A Monomeric Uncleaved Respiratory Syncytial Virus F Antigen Retains Prefusion-Specific Neutralizing Epitopes

Abstract

Respiratory syncytial virus (RSV) is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. There remains an unmet vaccine need despite decades of research. Insufficient potency, homogeneity, and stability of previous RSV fusion protein (F) subunit vaccine candidates have hampered vaccine development. RSV F and related parainfluenza virus (PIV) F proteins are cleaved by furin during intracellular maturation, producing disulfide-linked F1 and F2 fragments. During cell entry, the cleaved Fs rearrange from prefusion trimers to postfusion trimers. Using RSV F constructs with mutated furin cleavage sites, we isolated an uncleaved RSV F ectodomain that is predominantly monomeric and requires specific cleavage between F1 and F2 for self-association and rearrangement into stable postfusion trimers. The uncleaved RSV F monomer is folded and homogenous and displays at least two key RSV-neutralizing epitopes shared between the prefusion and postfusion conformations. Unlike the cleaved trimer, the uncleaved monomer binds the prefusion-specific monoclonal antibody D25 and human neutralizing immunoglobulins that do not bind to postfusion F. These observations suggest that the uncleaved RSV F monomer has a prefusion-like conformation and is a potential prefusion subunit vaccine candidate. IMPORTANCERSV is the leading infectious cause of severe respiratory disease in infants and a major cause of respiratory illness in the elderly. Development of an RSV vaccine was stymied when a clinical trial using a formalin-inactivated RSV virus made disease, following RSV infection, more severe. Recent studies have defined the structures that the RSV F envelope glycoprotein adopts before and after virus entry (prefusion and postfusion conformations, respectively). Key neutralization epitopes of prefusion and postfusion RSV F have been identified, and a number of current vaccine development efforts are focused on generating easily produced subunit antigens that retain these epitopes. Here we show that a simple modification in the F ectodomain results in a homogeneous protein that retains critical prefusion neutralizing epitopes. These results improve our understanding of RSV F protein folding and structure and can guide further vaccine design efforts.

Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation

“…As a result, the FP remains attached to the C terminus of F2, preventing a structural translocation of approximately 100 Å that would normally be required for the formation of the post-F 6HB. In line with results obtained by others, the removal of pep27 allowed the folding of the protein in its trimeric form (42,44,54). We have shown that the presence of pep27 and the two furin sites in the context of the Post-F-XC construct did not necessarily lead to a complete processing of the peptide.…”

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

“…As motavizumab has been previously shown to bind both cleaved and uncleaved RSV F with similar dissociation constants, quantification of cell surface expression should not be directly affected by processing (18). This experiment was also duplicated and confirmed with 18B2 (Argene), which targets an unknown epitope on F 1 that is exposed in both the pre-and postfusion forms (data not shown).…”

“…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).…”

Nonglycosylated G-Protein Vaccine Protects against Homologous and Heterologous Respiratory Syncytial Virus (RSV) Challenge, while Glycosylated G Enhances RSV Lung Pathology and Cytokine Levels

“…Most recent RSV vaccine development efforts are focused on the F protein, which is relatively conserved among strains (3) and can be expressed in mammalian and insect cells (14,18,19,41). The G attachment protein, expressed on the virus surface, also represents an important target of protective immunity.…”

“…A mixture of F, G, and M recombinant proteins was tested in individuals Ͼ65 years old and was found to induce Ͼ4-fold increases in serum neutralizing activity in 58% of subjects with low prevaccine titers (16). Recently, the structures of the F protective targets recognized by the monoclonal antibodies (MAbs) palivizumab and 101F were resolved, as well as the prefusion form of the F protein trimer, leading to the structure-based design of stabilized F protein vaccine candidates (17)(18)(19). The G protein was also evaluated as a vaccine candidate in preclinical studies.…”