Blake Martin, Peter E DeWitt, Seth Russell, Adit Anand, Katie R Bradwell, Carolyn Bremer, Davera Gabriel, Andrew T Girvin, Janos G Hajagos, Julie A McMurry, Andrew J Neumann, Emily R Pfaff, Anita Walden, Jacob T Wooldridge, Yun Jae Yoo, Joel Saltz, Ken R Gersing, Christopher G Chute, Melissa A Haendel, Richard Moffitt, Tellen D Bennett

Importance: SARS-CoV-2 Objective: To determine the characteristics, changes over time, outcomes, and severity risk factors of SARS-CoV-2 affected children within the National COVID Cohort Collaborative (N3C) Design: Prospective cohort study of encounters with end dates before May 27th, 2021. Setting: 45 N3C institutions Participants: Children < 19-years-old at initial SARS-CoV-2 testing Main Outcomes and Measures: Case incidence and severity over time, demographic and comorbidity severity risk factors, vital sign and laboratory trajectories, clinical outcomes, and acute COVID-19 vs MIS-C contrasts for children infected with SARS-CoV-2. Results: 728,047 children in the N3C were tested for SARS-CoV-2; of these, 91,865 (12.6%) were positive. Among the 5,213 (6%) hospitalized children, 685 (13%) met criteria for severe disease: mechanical ventilation (7%), vasopressor/inotropic support (7%), ECMO (0.6%), or death/discharge to hospice (1.1%). Male gender, African American race, older age, and several pediatric complex chronic condition (PCCC) subcategories were associated with higher clinical severity (p [&le;] 0.05). Vital signs (all p [&le;] 0.002) and many laboratory tests from the first day of hospitalization were predictive of peak disease severity. Children with severe (vs moderate) disease were more likely to receive antimicrobials (71% vs 32%, p < 0.001) and immunomodulatory medications (53% vs 16%, p < 0.001). Compared to those with acute COVID-19, children with MIS-C were more likely to be male, Black/African American, 1-to-12-years-old, and less likely to have asthma, diabetes, or a PCCC (p < 0.04). MIS-C cases demonstrated a more inflammatory laboratory profile and more severe clinical phenotype with higher rates of invasive ventilation (12% vs 6%) and need for vasoactive-inotropic support (31% vs 6%) compared to acute COVID-19 cases, respectively (p <0.03). Conclusions: In the largest U.S. SARS-CoV-2-positive pediatric cohort to date, we observed differences in demographics, pre-existing comorbidities, and initial vital sign and laboratory test values between severity subgroups. Taken together, these results suggest that early identification of children likely to progress to severe disease could be achieved using readily available data elements from the day of admission. Further work is needed to translate this knowledge into improved outcomes.

Renee WY Chan, Shaojun Liu, Jonathan Y Cheung, Joseph GS Tsun, Kate CC Chan, Kathy YY Chan, Genevieve PG Fung, Albert M Li, Hugh Simon Lam

Vaccines that elicit mucosal immune responses against SARS-CoV-2 could potentially be of exceptional importance in providing first line defense at the site of viral entry. The serological antibody response induced by SARS-CoV-2 vaccines have already been well characterized. In order to understand the mucosal immune response profiles of SARS-CoV-2 vaccines, we examined both the mucosal and systemic responses of subjects vaccinated by two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac). Serial nasal epithelial lining fluid (NELF) and peripheral blood samples were collected in ten subjects who had received CoronaVac and thirty-two subjects who had received Comirnaty. We quantified IgA and IgG specific to SARS-CoV-2 S1 protein by ELISA in NELF and plasma samples. The neutralization effect of these two sample types were evaluated by surrogate ACE-SARS-CoV-2 Spike protein ELISA. Only Comirnaty induced nasal SARS-CoV-2 S1 protein-specific (S1-specific) IgA and IgG responses, which were evident as early as on 14 days after the first dose. The NELF samples of 72% of subjects became IgA+IgG+, while in 62.5% of subjects the samples were neutralizing by 7 days after the second dose. In 45% of the subjects their NELF remained neutralizing 50 days after the booster of Comirnaty. In plasma, 91% and 100% Comirnaty subjects possessed S1-specific IgA+IgG+ on 14 days after the first dose and 7 days after booster, respectively. The plasma collected on 7 days after booster was 100% neutralizing. The induction of S1-specific antibody by CoronaVac was IgG dominant, and 70% of the subjects possessed S1-specific IgG by 7 days after booster and were all neutralizing. This study reveals that Comirnaty is able to induce S1-specific IgA and IgG response with neutralizing activity in the nasal mucosa in addition to a consistent systemic response. The clinical implications and the biological mechanism of an additional nasal immune response induced by vaccines such as Comirnaty warrant further investigation.

Ingrid H.C.H.M. Philippens, Kinga P. Boszormenyi, Jacqueline A. Wubben, Zahra C Fagrouch, Nikki van Driel, Amber Q. Mayenburg, Diana Lozovagia, Eva Roos, Bernadette Schurink, Marianna Bugiani, Ronald E Bontrop, Jinte Middeldorp, Willy M Bogers, Lioe-Fee de Geus-Oei, Jan A.M. Langermans, Marieke A Stammes, Babs E Verstrepen, Ernst J Verschoor

SARS-CoV-2 may cause acute respiratory disease, but the infection can also initiate neurological symptoms. Here we show that SARS-CoV-2 infection causes brain inflammation in the macaque model. An increased metabolic activity in the pituitary gland of two macaques was observed by longitudinal positron emission tomography-computed tomography (PET-CT). Post-mortem analysis demonstrated infiltration of T-cells and activated microglia in the brain, and viral RNA was detected in brain tissues from one animal. We observed Lewy bodies in brains of all rhesus macaques. These data emphasize the virus' capability to induce neuropathology in this nonhuman primate model for SARS-CoV-2 infection. As in humans, Lewy body formation is an indication for the development of Parkinson's disease, this data represents a warning for potential long-term neurological effects after SARS-CoV-2 infection.

Nabin K. Shrestha, Patrick C. Burke, Amy S. Nowacki, Paul Terpeluk, Steven M. Gordon

Background: There are good reasons to expect natural infection to provide protection against future infection with SARS-CoV-2. The purpose of this study was to evaluate the necessity of COVID-19 vaccination in persons previously infected with SARS-CoV-2. Methods: Employees of the Cleveland Clinic Health System working in Ohio on Dec 16, 2020, the day COVID-19 vaccination was started, were included. Any subject who tested positive for SARS-CoV-2 at least 42 days earlier was considered previously infected. One was considered vaccinated 14 days after receipt of the second dose of a SARS-CoV-2 mRNA vaccine. The cumulative incidence of SARS-CoV-2 infection over the next four months, among previously infected subjects who received the vaccine, was compared with those of previously infected subjects who remained unvaccinated, previously uninfected subjects who received the vaccine, and previously uninfected subjects who remained unvaccinated. Results: Among the 52238 included employees, 1220 (47%) of 2579 previously infected subjects received the vaccine, compared with 29461 (59%) of 49659 not previously infected. The cumulative incidence of SARS-CoV-2 infection did not differ among previously infected unvaccinated subjects, previously infected subjects who were vaccinated, and previously uninfected subjects who were vaccinated, and was much lower than that of previously uninfected subjects who remained unvaccinated. Not one of the 1359 previously infected subjects who remained unvaccinated had a SARS-CoV-2 infection over the duration of the study. Conclusion: Individuals who have had SARS-CoV-2 infection are unlikely to benefit from COVID-19 vaccination, and vaccines can be safely prioritized to those who have not been infected before.

Dami A Collier, Anna De Marco, Isabella A.T.M. Ferreira, Bo Meng, Rawlings Datir, Alexandra C Walls, Steven A. Kemp S, Jessica Bassi, Dora Pinto, Chiara Silacci Fregni, Siro Bianchi, M. Alejandra Tortorici, John Bowen, Katja Culap, Stefano Jaconi, Elisabetta Cameroni, Gyorgy Snell, Matteo S. Pizzuto, Alessandra Franzetti-Pellanda, Christian Garzoni, Agostino Riva, The CITIID-NIHR BioResource COVID-19 Collaboration, Anne Elmer, Nathalie Kingston, Barbara Graves, Laura E McCoy, Kenneth G.C. Smith, John R Bradley, Nigel James Temperton, Lourdes Ceron-Gutierrez L, Gabriela Barcenas-Morales, The COVID-19 Genomics UK (COG-UK) consortium, William Harvey, Herbert W Virgin, Antonio Lanzavecchia, Luca Piccoli, Rainer Doffinger, Mark Wills, David Veesler, Davide Corti, Ravindra K Gupta

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Scott Bolkan, Iris Stone, Lucas Pinto, Zoe Ashwood, Jorge Garcia, Alison Herman, Priyanka Singh, Akhil Bandi, Julia Cox, Christopher Zimmerman, Jounhong Cho, Ben Engelhard, Sue Koay, Jonathan Pillow, Ilana Witten

A classic view of the striatum holds that activity in direct and indirect pathways oppositely modulates motor output. Whether this involves direct control of movement, or reflects a cognitive process underlying movement, has remained unresolved. Here we find that strong, opponent control of behavior by the two pathways of the dorsomedial striatum (DMS) depends on a task's cognitive demands. Furthermore, a latent state model (a hidden markov model with generalized linear model observations) reveals that - even within a single task - the contribution of the two pathways to behavior is state-dependent. Specifically, the two pathways have large contributions in one of two states associated with a strategy of evidence accumulation, compared to a state associated with a strategy of repeating previous choices. Thus, both the cognitive demands imposed by a task, as well as the strategy that mice pursue within a task, determine whether DMS pathways provide strong and opponent control of behavior.

M. Gabriela M. Gomes, Marcelo U Ferreira, Maria Chikina, Wesley Pegden, Ricardo Aguas

Individual variation in susceptibility and exposure is subject to selection by force of infection, accelerating the natural acquisition of immunity, and reducing herd immunity thresholds and epidemic final sizes. This is a manifestation of a wider population phenomenon known as "frailty variation" in demography. Despite this theoretical understanding, public health policies continue to be guided by mathematical models that leave out most of the relevant variation and as a result inflate projected infection burdens. Here we focus on the trajectories of the coronavirus disease (COVID-19) pandemic in England and Scotland. We fit models to series of daily deaths and estimate relevant epidemiological parameters, including coefficients of variation which we find in agreement with direct measurements based on published contact surveys. Our estimates are robust to whether the data series encompass one or two pandemic waves. We conclude that herd immunity thresholds are being reached with a larger contribution of vaccination in Scotland than in England, where naturally acquired immunity is higher. These results are relevant to global vaccination policies.

Gwenaëlle Douaud, Soojin Lee, Fidel Alfaro-Almagro, Christoph Arthofer, Chaoyue Wang, Frederik Lange, Jesper L.R. Andersson, Ludovica Griffanti, Eugene Duff, Saad Jbabdi, Bernd Taschler, Anderson Winkler, Thomas Nichols, Rory Collins, Paul M. Matthews, Naomi Allen, Karla L Miller, Stephen M Smith

There is strong evidence for brain-related pathologies in COVID-19, some of which could be a consequence of viral neurotropism. The vast majority of brain imaging studies so far have focused on qualitative, gross pathology of moderate to severe cases, often carried out on hospitalised patients. It remains unknown however whether the impact of COVID-19 can be detected in milder cases, in a quantitative and automated manner, and whether this can reveal a possible mechanism for the spread of the disease. UK Biobank scanned over 40,000 participants before the start of the COVID-19 pandemic, making it possible to invite back in 2021 hundreds of previously-imaged participants for a second imaging visit. Here, we studied the effects of the disease in the brain using multimodal data from 782 participants from the UK Biobank COVID-19 re-imaging study, with 394 participants having tested positive for SARS-CoV-2 infection between their two scans. We used structural and functional brain scans from before and after infection, to compare longitudinal brain changes between these 394 COVID-19 patients and 388 controls who were matched for age, sex, ethnicity and interval between scans. We identified significant effects of COVID-19 in the brain with a loss of grey matter in the left parahippocampal gyrus, the left lateral orbitofrontal cortex and the left insula. When looking over the entire cortical surface, these results extended to the anterior cingulate cortex, supramarginal gyrus and temporal pole. We further compared COVID-19 patients who had been hospitalised (n=15) with those who had not (n=379), and while results were not significant, we found comparatively similar findings to the COVID-19 vs control group comparison, with, in addition, a greater loss of grey matter in the cingulate cortex, central nucleus of the amygdala and hippocampal cornu ammonis (all |Z|>3). Our findings thus consistently relate to loss of grey matter in limbic cortical areas directly linked to the primary olfactory and gustatory system. Unlike in post hoc disease studies, the availability of pre-infection imaging data helps avoid the danger of pre-existing risk factors or clinical conditions being mis-interpreted as disease effects. Since a possible entry point of the virus to the central nervous system might be via the olfactory mucosa and the olfactory bulb, these brain imaging results might be the in vivo hallmark of the spread of the disease (or the virus itself) via olfactory and gustatory pathways.

Omaya Dudin, Sébastien Wielgoss, Aaron M New, Iñaki Ruiz-Trillo

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits which control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves, are still poorly understood. Here, using isolates of the ichthyosporean Sphaeroforma genus - close unicellular relatives of animals with brief transient multicellular life stages - we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least two distinct physical mechanisms. We first find a dramatic >300x variation in sedimentation rate for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Using experimental evolution with sedimentation rate as a focal trait, we readily obtained fast settling S. arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Additionally, density increases arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in four additional species from the Sphaeroforma genus, suggesting variation in these traits might be widespread in the marine habitat. By sequencing evolved isolates, we identified mutations in regulators of cytokinesis, plasma membrane remodelling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the evolution of multicellularity.

Sharifa Nasreen, Hannah Chung, Siyi He, Kevin A. Brown, Jonathan B Gubbay, Sarah A Buchan, Deshayne B Fell, Peter C Austin, Kevin L Schwartz, Maria E. Sundaram, Andrew Calzavara, Branson Chen, Mina Tadrous, Kumanan Wilson, Sarah E. Wilson, Jeffrey C Kwong

Background: SARS-CoV-2 variants of concern (VOC) are more transmissible and have the potential for increased disease severity and decreased vaccine effectiveness. We sought to estimate the effectiveness of BNT162b2 (Pfizer-BioNTech), mRNA-1273 (Moderna), and ChAdOx1 (AstraZeneca) vaccines against symptomatic SARS-CoV-2 infection and severe outcomes (COVID-19 hospitalization or death) caused by the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) VOCs during December 2020 to May 2021. Methods: We conducted a test-negative design study using linked population-wide vaccination, laboratory testing, and health administrative databases in Ontario, Canada. Findings: Against symptomatic infection caused by Alpha, vaccine effectiveness with partial vaccination ([&ge;]14 days after dose 1) was higher for mRNA-1273 (83%) than BNT162b2 (66%) and ChAdOx1 (64%), and full vaccination ([&ge;]7 days after dose 2) increased vaccine effectiveness for BNT162b2 (89%) and mRNA-1273 (92%). Protection against symptomatic infection caused by Beta/Gamma was also higher with partial vaccination for mRNA-1273 (77%) than BNT162b2 (60%) and ChAdOx1 (48%), and full vaccination increased effectiveness for BNT162b2 (84%). Against Delta, vaccine effectiveness after partial vaccination tended to be lower compared to Alpha for mRNA-1273 (72% vs. 83%) and BNT162b2 (56% vs. 66%), but was similar to Alpha for ChAdOx1 (67% vs. 64%). Full vaccination with BNT162b2 increased protection against Delta (87%) to levels comparable to Alpha (89%) and Beta/Gamma (84%). Vaccine effectiveness against hospitalization or death caused by all VOCs was generally higher than for symptomatic infection after partial vaccination for all three vaccines. Interpretation: Our findings suggest that even a single dose of these 3 vaccines provide substantial protection against these 4 VOCs, and 2 doses likely provide higher protection. Jurisdictions facing vaccine supply constraints might consider delaying second doses to more rapidly achieve greater overall population protection.

F. Konstantin Föhse, Büsranur Geckin, Gijs Overheul, Josephine van de Maat, Gizem Kilic, Ozlem Bulut, Helga Dijkstra, Heidi Lemmers, S. Andrei Sarlea, Maartje Reijnders, Jacobien Hoogerwerf, Jaap ten Oever, Elles Simonetti, Frank L van de Veerdonk, Leo A.B. Joosten, Bart L. Haagmans, Reinout van Crevel, Yang Li, Ronald P. van Rij, Corine GeurtsvanKessel, Marien I. de Jonge, Jorge Domínguez-Andrés, Mihai G. Netea

The mRNA-based BNT162b2 vaccine from Pfizer/BioNTech was the first registered COVID-19 vaccine and has been shown to be up to 95% effective in preventing SARS-CoV-2 infections. Little is known about the broad effects of the new class of mRNA vaccines, especially whether they have combined effects on innate and adaptive immune responses. Here we confirmed that BNT162b2 vaccination of healthy individuals induced effective humoral and cellular immunity against several SARS-CoV-2 variants. Interestingly, however, the BNT162b2 vaccine also modulated the production of inflammatory cytokines by innate immune cells upon stimulation with both specific (SARS-CoV-2) and non-specific (viral, fungal and bacterial) stimuli. The response of innate immune cells to TLR4 and TLR7/8 ligands was lower after BNT162b2 vaccination, while fungi-induced cytokine responses were stronger. In conclusion, the mRNA BNT162b2 vaccine induces complex functional reprogramming of innate immune responses, which should be considered in the development and use of this new class of vaccines.

Thomas Radtke, Agne Ulyte, Milo Alan Puhan, Susi Kriemler

Although nobody doubts the existence of long COVID in children, it is still unclear to what extent children are affected. The Ciao Corona study is a longitudinal cohort investigating SARS-CoV-2 seroprevalence and clustering of cases among around 2500 children from 55 randomly selected primary and secondary schools in the canton of Zurich in Switzerland. Between June 2020 and April 2021, we completed three testing phases where we collected venous blood for serological analysis (ABCORA 2.0 test) and asked about symptoms with online questionnaires. We compared children who tested positive for SARS-CoV-2 antibodies in October/November 2020 with those who tested negative. Children who were seronegative in October/November 2020 and seroconverted or were not retested by March/April 2021 were excluded from the analysis (n=256). In March-May 2021 we assessed the presence of symptoms occurring since October 2020, lasting for at least 4 weeks, and persisting for either >4 weeks or >12 weeks. Overall, 1355 of 2503 children with a serology result in October/November 2020 and follow up questionnaire in March/April 2021 were included. Among seropositive and seronegative 6-to 16-year-old children and adolescents, 9% versus 10% reported at least one symptom beyond 4 weeks, and 4% versus 2% at least one symptom beyond 12 weeks. None of the seropositive children reported hospitalization after October 2020. Seropositive children, all with a history of pauci-symptomatic SARS-CoV-2 infection, did not report long COVID more frequently than seronegative children. This study suggests a very low prevalence of long COVID in a randomly selected population-based cohort of children followed over 6 months after serological testing.

Baisheng Li, Aiping Deng, Kuibiao Li, Yao Hu, Zhencui Li, Qianling Xiong, Zhe Liu, Qianfang Guo, Lirong Zou, Huan Zhang, Meng Zhang, Fangzhu Ouyang, Juan Su, Wenzhe Su, Jing Xu, Huifang Lin, Jing Sun, Jinju Peng, Huimin Jiang, Pingping Zhou, Ting Hu, Min Luo, Yingtao Zhang, Huanying Zheng, Jianpeng Xiao, Tao Liu, Rongfei Che, Hanri Zeng, Zhonghua Zheng, Yushi Huang, Jianxiang Yu, Lina Yi, Jie Wu, Jingdiao Chen, Haojie Zhong, Xiaoling Deng, min Kang, Oliver G. Pybus, Matthew Hall, Katrina A Lythgoe, Yan Li, Jun Yuan, Jianfeng He, Jing Lu

We report the first local transmission of the SARS-CoV-2 Delta variant in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of the quarantined subjects indicated that the viral loads of Delta infections, when they first become PCR+, were on average ~1000 times greater compared to A/B lineage infections during initial epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. We performed high-quality sequencing on samples from 126 individuals. Reliable epidemiological data meant that, for 111 transmission events, the donor and recipient cases were known. The estimated transmission bottleneck size was 1-3 virions with most minor intra-host single nucleotide variants (iSNVs) failing to transmit to the recipients. However, transmission heterogeneity of SARS-CoV-2 was also observed. The transmission of minor iSNVs resulted in at least 4 of the 30 substitutions identified in the outbreak, highlighting the contribution of intra-host variants to population level viral diversity during rapid spread. Disease control activities, such as the frequency of population testing, quarantine during pre-symptomatic infection, and level of virus genomic surveillance should be adjusted in order to account for the increasing prevalence of the Delta variant worldwide.

Yair Goldberg, Micha Mandel, Yonatan Woodbridge, Ronen Fluss, Ilya Novikov, Rami Yaari, Arnona Ziv, Laurence Freedman, Amit Huppert

Worldwide shortage of vaccination against SARS-CoV-2 infection while the pandemic is still uncontrolled leads many states to the dilemma whether or not to vaccinate previously infected persons. Understanding the level of protection of previous infection compared to that of vaccination is critical for policy making. We analyze an updated individual-level database of the entire population of Israel to assess the protection efficacy of both prior infection and vaccination in preventing subsequent SARS-CoV-2 infection, hospitalization with COVID-19, severe disease, and death due to COVID-19. Vaccination was highly effective with overall estimated efficacy for documented infection of 92.8% (CI: [92.6, 93.0]); hospitalization 94.2% (CI: [93.6, 94.7]); severe illness 94.4% (CI: [93.6, 95.0]); and death 93.7% (CI: [92.5, 94.7]). Similarly, the overall estimated level of protection from prior SARS-CoV-2 infection for documented infection is 94.8% (CI: [94.4, 95.1]); hospitalization 94.1% (CI: [91.9, 95.7]); and severe illness 96.4% (CI: [92.5, 98.3]). Our results question the need to vaccinate previously-infected individuals.

Takuya Tada, Hao Zhou, Marie I. Samanovic, Belinda M Dcosta, Amber Cornelius, Mark J Mulligan, Nathaniel R Landau

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC50 <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

Liam J. Revell

In recent years it has become increasingly popular to use phylogenetic comparative methods to investigate heterogeneity in the rate or process of quantitative trait evolution across the branches or clades of a phylogenetic tree. Here, I present a new method for modeling variability in the rate of evolution of a continuously-valued character trait on a reconstructed phylogeny. The underlying model of evolution is stochastic diffusion (Brownian motion), but in which the instantaneous diffusion rate ({sigma}2) also evolves by Brownian motion on a logarithmic scale. Unfortunately, it's not possible to simultaneously estimate the rates of evolution along each edge of the tree and the rate of evolution of {sigma}2 itself using Maximum Likelihood. As such, I propose a penalized-likelihood method in which the penalty term is equal to the log-transformed probability density of the rates under a Brownian model, multiplied by a 'smoothing' coefficient, {lambda}, selected by the user. {lambda} determines the magnitude of penalty that's applied to rate variation between edges. Lower values of {lambda} penalize rate variation relatively little; whereas larger {lambda} values result in minimal rate variation among edges of the tree in the fitted model, eventually converging on a single value of {sigma}2 for all of the branches of the tree. In addition to presenting this model here, I have also implemented it as part of my phytools R package in the function multirateBM. Using different values of the penalty coefficient, {lambda}, I fit the model to simulated data with: Brownian rate variation among edges (the model assumption); uncorrelated rate variation; rate changes that occur in discrete places on the tree; and no rate variation at all among the branches of the phylogeny. I then compare the estimated values of {sigma}2 to their known true values. In addition, I use the method to analyze a simple empirical dataset of body mass evolution in mammals. Finally, I discuss the relationship between the method of this article and other models from the phylogenetic comparative methods and finance literature, as well as some applications and limitations of the approach.

Flavio A Cadegiani, Daniel N Fonseca, John McCoy, Ricardo A. Zimerman, Fatima N Mirza, Michael N Correia, Renan N Barros, Dirce C Onety, Karla Cristina P Israel, Brenda G Almeida, Emilyn O Guerreiro, Jose Enrique M Medeiros, Raquel N Nicolau, Luiza FM Nicolau, Rafael X Cunha, Maria Fernanda R Barroco, Patricia S da Silva, Gabriel S. Ferreira, Flavio Renan PC Alcantara, Angelo M Ribeiro, Felipe O de Almeida, Adailson A de Souza, Suzyane S do Rosario, Raysa WS Paulain, Alessandra Reis, Marissa Li, Claudia E Thompson, Gerald J Nau, Carlos Gustavo Wambier, Andy Goren

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is mediated by the androgen-promoted protease, transmembrane protease, serine 2 (TMPRSS2). Previously, we have shown that treatment with proxalutamide, a non-steroidal androgen receptor antagonist, accelerates viral clearance and clinical remission in outpatients with coronavirus disease 2019 (COVID-19) compared to placebo. The effects in hospitalized COVID-19 patients were unknown. Methods: Men and women hospitalized but not requiring mechanical ventilation were randomized (1:1 ratio) to receive 300 mg of proxalutamide per day or placebo for 14 days. The study was conducted at eight sites in the state of Amazonas, Brazil. The primary outcome measure was the clinical status (8-point ordinal scale) at 14-days post-randomization. The primary efficacy endpoint was the 14-day recovery ratio (alive hospital discharge [scores 1, 2]). Findings: A total of 645 patients were randomized (317 received proxalutamide, 328 placebo) and underwent intention-to-treat analysis. The 14-day median ordinal scale score in the proxalutamide group was 1 (interquartile range [IQR]=1-2) versus 7 (IQR=2-8) for placebo, P<0.001. The 14-day recovery rate was 81.4% for proxalutamide and 35.7% for placebo (recovery ratio, 2.28; 95% CI 1.95-2.66 [P<0.001]). The 28-day all-cause mortality rate was 11.0% for proxalutamide versus 49.4% for placebo (hazard ratio, 0.16; 95% CI 0.11-0.24). The median post-randomization time to recovery was 5 days (IQR=3-8) for proxalutamide versus 10 days (IQR=6-15) for placebo. Interpretation: Hospitalized COVID-19 patients not requiring mechanical ventilation receiving proxalutamide had a 128% higher recovery rate than those treated with placebo. All-cause mortality was reduced by 77.7% over 28 days. (ClinicalTrials.gov number, NCT04728802).

Noel Blanco-Tourinan, David Esteve-Bruna, Antonio Serrano-Mislata, Rosa M Esquinas, Francesca Resentini, Javier Forment, Cristian Carrasco-Lopez, Claudio Novella-Rausell, Alberto Palacios, Pedro Carrasco, Julio Salinas, Miguel A. Blazquez, David Alabadi

The prefoldin complex (PFDc) was identified in humans as co-chaperone of the cytosolic chaperonin TRiC/CCT. It is conserved in eukaryotes and is composed of subunits PFD1 to 6. PFDc-TRiC/CCT operates folding actin and tubulins. In addition to this function, PFDs participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction cause developmental alterations and disease in animals, and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all functions performed by PFDs require the participation of the canonical complex. The lack of systematic genetic analyses in higher eukaryotes makes it difficult to discern whether PFDs participate in a particular process as canonical complex or in alternative configurations, i.e. as individual subunits or in other complexes. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we have prepared an Arabidopsis mutant deficient in the six prefoldins, and compared various growth and environmental responses with those of the individual pfd. In this way, we demonstrate that the PFDc is required to delay flowering, for seed germination, or to respond to high salt stress, whereas two or more PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant has identified several transcription factors, such as ABI5 or PIF4, acting downstream of PFDs. Furthermore, it has made possible to assign novel roles for PFDs, for instance, in the response to warm temperature.

Liguo Zhang, Alexsia Richards, Andrew Khalil, Emile Wogram, Haiting Ma, Richard A. Young, Rudolf Jaenisch

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.

Mariano Carossino, Paige Montanaro, Aoife O'Connell, Devin Kenney, Hans Gertje, Kyle Grosz, Susanna Kurnick, Markus Bosmann, Mohsan Saeed, Udeni Balasuriya, Florian Douam, Nicholas A. Crossland

Animal models recapitulating the distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. However, the cause(s) and mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate inflammation in the lungs, lethality was invariably associated with viral neuroinvasion and neuronal damage (including spinal motor neurons). Neuroinvasion occurred following virus transport through the olfactory neuroepithelium in a manner that was only partially dependent on hACE2. Interestingly, SARS-CoV-2 tropism was overall neither widespread among nor restricted to only ACE2-expressing cells. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis. SUMMARYCOVID-19 is a respiratory disease caused by SARS-CoV-2, a betacoronavirus. Here, we show that in a widely used transgenic mouse model of COVID-19, lethality is invariably associated with viral neuroinvasion and the ensuing neuronal disease, while lung inflammation remains moderate.