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

Abstract

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. IMPORTANCEProtection 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. H uman respiratory syncytial virus (RSV) (hRSV) is the most frequent cause of severe lower respiratory tract infections (bronchiolitis and pneumonia) in infants and young children throughout the world. It is estimated that each year, the virus causes severe disease in ϳ34 million children Ͻ5 years of age, with Ͼ3.5 million requiring hospitalization, and is responsible for 66,000 to 199,000 deaths, mainly in developing ...

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

“…pRB21/prefusion hRSV F (Pre‐hRSV F): This plasmid encodes the ectodomain of hRSV F (residues 1–524) from the Long strain, stabilized in its prefusion conformation by incorporating the mutations described by McLellan et al (McLellan et al , 2013a) for the DS‐Cav1 protein, which included addition of both an intraprotomer disulfide bridge (S155C‐S290C) and two cavity‐filling substitutions (S190F and V207L) (Palomo et al , 2016). …”

“…pRB21/postfusion hRSV F (Post‐hRSV F): This plasmid encodes residues 1–524 of the hRSV F ectodomain (Long strain) with a deletion of the fusion peptide (residues 137–146) to avoid aggregation (McLellan et al , 2011b; Palomo et al , 2016). …”

“…Stabilized soluble forms of prefusion hRSV F were shown to induce higher levels of neutralizing antibodies than soluble postfusion hRSV F in mice, cotton rats, and non‐human primates (McLellan et al , 2013a; Krarup et al , 2015; Palomo et al , 2016). However, postfusion F, which has the advantage of being highly stable, can induce sizeable levels of neutralizing antibodies and afford protection against hRSV because it shares certain epitopes with prefusion F (Swanson et al , 2011).…”

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

“…Purity and integrity of the different proteins were checked by SDS-PAGE, and their conformation properties were confirmed by electron microscopy and by reactivity in ELISAs with MAb D25, which is specific for the prefusion conformation (33), and MAb 114F, which is specific for the postfusion conformation (53). These ELISAs were performed by capturing serial dilutions of the purified proteins with MAbs bound to 96-well microtiter plates and revealing the amount of protein to each well with an anti-His MAb, as described previously (49).…”

“…Soluble forms of the wild-type hRSV F glycoprotein (Long strain) folded in either the prefusion or postfusion conformation were obtained as described previously (49). Briefly, recombinant vaccinia viruses were obtained that expressed the ectodomain (amino acids [aa] 1 to 524) of the F protein followed by the foldon trimerization domain (50) and a 6ϫHis tag.…”