Structure-based Vaccine Design: Lessons from RSV F

The use of atomic-level information in the design of immunogens has created new candidate vaccines against important pathogens. From our work on the fusion glycoprotein from respiratory syncytial virus (RSV), we have learned several important lessons that may be generally applicable to other structure-based vaccine-design efforts. This talk will summarize these lessons and the latest advances in the field, as well as highlight opportunities for future development

Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development

Extraordinary progress in the structure and immunobiology of the human respiratory syncytial virus glycoproteins has been accomplished during the last few years. Determination of the fusion (F) glycoprotein structure folded in either the prefusion or the postfusion conformation was an inspiring breakthrough not only to understand the structural changes associated with the membrane fusion process but additionally to appreciate the antigenic intricacies of the F protein. Furthermore, these developments have opened new avenues for structure-based designs of promising hRSV vaccine candidates. Finally, recent advances in our knowledge of the attachment (G) glycoprotein and its interaction with cell-surface receptors have revitalized interest in this molecule as a vaccine, as well as its role in hRSV immunobiology.Work in the Madrid lab is currently funded by grant SAF2015-67033-R from Plan Nacional de I+D+I. J.S.M is supported in part by award P20GM113132 from the National Institute of General Medical Sciences of the National Institutes of Health.S

Mental Health and Substance Use in NCAA Athletes in the Context of the COVID-19 Pandemic and Lockdown.

Background Although the coronavirus disease 2019 (COVID-19) pandemic had pervasive effects on the lives of individuals, its influence on the mental health of collegiate athletes remains unknown. This study aimed to assess changes in mental health and substance use in National Collegiate Athlete Association (NCAA) Division I athletes in Southern California during the COVID-19 pandemic. Methodology An online survey was created using the Qualtrics software (Qualtrics, Provo, Utah). NCAA Division I athletes in Southern California completed preseason surveys querying indices of mental health, substance use, and injury in the year before the COVID-19 pandemic (March 2019 to March 2020) and during the pandemic (March 2020 to March 2021). The athletes filled out the survey from June 2021 to September 2021. Participants were asked how likely they were to agree with the following statements: I have felt physically prepared for athletic competitions, I have been satisfied with my mental health, and I have had adequate sleep. Participants were also asked to compare their substance use between the two time periods. Sociodemographic information regarding participants age, gender, sports team, as well as year in sport and school was also collected. Group comparison analyses were performed using Fishers exact test. Correlations between mental health measures and other variables were examined using Spearmans correlation coefficients. Results A total of 189 athletes completed the survey (out of the 259 surveys that were started). Females were significantly less likely to feel satisfied with mental health (p < 0.01) and physically prepared for sport (p < 0.01). Across all respondents, satisfaction with mental health was positively correlated with adequate sleep (p < 0.01) and physical preparedness for sport (p < 0.01) and negatively correlated with injury (p < 0.05). There was no significant correlation between mental health status and history of COVID-19 infection (p = 0.84). The vast majority of athletes reported no significant change in substance use pre- to post-pandemic, with no differences according to sex. Conclusions The COVID-19 pandemic had a differential impact on the mental health of female versus male NCAA athletes. Mental health was correlated with sleep, physical preparedness, and being injury-free but not with a history of COVID-19 infection. Despite reports indicating increased substance use in the general population, athletes in this group reported no change in licit and illicit substance use

Lake Roosevelt White Sturgeon Recovery Project : Annual Progress Report, January 2003 – March 2004.

This report summarizes catch data collected from white sturgeon Acipenser transmontanus in Lake Roosevelt during limited setlining and gill netting activities in the fall of 2003, and documents progress toward development of a U.S. white sturgeon conservation aquaculture program for Lake Roosevelt. From 27-30 October, 42 overnight small mesh gill net sets were made between Marcus and Northport, WA for a total catch of 15 juvenile white sturgeon (275-488 mm FL). All sturgeon captured were of Canadian hatchery origin. These fish had been previously released as sub-yearlings into the Canadian portion (Keenleyside Reach) of the Transboundary Reach of the Columbia River during 2002 and 2003. Most sturgeon (n=14) were caught in the most upstream area sampled (Northport) in low velocity eddy areas. Five fish exhibited pectoral fin deformities (curled or stunted). Growth rates were less than for juvenile sturgeon captured in the Keenleyside Reach but condition factor was similar. Condition factor was also similar to that observed in juvenile sturgeon (ages 1-8) captured in the unimpounded Columbia River below Bonneville Dam between 1987-92. From 10-14 November, 28 overnight setline sets were made in the Roosevelt Reach between the confluence of the Spokane River and Marcus Island for a total catch of 17 white sturgeon (94-213 cm FL). Catch was greatest in the most upstream areas sampled, a distribution similar to that observed during a WDFW setline survey in Lake Roosevelt in 1998. The mean W{sub r} index of 110% for fish captured this year was higher than the mean W{sub r} of 91% for fish captured in 1998. Excellent fish condition hindered surgical examination of gonads as lipid deposits made the ventral body wall very thick and difficult to penetrate with available otoscope specula. Acoustic tags (Vemco model V16 coded pingers, 69 kHz, 48-month life expectancy) were internally applied to 15 fish for subsequent telemetry investigations of seasonal and reproductively motivated movements. In August 2003, three Vemco VR2 fixed station acoustic receivers, supplied by the UCWSRI Transboundary Telemetry Project, were deployed in the vicinities of Kettle Falls Bridge, Marcus Island, and Northport, WA. Data downloaded from these receivers through December 2003 confirmed the findings of a previous telemetry study that the Marcus area is an important overwintering habitat for white sturgeon. On 18 February 2004, juvenile white sturgeon (n=2,000) were transported from Kootenay Sturgeon Hatchery in British Columbia to WDFW Columbia Basin Hatchery (CBH) in Moses Lake, WA. Fish were reared at CBH to approximately 30 g and individually outfitted with PIT tags and scute marked. On 11 May 2004, fish were released into Lake Roosevelt in the vicinities of Kettle Falls Bridge, North Gorge, and Northport

Lake Roosevelt Fisheries Evaluation Program : Lake Whatcom Kokanee Salmon (Oncorhynchus nerka kennerlyi) : Investigations in Lake Roosevelt Annual Report 1999-2000.

Lake Whatcom stock kokanee have been planted in Lake Roosevelt since 1988 with the primary goal of establishing a self-sustaining fishery. Returns of hatchery kokanee to egg collection facilities and recruitment to the creel have been minimal. Therefore, four experiments were conducted to determine the most appropriate release strategy that would increase kokanee returns. The first experiment compared morpholine and non-morpholine imprinted kokanee return rates, the second experiment compared early and middle run Whatcom kokanee, the third experiment compared early and late release dates, and the fourth experiment compared three net pen release strategies: Sherman Creek hatchery vs. Sherman Creek net pens, Colville River net pens vs. Sherman Creek net pens, and upper vs. lower reservoir net pen releases. Each experiment was tested in three ways: (1) returns to Sherman Creek, (2) returns to other tributaries throughout the reservoir, and (3) returns to the creel. Chi-square analysis of hatchery and tributary returns indicated no significant difference between morpholine imprinted and non-imprinted fish, early run fish outperformed middle run fish, early release date outperformed late release fish, and the hatchery outperformed all net pen releases. Hatchery kokanee harvest was estimated at 3,323 fish, which was 33% of the total harvest. Return rates (1998 = 0.52%) of Whatcom kokanee were low indicating an overall low performance that could be caused by high entrainment, predation, and precocity. A kokanee stock native to the upper Columbia, as opposed to the coastal Whatcom stock, may perform better in Lake Roosevelt

Temporary Restoration of Bull Trout Passage at Albeni Falls Dam

This study was designed to monitor movements of bull trout that were provided passage above Albeni Falls Dam, Pend Oreille River. Electrofishing and angling were used to collect bull trout below the dam. Tissue samples were collected from each bull trout and sent to the U. S. Fish and Wildlife Service Abernathy Fish Technology Center Conservation Genetics Lab, Washington. The DNA extracted from tissue samples were compared to a catalog of bull trout population DNA from the Priest River drainage, Lake Pend Oreille tributaries, and the Clark Fork drainage to determine the most probable tributary of origin. A combined acoustic radio or radio tag was implanted in each fish prior to being transported and released above the dam. Bull trout relocated above the dam were able to volitionally migrate into their natal tributary, drop back downstream, or migrate upstream to the next dam. A combination of stationary radio receiving stations and tracking via aircraft, boat, and vehicle were used to monitor the movement of tagged fish to determine if the spawning tributary it selected matched the tributary assigned from the genetic analysis. Seven bull trout were captured during electrofishing surveys in 2008. Of these seven, four were tagged and relocated above the dam. Two were tagged and left below the dam as part of a study monitoring movements below the dam. One was immature and too small at the time of capture to implant a tracking tag. All four fish released above the dam passed by stationary receivers stations leading into Lake Pend Oreille and no fish dropped back below the dam. One of the radio tags was recovered in the tributary corresponding with the results of the genetic test. Another fish was located in the vicinity of its assigned tributary, which was impassable due to low water discharge at its mouth. Two fish have not been located since entering the lake. Of these fish, one was immature and not expected to enter its natal tributary in the fall of 2008. The other fish was large enough to be mature, but at the time of capture its sex was unable to be determined, indicating it may not have been mature at the time of capture. These fish are expected to enter their natal tributaries in early summer or fall of 2009

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV

Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins

The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions. Through combinatorial addition of disulfide bonds, cavity-filling substitutions, and improved electrostatic interactions, we describe a prefusion-stabilized F protein (DS-CavEs2) that expresses at 15 mg/L and has a melting temperature of 71.9 °C. Crystal structures of two prefusion-stabilized hMPV F variants reveal that antigenic surfaces are largely unperturbed. Importantly, immunization of mice with DS-CavEs2 elicits significantly higher neutralizing antibody titers against hMPV A1 and B1 viruses than postfusion F. The improved properties of DS-CavEs2 will advance the development of hMPV vaccines and the isolation of therapeutic antibodies.This work was funded in part by Welch Foundation grant number F-0003-19620604 (J.S.M). Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy (DOE), Office of Biological and Environmental Research under Contract DE-AC02-06CH11357.S

The impacts of COVID-19 vaccine timing, number of doses, and risk prioritization on mortality in the US

As COVID-19 vaccination begins worldwide, policymakers face critical trade-offs. Using a mathematical model of COVID-19 transmission, we find that timing of the rollout is expected to have a substantially greater impact on mortality than risk-based prioritization and uptake and that prioritizing first doses over second doses may be life saving.This research was supported by NIH Grant R01 AI151176 and CDC Grant U01 IP00136.Molecular BiosciencesIntegrative Biolog

A Highly Stable Prefusion Rsv F Vaccine Derived from Structural Analysis of the Fusion Mechanism

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain