Antigenic and sequence variability of the human respiratory syncytial virus F glycoprotein compared to related viruses in a comprehensive dataset.

A comprehensive analysis of sequence variation was carried out comparing the fusion (F) protein of human respiratory syncytial viruses (hRSV) from antigenic groups A and B with the prototype sequence of the A2 strain, also belonging to antigenic group A. The limited number of full bovine RSV F sequences available were included, as well as an extensive set of F sequences from the related human metapneumovirus (hMPV). The results were analysed in the context of the recently determined three dimensional F protein structures, with antigenic sites mapped to these. Although a high degree of sequence conservation in hRSV F exists, and sequence changes did not correlate with location of antigenic sites, preferential accumulation of amino acid changes in certain antigenic sites was noted. When the analysis was extended to hMPV F, a high number of changes was noticed, in agreement with the limited degree of sequence conservation. However, some conserved regions were noted, which may account for the limited number of cross-reactive monoclonal antibodies described between hRSV F and hMPV F. These results provide information about the degree of sequence and antigenic variation currently found in the F protein of circulating viruses. They highlight the importance of establishing a baseline dataset to monitor for future changes that might evolve should preventative immunological measures be made widely available.

The respiratory syncytial virus vaccine landscape: lessons from the graveyard and promising candidates.

The global burden of disease caused by respiratory syncytial virus (RSV) is increasingly recognised, not only in infants, but also in older adults (aged ≥65 years). Advances in knowledge of the structural biology of the RSV surface fusion glycoprotein have revolutionised RSV vaccine development by providing a new target for preventive interventions. The RSV vaccine landscape has rapidly expanded to include 19 vaccine candidates and monoclonal antibodies (mAbs) in clinical trials, reflecting the urgency of reducing this global health problem and hence the prioritisation of RSV vaccine development. The candidates include mAbs and vaccines using four approaches: (1) particle-based, (2) live-attenuated or chimeric, (3) subunit, (4) vector-based. Late-phase RSV vaccine trial failures highlight gaps in knowledge regarding immunological protection and provide lessons for future development. In this Review, we highlight promising new approaches for RSV vaccine design and provide a comprehensive overview of RSV vaccine candidates and mAbs in clinical development to prevent one of the most common and severe infectious diseases in young children and older adults worldwide.

Chimeric Pneumoviridae fusion proteins as immunogens to induce cross-neutralizing antibody responses.

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV However, no cross-neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well-characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus-specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross-neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross-neutralizing antibody and protective responses against more than one human pneumovirus.

The Complexity of Antibody Responses Elicited against the Respiratory Syncytial Virus Glycoproteins in Hospitalized Children Younger than 2 Years.

The influence of age and maternal antibodies on the antibody responses to human respiratory syncytial virus (hRSV) glycoproteins in very young children has been a matter of controversy. Both, immaturity of the immune system at very early age and suppression of the host immune response by high level of maternal antibodies have been claimed to limit the host antibody response to virus infection and to jeopardize the use of hRSV vaccines under development in that age group. Hence, the antibody responses to the two major hRSV glycoproteins (F and G) were evaluated in children younger than 2 years, hospitalized with laboratory confirmed hRSV bronchiolitis. A strong negative correlation was found between the titre of circulating ELISA antibodies directed against either prefusion or postfusion F in the acute phase, but not age, and their fold change at convalescence. These changes correlated also with the level of circulating neutralizing antibodies in sera. As reported in adults, most neutralizing antibodies in a subset of tested sera could not be depleted with postfusion F, suggesting that they were mostly directed against prefusion-specific epitopes. In contrast, a weak negative association was found for group-specific anti-G antibodies in the acute phase and their fold change at convalescence only after correcting for the antigenic group of the infecting virus. In addition, large discrepancies were observed in some individuals between the antibody responses specific for F and G glycoproteins. These results illustrate the complexity of the anti-hRSV antibody responses in children experiencing a primary severe infection and the influence of preexisting maternal antibodies on the host response, factors that should influence hRSV serological studies as well as vaccine development.

Corrigendum: Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state.

This corrects the article DOI: 10.1038/ncomms14158.

Structure and immunogenicity of pre-fusion-stabilized human metapneumovirus F glycoprotein.

Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus-cell membrane fusion and is the primary target of neutralizing antibodies. The inability to produce recombinant hMPV F glycoprotein in the metastable pre-fusion conformation has hindered structural and immunological studies. Here, we engineer a pre-fusion-stabilized hMPV F ectodomain and determine its crystal structure to 2.6 Å resolution. This structure reveals molecular determinants of strain-dependent acid-induced fusion, as well as insights into refolding from pre- to post-fusion conformations. A dense glycan shield at the apex of pre-fusion hMPV F suggests that antibodies against this site may not be elicited by host immune responses, which is confirmed by depletion studies of human immunoglobulins and by mouse immunizations. This is a major difference with pre-fusion F from human respiratory syncytial virus (hRSV), and collectively our results should facilitate development of effective hMPV vaccine candidates.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state.

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV.

Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development.

Extraordinary progress in the structure and immunobiology of the human respiratory syncytial virus glycoproteins has been accomplished during the last few years. Determination of the fusion (F) glycoprotein structure folded in either the prefusion or the postfusion conformation was an inspiring breakthrough not only to understand the structural changes associated with the membrane fusion process but additionally to appreciate the antigenic intricacies of the F protein. Furthermore, these developments have opened new avenues for structure-based designs of promising hRSV vaccine candidates. Finally, recent advances in our knowledge of the attachment (G) glycoprotein and its interaction with cell-surface receptors have revitalized interest in this molecule as a vaccine, as well as its role in hRSV immunobiology.

Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation.

Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses.

Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection.

ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.

Influence of antigen conformation and mode of presentation on the antibody and protective responses against human respiratory syncytial virus: relevance for vaccine development.

Human respiratory syncytial virus (hRSV) remains one of the most prevalent human pathogens for which a vaccine is still missing. After several decades of hesitant efforts, particularly after the harmful effects of a formalin-inactivated hRSV vaccine trial in the 1960s, hRSV vaccine development has received new impetus from structure-based studies of its main protective antigen: the fusion (F) glycoprotein. This article reviews studies done with hRSV F, either in pieces (e.g. epitopes) or as soluble or membrane-anchored molecules folded in different conformations or presented under different forms. Knowledge gained from these studies has provided the basis for novel vaccines that are now in different phases of development and has generated tools and reagents for developing other control measures such as prophylactic or therapeutic antibodies against this virus, which remains the most important cause of hospitalization in infants and one of the leading global causes of infant mortality.

Influence of Respiratory Syncytial Virus F Glycoprotein Conformation on Induction of Protective Immune Responses.

UNLABELLED: Human respiratory syncytial virus (hRSV) vaccine development has received new impetus from structure-based studies of its main protective antigen, the fusion (F) glycoprotein. Three soluble forms of F have been described: monomeric, trimeric prefusion, and trimeric postfusion. Most human neutralizing antibodies recognize epitopes found exclusively in prefusion F. Although prefusion F induces higher levels of neutralizing antibodies than does postfusion F, postfusion F can also induce protection against virus challenge in animals. However, the immunogenicity and protective efficacy of the three forms of F have not hitherto been directly compared. Hence, BALB/c mice were immunized with a single dose of the three proteins adjuvanted with CpG and challenged 4 weeks later with virus. Serum antibodies, lung virus titers, weight loss, and pulmonary pathology were evaluated after challenge. Whereas small amounts of postfusion F were sufficient to protect mice, larger amounts of monomeric and prefusion F proteins were required for protection. However, postfusion and monomeric F proteins were associated with more pathology after challenge than was prefusion F. Antibodies induced by all doses of prefusion F, in contrast to other F protein forms, reacted predominantly with the prefusion F conformation. At high doses, prefusion F also induced the highest titers of neutralizing antibodies, and all mice were protected, yet at low doses of the immunogen, these antibodies neutralized virus poorly, and mice were not protected. These findings should be considered when developing new hRSV vaccine candidates. IMPORTANCE: Protection against hRSV infection is afforded mainly by neutralizing antibodies, which recognize mostly epitopes found exclusively in the viral fusion (F) glycoprotein trimer, folded in its prefusion conformation, i.e., before activation for membrane fusion. Although prefusion F is able to induce high levels of neutralizing antibodies, highly stable postfusion F (found after membrane fusion) is also able to induce neutralizing antibodies and protect against infection. In addition, a monomeric form of hRSV F that shares epitopes with prefusion F was recently reported. Since each of the indicated forms of hRSV F may have advantages and disadvantages for the development of safe and efficacious subunit vaccines, a direct comparison of the immunogenic properties and protective efficacies of the different forms of hRSV F was made in a mouse model. The results obtained show important differences between the noted immunogens that should be borne in mind when considering the development of hRSV vaccines.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation.

UNLABELLED: Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15(-/-)) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE: At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

Rapid profiling of RSV antibody repertoires from the memory B cells of naturally infected adult donors.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in young children and the elderly. There are currently no licensed RSV vaccines, and passive prophylaxis with the monoclonal antibody palivizumab is restricted to high-risk infants in part due to its modest efficacy. Although it is widely agreed that an effective RSV vaccine will require the induction of a potent neutralizing antibody response against the RSV fusion (F) glycoprotein, little is known about the specificities and functional activities of RSV F-specific antibodies induced by natural infection. Here, we have comprehensively profiled the human antibody response to RSV F by isolating and characterizing 364 RSV F-specific monoclonal antibodies from the memory B cells of three healthy adult donors. In all donors, the antibody response to RSV F is comprised of a broad diversity of clones that target several antigenic sites. Nearly half of the most potent antibodies target a previously undefined site of vulnerability near the apex of the prefusion conformation of RSV F (preF), providing strong support for the development of RSV vaccine candidates that preserve the membrane-distal hemisphere of the preF protein. Additionally, the antibodies targeting this new site display convergent sequence features, thus providing a future means to rapidly detect the presence of these antibodies in human vaccine samples. Many of the antibodies that bind preF-specific surfaces are over 100 times more potent than palivizumab, and several cross-neutralize human metapneumovirus (HMPV). Taken together, the results have implications for the design and evaluation of RSV vaccine candidates and offer new options for passive prophylaxis.

Generation of monoclonal antibodies specific of the postfusion conformation of the Pneumovirinae fusion (F) protein.

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins, the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins, and as extension of previous work (Palomo et al., 2014), a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion.

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics.

The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.

Conservation of G-Protein Epitopes in Respiratory Syncytial Virus (Group A) Despite Broad Genetic Diversity: Is Antibody Selection Involved in Virus Evolution?

UNLABELLED: Worldwide G-glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than 50 years of recorded history. Multiple genotypes cocirculated during prolonged periods of time, but recent dominance of the GA2 genotype was noticed in several studies, and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with monoclonal antibodies (MAbs) that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated with the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as a major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV G-protein variability. IMPORTANCE: An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of nonsynonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS values at certain sites predictive of positive selection. Since these sites cluster preferentially in the C-terminal third of the G protein, like certain epitopes recognized by murine antibodies, it was proposed that immune (antibody) selection might be driving the apparent positive selection, analogous to the antigenic drift observed in the influenza virus hemagglutinin (HA). However, careful antigenic and genetic comparison of the G glycoprotein does not provide evidence of antigenic drift in the G molecule, in agreement with recently published data which did not indicate antigenic drift in the G protein with human sera. Alternative explanations to the immune-driven selection hypothesis are offered to account for the high level of G-protein genetic diversity highlighted in this study.