 , and was purified from influenza virions (4). A postfusion conformation of HA 1 and HA 2 were obtained from prefusion viral HA th...

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“…The discovery of conserved epitopes in the HA stem region has spurred great efforts on development of stem-based "universal" vaccines (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Basically, there are two approaches.…”

mentioning

confidence: 99%

“…Sequential prime-boosts with cHA containing a common H1 stem were able to cross-protect mice from lethal challenge of H5N1 and H6N1 viruses (group 1) and to cross-neutralize H2N2 virus (group 1) (9), whereas sequential prime-boosts with cHA containing a common H3 stem were able to cross-protect mice from lethal challenge of H7N1 and H7N9 (group 2) (10). Another approach was to develop stem-only (or headless) HA (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). For example, two groups recently reported the construction of correctly folded H1 stem trimer (20) and self-assembled H1 stem-containing nanoparticle (21).…”

mentioning

confidence: 99%

A Bivalent Heterologous DNA Virus-Like-Particle Prime-Boost Vaccine Elicits Broad Protection against both Group 1 and 2 Influenza A Viruses

Jiang

Wang

Chen

et al. 2017

J Virol

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Current seasonal influenza vaccines are efficacious when vaccine strains are matched with circulating strains. However, they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Thus, there is a critical need for developing so-called "universal" vaccines that protect against all influenza viruses. In the present study, we developed a bivalent heterologous DNA virus-like particle prime-boost vaccine strategy. We show that mice immunized with this vaccine were broadly protected against lethal challenge from group 1 (H1, H5, and H9) and group 2 (H3 and H7) viruses, with 94% aggregate survival. To determine the immune correlates of protection, we performed passive immunizations and in vitro assays. We show that this vaccine elicited antibody responses that bound HA from group 1 (H1, H2, H5, H6, H8, H9, H11, and H12) and group 2 (H3, H4, H7, H10, H14, and H15) and neutralized homologous and intrasubtypic H5 and H7 and heterosubtypic H1 viruses and hemagglutinin-specific CD4 and CD8 T cell responses. As a result, passive immunization with immune sera fully protected mice against H5, H7, and H1 challenge, whereas with both immune sera and T cells the mice survived heterosubtypic H3 and H9 challenge. Thus, it appears that (i) neutralizing antibodies alone fully protect against homologous and intrasubtypic H5 and H7 and (ii) neutralizing and binding antibodies are sufficient to protect against heterosubtypic H1, (iii) but against heterosubtypic H3 and H9, binding antibodies and T cells are required for complete survival. We believe that this vaccine regimen could potentially be a candidate for a "universal" influenza vaccine.IMPORTANCE Influenza virus infection is global health problem. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. However, these vaccines do not protect antigenic variants and newly emerging pandemic and outbreak strains. Because of this, there is an urgent need to develop so-called "universal" influenza vaccines that can protect against both current and future influenza strains. In the present study, we developed a bivalent heterologous prime-boost vaccine strategy. We show that a bivalent vaccine regimen elicited broad binding and neutralizing antibody and T cell responses that conferred broad protection against diverse challenge viruses in mice, suggesting that this bivalent prime-boost strategy could practically be a candidate for a "universal" influenza vaccine. Citation Jiang W, Wang S, Chen H, Ren H, Huang X, Wang G, Chen Z, Chen L, Chen Z, Zhou P. 2017. A bivalent heterologous DNA virus-like-particle prime-boost vaccine elicits broad protection against both group 1 and 2 influenza A viruses. J Virol 91:e02052-16. KEYWORDS influenza vaccine

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Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines

Cited by 191 publications

References 30 publications

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

Structural Basis for the Development of Avian Virus Capsids That Display Influenza Virus Proteins and Induce Protective Immunity

Order and disorder control the functional rearrangement of influenza hemagglutinin

A Bivalent Heterologous DNA Virus-Like-Particle Prime-Boost Vaccine Elicits Broad Protection against both Group 1 and 2 Influenza A Viruses

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