Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies

Abstract

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

Respiratory syncytial virus entry and how to block it

“…However, a direct interaction using purified components has not been demonstrated. Moreover, the structures of fractalkine and the G cystine noose do not adopt the same fold 52 , and the disulfide-bond arrangement in fractalkine (1-3, 2-4) is different from that in G (1-4, 2-3).…”

“…The cystine noose present in these structures adopts a conserved fold that is also observed in the fourth subdomain of the tumour necrosis factor receptor (TNFR) 51 . In the past year, two groups determined four high-resolution X-ray crystal structures of neutralizing antibodies bound to human RSV G-derived peptides 52,53 . Although the cystine noose was structurally conserved with minor flexibility at the tip of the noose, multiple conformations of the highly conserved amino-terminal flanking region were observed, suggesting that this region is flexible and generally unstructured (FIg.…”

“…However, a different prefusion-specific antibody, REGN2222, which recognizes antigenic site V, recently failed to reach its clinical end points in a phase III study 135 , and the reasons for the failure are not yet known. Other prefusion-specific antibodies are also in preclinical development, as are antibodies directed against the G protein that inhibit viral attachment 52,53 . Cocktails of antibodies against F and G, or against two different antigenic sites on F, are also being considered and may lead to synergistic effects and reduced viral escape.…”

Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes

“…While both RSV F and G are immunogenic and are targeted by neutralizing antibodies, the majority of neutralizing antibodies in human sera target RSV F (8,9). As such, most RSV vaccine candidates and therapeutic antibodies currently in development focus on RSV F. However, RSV that does not express the G protein is highly attenuated in vivo (10), and monoclonal antibodies that target RSV G are protective in vivo (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). In humans, anti-G antibodies are associated with lower clinical disease severity scores, despite an abundance in sera more than 30 times lower than anti-F antibodies (8).…”

“…Most of the anti-G bnmAbs that have been studied to date bind with high affinity to RSV G (K D [binding dissociation constant] ϭ 1.1 pM to 3.3 nM) and bind to linear epitopes within the RSV G CCD as determined by linear epitope mapping techniques (17,21,40,45). Recently, two studies elucidated four high-resolution crystal structures of antibody-RSV G CCD complexes (16,46). Unexpectedly, all four antibodies have additional interactions outside their linear epitopes, revealing a previously unappreciated role of the disulfide-stabilized cysteine noose in forming conformational epitopes and contributing to high-affinity antibody binding.…”