Uncovering Structural Plasticity of Enterovirus A through Deep Insertional and Deletional Scanning.

Insertions and deletions (InDels) are essential sources of novelty in protein evolution. In RNA viruses, InDels cause dramatic phenotypic changes contributing to the emergence of viruses with altered immune profiles and host engagement. This work aimed to expand our current understanding of viral evolution and explore the mutational tolerance of RNA viruses to InDels, focusing on Enterovirus A71 (EV-A71) as a prototype for Enterovirus A species (EV-A). Using newly described deep InDel scanning approaches, we engineered approximately 45,000 insertions and 6,000 deletions at every site across the viral proteome, quantifying their effects on viral fitness. As a general trend, most InDels were lethal to the virus. However, our screen reproducibly identified a set of InDel-tolerant regions, demonstrating our ability to comprehensively map tolerance to these mutations. Tolerant sites highlighted structurally flexible and mutationally plastic regions of viral proteins that avoid core structural and functional elements. Phylogenetic analysis on EV-A species infecting diverse mammalian hosts revealed that the experimentally-identified hotspots overlapped with sites of InDels across the EV-A species, suggesting structural plasticity at these sites is an important function for InDels in EV speciation. Our work reveals the fitness effects of InDels across EV-A71, identifying regions of evolutionary capacity that require further monitoring, which could guide the development of Enterovirus vaccines.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

RBD-based high affinity ACE2 antagonist limits SARS-CoV-2 replication in upper and lower airways.

SARS-CoV-2 has the capacity to evolve mutations to escape vaccine-and infection-acquired immunity and antiviral drugs. A variant-agnostic therapeutic agent that protects against severe disease without putting selective pressure on the virus would thus be a valuable biomedical tool. Here, we challenged rhesus macaques with SARS-CoV-2 Delta and simultaneously treated them with aerosolized RBD-62, a protein developed through multiple rounds of in vitro evolution of SARS-CoV-2 RBD to acquire 1000-fold enhanced ACE2 binding affinity. RBD-62 treatment gave equivalent protection in upper and lower airways, a phenomenon not previously observed with clinically approved vaccines. Importantly, RBD-62 did not block the development of memory responses to Delta and did not elicit anti-drug immunity. These data provide proof-of-concept that RBD-62 can prevent severe disease from a highly virulent variant.

Impact of malaria diagnostic choice on monitoring of Plasmodium falciparum prevalence estimates in the Democratic Republic of the Congo and relevance to control programs in high-burden countries.

Malaria programs rely upon a variety of diagnostic assays, including rapid diagnostic tests (RDTs), microscopy, polymerase chain reaction (PCR), and bead-based immunoassays (BBA), to monitor malaria prevalence and support control and elimination efforts. Data comparing these assays are limited, especially from high-burden countries like the Democratic Republic of the Congo (DRC). Using cross-sectional and routine data, we compared diagnostic performance and Plasmodium falciparum prevalence estimates across health areas of varying transmission intensity to illustrate the relevance of assay performance to malaria control programs. Data and samples were collected between March-June 2018 during a cross-sectional household survey across three health areas with low, moderate, and high transmission intensities within Kinshasa Province, DRC. Samples from 1,431 participants were evaluated using RDT, microscopy, PCR, and BBA. P. falciparum parasite prevalence varied between diagnostic methods across all health areas, with the highest prevalence estimates observed in Bu (57.4-72.4% across assays), followed by Kimpoko (32.6-53.2%), and Voix du Peuple (3.1-8.4%). Using latent class analysis to compare these diagnostic methods against an "alloyed gold standard," the most sensitive diagnostic method was BBA in Bu (high prevalence) and Voix du Peuple (low prevalence), while PCR diagnosis was most sensitive in Kimpoko (moderate prevalence). RDTs were consistently the most specific diagnostic method in all health areas. Among 9.0 million people residing in Kinshasa Province in 2018, the estimated P. falciparum prevalence by microscopy, PCR, and BBA were nearly double that of RDT. Comparison of malaria RDT, microscopy, PCR, and BBA results confirmed differences in sensitivity and specificity that varied by endemicity, with PCR and BBA performing best for detecting any P. falciparum infection. Prevalence estimates varied widely depending on assay type for parasite detection. Inherent differences in assay performance should be carefully considered when using community survey and surveillance data to guide policy decisions.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

From a genome-wide screen of RNAi molecules against SARS-CoV-2 to a validated broad-spectrum and potent prophylaxis.

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen of over 16,000 RNAi triggers against the SARS-CoV-2 genome, using a massively parallel assay to identify hyper-potent siRNAs. We selected Ten candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity (IC50 < 20 pM) and strong blockade of infectivity in live-virus experiments. We further enhanced this activity by combinatorial pairing of the siRNA candidates and identified cocktails that were active against multiple types of variants of concern (VOC). We then examined over 2,000 possible mutations in the siRNA target sites by using saturation mutagenesis and confirmed broad protection of the leading cocktail against future variants. Finally, we demonstrated that intranasal administration of this siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the gold-standard Syrian hamster model. Our results pave the way for the development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

Recommending COVID-19 vaccination for adolescents in primary care.

BACKGROUND: COVID-19 vaccines are available for adolescents in the United States, but many parents are hesitant to have their children vaccinated. The advice of primary care professionals strongly influences vaccine uptake. OBJECTIVE: We examined the willingness of primary care professionals (PCPs) to recommend COVID-19 vaccination for adolescents. METHODS: Participants were a national sample of 1,047 US adolescent primary care professionals. They participated in an online survey in early 2021, after a COVID-19 vaccine had been approved for adults but before approval for adolescents. Respondents included physicians (71%), advanced practice providers (17%), and nurses (12%). We identified correlates of willingness to recommend COVID-19 vaccination for adolescents using logistic regression. RESULTS: The majority (89%) of respondents were willing to recommend COVID-19 vaccination for adolescents, with advanced practice providers and nurses being less likely than paediatricians to recommend vaccination (84% vs. 94%, aOR 0.47, 95% CI 0.23-0.92). Respondents who had received at least one dose of a COVID-19 vaccine were more likely to recommend adolescent vaccination (92% vs. 69%, aOR 4.20, 95% CI 2.56-6.87) as were those with more years in practice (94% vs. 88%, aOR 2.93, 95% CI 1.79-4.99). Most respondents (96%) said they would need some measure of support in order to provide COVID-19 vaccination to adolescents, with vaccine safety and efficacy information being the most commonly cited need (80%). CONCLUSION: Adolescent primary care professionals were generally willing to recommend COVID-19 vaccination. However, most indicated a need for additional resources to be able to administer COVID-19 vaccines at their clinic.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis.

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αß-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αß-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

High Mortality among 30-Day Readmission after Stroke: Predictors and Etiologies of Readmission.

BACKGROUND: Although some risk factors for stroke readmission have been reported, the mortality risk is unclear. We sought to evaluate etiologies and predictors of 30-day readmissions and determine the associated mortality risk. METHODS: This is a retrospective case-control study evaluating 1,544 patients admitted for stroke (hemorrhagic, ischemic, or TIA) from January 2013 to December 2014. Of these, 134 patients readmitted within 30 days were identified as cases; 1,418 other patients, with no readmissions were identified as controls. Patients readmitted for hospice or elective surgery were excluded. An additional 248 patients deceased on index admission were included for only a comparison of mortality rates. Factors explored included socio-demographic characteristics, clinical comorbidities, stroke characteristics, and length of stay. Chi-square test of proportions and multivariable logistic regression were used to identify independent predictors of 30-day stroke readmissions. Mortality rates were compared for index admission and readmission and among readmission diagnoses. RESULTS: Among the 1,544 patients in the main analysis, 67% of index stroke admissions were ischemic, 22% hemorrhagic, and 11% TIA. The 30-day readmission rate was 8.7%. The most common etiologies for readmission were infection (30%), recurrent stroke and TIA (20%), and cardiac complications (14%). Significantly higher proportion of those readmitted for recurrent strokes and TIAs presented within the first week (p = 0.039) and had a shorter index admission length of stay (p = 0.027). Risk factors for 30-day readmission included age >75 (p = 0.02), living in a facility prior to index stroke (p = 0.01), history of prior stroke (p = 0.03), diabetes (p = 0.03), chronic heart failure (p ≤ 0.001), atrial fibrillation (p = 0.03), index admission to non-neurology service (p < 0.01), and discharge to other than home (p < 0.01). On multivariate analysis, index admission to a non-neurology service was an independent predictor of 30-day readmission (p ≤ 0.01). The mortality after a within 30-day readmission after stroke was higher than index admission (36.6 vs. 13.8% p ≤ 0.001) (OR 3.6 95% CI 2.5-5.3). Among those readmitted, mortality was significantly higher for those admitted for a recurrent stroke (p = 0.006). CONCLUSION: Approximately one-third of 30-day readmissions were infection related and one-fifth returned with recurrent stroke or TIA. Index admission to non-neurology service was an independent risk factor of 30-day readmissions. The mortality rate for 30-day readmission after stroke is more than 2.5 times greater than index admissions and highest among those readmitted for recurrent stroke. Identifying high-risk patients for readmission, ensuring appropriate level of service, and early outpatient follow-up may help reduce 30-day readmission and the high associated risk of mortality.