The thermal melting curve of 1 1 M 4C-stored DS-Cav1 may also show a similar early transition (Fig 6B), again suggesting that, while the DS-Cav1 stored at 4C is more stable than freshly thawed DS-Cav1, its likely not as stable as postfusion F, for which no second transition temperature was detected at any of the protein concentrations tested. protein concentration. (C) The integral between 50C and 75C (area of Tm1) of F350/F330 DSF unfolding curves for freshly thawed DS-Cav1 at 35 M, 17.5 M, 4.4 M, 2.2 M, 1.1 M, 0.5 M and 0.3 M was plotted against the protein concentration. The data points are fitted with a sigmoidal curve. The midpoint of the sigmoidal curve is at 2.8 M.(PDF) pone.0164789.s003.pdf (748K) GUID:?C41F341E-0C5C-4D4D-8762-BBB6E9ACFADC Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Contamination with Respiratory Syncytial Computer virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the contamination process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an designed RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4C or higher is usually a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site ?, and a novel antibody 4D7, which was found to bind antigenic site I around the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly drop the site ? epitope and gain the ability to bind 4D7. Introduction Respiratory Syncytial Computer virus (RSV) infections are common and generally cause moderate, cold-like symptoms in healthy adults and older children. However, in premature babies, infants, older adults and immunocompromised individuals, RSV can lead to more severe lower respiratory tract disease, causing pneumonia or bronchiolitis, and may be life-threatening [1C4]. Despite extensive research effort, there is no vaccine available to prevent RSV contamination or disease. Passive prophylaxis with palivizumab (Synagis?), however, is usually approved for use in a subset of preterm infants that are at best risk for developing severe RSV-induced lung disease. Palivizumab is usually a humanized monoclonal antibody that binds OAC1 one of the RSV surface-exposed envelope glycoproteins, the fusion protein F [5, 6]. The clinical efficacy of palivizumab, a reduction in RSV-related hospitalization [7, 8], provides proof of concept that a vaccine that can elicit an anti-F neutralizing antibody response would show effective against RSV-induced disease. Targeting RSV F as a vaccine antigen is usually OAC1 complicated by the fact that this protein can adopt multiple conformations. On the computer virus surface, RSV F can exist in a metastable prefusion conformation that, during the contamination process, rearranges to a more stable postfusion form (Fig 1), to enable computer virus entry into the host cell. At least two antigenic sites uncovered on both the prefusion and postfusion forms of F (sites II and IV) are recognized by antibodies with neutralizing activity (Fig 1) [9C13]. However, depleting postfusion F-binding antibodies from convalescent human serum only OAC1 modestly reduces the ability of the sample to neutralize RSV [14C16]. Adsorption of antibodies that bind the Sfpi1 prefusion conformation of F, in contrast, removes almost all of the serum neutralizing activity . Taken together, these.