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

The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.

Evaluation of photoreactivation of Escherichia coli and Enterococci after UV disinfection of municipal wastewater.

Because chlorine disinfection is not permitted in the province of Quebec, wastewater disinfection by ultraviolet (UV) light has been used for years in wastewater treatment plants. Thermotolerant coliforms discharge criteria are set for each plant and are adjusted by a factor of 1 log to compensate for photoreactivation in UV-disinfected effluents. The current study evaluated levels of Escherichia coli and enterococci photoreactivation from disinfected wastewater under varying temperature, visible light, and type of UV lamps. Escherichia coli photoreactivation increased significantly after exposure to 5600 lx compared with 1600 lx of visible light. This increase was significantly higher in warm water (25 degrees C) than cold water (4 degrees C). The level of photoreactivation of E. coli was also higher after wastewater disinfection by low-pressure UV lamps as opposed to medium-pressure UV lamps. Enterococci, however, were not photoreactivated under any test conditions. This result suggests that enterococci could be a better indicator than thermotolerant coliforms or E. coli. The use of enterococci would also eliminate the requirement to set different discharge criteria based on disinfection type (UV or chemical) and would also provide a better assessment of treatment efficiency for more resistant microorganisms.