Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract illness in infants and young children. The significant morbidity and mortality rates associated with RSV infection make an effective RSV vaccine development a priority. Two neutralising antibody binding sites, Ø and II, located on the pre-fusion RSV F glycoprotein are prime candidates for epitope-focused vaccine design. We report on a vaccine strategy that utilises a lipid core peptide (LCP) delivery system with self-adjuvanting properties in conjunction with either the antigenic site Ø or II (B cell epitopes) along with PADRE as a T helper cell epitope. These LCP constructs adopted the desired helical conformation in solution and were recognised by their cognate antibodies D25 and Motavizumab, specific for site Ø and II on RSV F protein, respectively. The LCP constructs were capable of eliciting higher levels of antigen specific antibodies than those induced by antigens administered with complete Freund’s adjuvant, demonstrating the potent adjuvanting properties of LCP delivery. However, the antibodies induced failed to recognise native F protein or neutralise virus infectivity. These results provide a note of caution in assuming that peptide vaccines, successfully designed to structurally mimic minimal linear B cell epitopes, will necessarily elicit the desired immune response.

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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