A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism

Krarup, Anders; Truan, Daphné; Furmanova-Hollenstein, Polina; Bogaert, Lies; Bouchier, Pascale; Bisschop, Ilona J.M.; Widjojoatmodjo, Myra N.; Zahn, Roland; Schuitemaker, Hanneke; McLellan, Jason S.; Langedijk, Johannes P. M.

doi:10.1038/ncomms9143

Cited by 290 publications

(362 citation statements)

References 35 publications

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“…Mab 13A8 possessed potency similar to that of motavizumab and D25. mAbs were tested for binding by ELISA to postfusion or prefusion-stabilized disulfide-cavity filling (DsCav1) or single chain-triple mutant (SC-TM) RSV strain A2 F proteins (17,18) and postfusion F from RSV strain 18537 B (Table 1 and Fig. S2).…”

Section: Resultsmentioning

confidence: 99%

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Mousa

Sauer

Sevy

et al. 2016

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

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Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helixloop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.F protein | human respiratory syncytial virus | neutralizing antibodies R espiratory syncytial virus (RSV) is a highly contagious human pathogen, infecting the majority of infants before age 2 y, and is the leading cause of viral bronchiolitis and viral pneumonia in infants and children (1, 2). RSV remains a top priority for vaccine development, as thousands of deaths are recorded worldwide each year because of complications from infection (3). To date, there is no licensed RSV vaccine. A major focus of RSV vaccine development has been inclusion of the RSV fusion (F) protein, a class I fusion glycoprotein that is synthesized as a precursor and cleaved into two disulfide-linked fragments upon maturation into a trimer (4). Although the RSV virion contains two additional surface proteins, the highly-glycosylated attachment (G) protein and the small hydrophobic protein, the F protein is highly conserved among strains of RSV strains and is the major target of protective neutralizing antibodies.The F protein is known to adopt at least two major conformations: the metastable prefusion conformation and the postfusion conformation. Following attachment of the virion to a cell by the G protein, the F protein undergoes a dramatic structural rearrangement, resulting in fusion of the viral and cell membranes, and in cultured cells causes...

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Section: Resultsmentioning

confidence: 99%

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Mousa

Sauer

Sevy

et al. 2016

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

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“…Changes to the HRC (aa 75 to 97) region could be expected to affect processing as it is located N-terminal to the first cleavage site between amino acids 109 and 110. While there are currently no crystal structures of RSV F in its uncleaved state, it has previously been shown that the F protein is likely monomeric prior to cleavage and p27 removal (18,27). Changes in side chain packing may alter helical positioning within monomeric F, thereby affecting presentation of cleavage sites to the required protease(s).…”

Section: Discussionmentioning

confidence: 99%

“…This mutation was proposed to increase hydrophobic interactions between the loop, HRC, and part of HRA and effectively "stick" this loop to these domains. Another mutation at position 215 acted to reduce repulsion forces in this apex region where the HRA, HRD, and HRC helices meet (27).…”

Section: Discussionmentioning

confidence: 99%

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

Bermingham

Chappell

Watterson

et al. 2018

J Virol

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Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F 1 and F 2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic ␣-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F 2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F 2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.KEYWORDS respiratory syncytial virus, fusion, membrane fusion, mutagenesis, heptad repeat R espiratory syncytial virus (RSV) is widely considered the most significant viral pediatric pathogen worldwide. Belonging to the Pneumovirinae family, RSV causes substantial morbidity and mortality worldwide, with an estimated 33.8 million acute lower respiratory tract infections per year in children under 5 years of age, and has been linked to almost 200,000 deaths annually, 99% of which are in developing countries (1, 2). RSV is also recognized as an important pathogen of high-risk adult and elderly populations (3). Despite this, no targeted drug or vaccin...

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“…46 Both of the F protein forms, particularly the pre-fusion form, induce potent neutralizing antibodies. [47][48][49] The identification and quantitative measurement of both the F protein forms were not investigated in this study because both forms induce powerful immune responses. However, we expect that a mixture of pre-and postfusion forms exist in the transfected cells, with the post-fusion form dominating due to its higher stability.…”

Section: Discussionmentioning

confidence: 99%

Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency

Farrag

Amer

Öhlschläger

et al. 2017

Human Vaccines & Immunotherapeutics

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The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codonoptimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M2 82-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8 C T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

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A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism

Cited by 290 publications

References 35 publications

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency

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