Eric Song, Ce Zhang, Benjamin Israelow, Alice Lu-Culligan, Alba Vieites Prado, Sophie Skriabine, Peiwen Lu, Orr-El Weizman, Feimei Liu, Yile Dai, Klara Szigeti-Buck, Yuki Yasumoto, Guilin Wang, Christopher Castaldi, Jaime Heltke, Evelyn Ng, John Wheeler, Mia Madel Alfajaro, Etienne Levavasseur, Benjamin Fontes, Neal G. Ravindra, David Van Dijk, Shrikant Mane, Murat Gunel, Aaron Ring, Syed A. Jaffar Kazmi, Kai Zhang, Craig B. Wilen, Tamas L Horvath, Isabelle Plu, Stephane Haik, Jean-Leon Thomas, Angeliki Louvi, Shelli F. Farhadian, Anita Huttner, Danielle Seilhean, Nicolas Renier, Kaya Bilguvar, Akiko Iwasaki

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2. ### Competing Interest Statement The authors have declared no competing interest.

Anthony Levasseur, Jeremy Delerce, Aurelia Caputo, Ludivine Brechard, Philippe Colson, Jean-Christophe Lagier, Pierre-Edouard Fournier, Didier Raoult

The novel coronavirus (SARS-CoV-2) causes pandemic of viral pneumonia. The evolution and mutational events of the SARS-CoV-2 genomes are critical for controlling virulence, transmissibility, infectivity, severity of symptoms and mortality associated to this infectious disease. We collected and investigated 309 SARS-CoV-2 genomes from patients infected in France. Detailed genome cartography of all mutational events (SNPs, indels) was reported and correlated to clinical features of patients. A comparative analysis between our 309 SARS-CoV-2 genomes from French patients and the reference Wuhan coronavirus genome revealed 315 substitution mutations and six deletion events: ten were in 5’/3’ UTR, 178 were nonsynonymous, 126 were synonymous and one generated a stop codon. Six different deleted areas were also identified in nine viral variants. In particular, 30 substitution mutations (18 nonsynonymous) and one deletion (Δ21765-21770) concerned the spike S glycoprotein. An average of 7.8 mutational events (+/- 1.7 SD) and a median of 8 (range, 7-9) were reported per viral isolate. Comparative analyses and clustering of specific mutational signatures in 309 genomes disclose several divisions in groups and subgroups combining their geographical and phylogenetic origin. Clinical outcomes of the 309 COVID-19-infected patients were investigated according to the mutational signatures of viral variants. These findings highlight the genome dynamics of the coronavirus 2019-20 and shed light on the mutational landscape and evolution of this virus. Inclusion of the French cohort enabled us to identify 161 novel mutations never reported in SARS-CoV-2 genomes collected worldwide. These results support a global and continuing surveillance of the emerging variants of the coronavirus SARS-CoV-2. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations: AA : Amino Acid GO : good clinical outcome Indel : Insertion and deletion ORF : Open Reading Frame PClinO : poor clinical outcome PVirO : poor virological outcome RBD : Receptor Binding Domain SD : Standard Deviation SNP : Single Nucleotide Polymorphism UTR : Untranslated Transcribed Region

Phil R. Bell, Michael Pittman, Thomas G. Kaye, Christophe Hendrickx

Our knowledge of the reproductive biology of dinosaurs covers a range of aspects, from brooding behaviour to nesting style and the timing of sexual maturity. Yet, the basic anatomy and function of the cloaca in non-avialan dinosaurs remains unknown. Here, we describe the outer morphology of the only known non-avialan dinosaur cloaca, preserved in an exceptional specimen of the early-diverging ceratopsian dinosaur Psittacosaurus . We clarify the position of the cloaca with respect to the ischia and caudal vertebrae and document the scales immediately adjacent to the abdomen and tail. We find that the cloaca is from a near-sexually mature subadult individual and is most similar to the cloaca of crocodylians, to the exclusion of lepidosaurians and birds. However, the sex of SMF R 4970 could not be determined as the cloaca and the rest of the specimen does not yield any sexually dimorphic information. This study highlights the ongoing role of exceptional specimens in providing rare soft tissues that help to bridge longstanding gaps in our knowledge of the basic biology of dinosaurs and other extinct reptiles. ### Competing Interest Statement The authors have declared no competing interest.

Prashant Pradhan, Ashutosh Kumar Pandey, Akhilesh Mishra, Parul Gupta, Praveen Kumar Tripathi, Manoj Balakrishnan Menon, James Gomes, Perumal Vivekanandan, Bishwajit Kundu

This paper has been withdrawn by its authors. They intend to revise it in response to comments received from the research community on their technical approach and their interpretation of the results. If you have any questions, please contact the corresponding author.

B Korber, WM Fischer, S Gnanakaran, H Yoon, J Theiler, W Abfalterer, B Foley, EE Giorgi, T Bhattacharya, MD Parker, DG Partridge, CM Evans, TM Freeman, TI de Silva, on behalf of the Sheffield COVID-19 Genomics Group, CC LaBranche, DC Montefiori

We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified fourteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; after beginning to spread in Europe in early February, when introduced to new regions it repeatedly and rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions. ### Competing Interest Statement The authors have declared no competing interest.

Aubin Moutal, Laurent F. Martin, Lisa Boinon, Kimberly Gomez, Dongzhi Ran, Yuan Zhou, Harrison J. Stratton, Song Cai, Shizhen Luo, Kerry Beth Gonzalez, Samantha Perez-Miller, Amol Patwardhan, Mohab M. Ibrahim, Rajesh Khanna

Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues unabated. Binding of SARS-CoV-2’s Spike protein to host angiotensin converting enzyme 2 triggers viral entry, but other proteins may participate, including neuropilin-1 receptor (NRP-1). As both Spike protein and vascular endothelial growth factor-A (VEGF-A) – a pro-nociceptive and angiogenic factor, bind NRP-1, we tested if Spike could block VEGF-A/NRP-1 signaling. VEGF-A–triggered sensory neuronal firing was blocked by Spike protein and NRP-1 inhibitor EG00229. Pro-nociceptive behaviors of VEGF-A were similarly blocked via suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A ‘silencing’ of pain via subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals. ### Competing Interest Statement R. Khanna is the co-founder of Regulonix LLC, a company developing non-opioids drugs for chronic pain. In addition, R. Khanna has patents US10287334 and US10441586 issued to Regulonix LLC. The other authors declare no competing financial interests. * ACE2 : Angiotensin converting enzyme 2 AUC : area under the curve CaV2.2 : N-type voltage-gated calcium channel COVID-19 : coronavirus disease 2019 DRG : dorsal root ganglia MEA : multi-well microelectrode array NaV1.7 : voltage-gated sodium channel isoform 7 NRP-1 : Neuropilin-1 PWTs : paw withdrawal thresholds SARS-CoV-2 : Severe acute respiratory syndrome coronavirus 2 sEPSCs : spontaneous excitatory postsynaptic currents SNI : spared nerve injury VEGF-A : vascular endothelial growth factor-A

Alina Baum, Richard Copin, Dharani Ajithdoss, Anbo Zhou, Kathryn Lanza, Nicole Negron, Min Ni, Yi Wei, Gurinder S. Atwal, Adelekan Oyejide, Yenny Goez-Gazi, John Dutton, Elizabeth Clemmons, Hilary M Staples, Carmen Bartley, Benjamin Klaffke, Kendra Alfson, Michal Gazi, Olga Gonzales, Edward Dick, Ricardo Carrion, Laurent Pessaint, Maciel Porto, Anthony Cook, Renita Brown, Vaneesha Ali, Jack Greenhouse, Tammy Taylor, Hanne Andersen, Mark G Lewis, Neil Stahl, Andrew J Murphy, George D Yancopoulos, Christos A. Kyratsous

An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail. ### Competing Interest Statement Regeneron authors own options and/or stock of the company. This work has been described in one or more pending provisional patent applications. N.S, A.J.M., G.D.Y. and C.A.K. are officers of Regeneron.

Manman Dai, Huanan Li, Nan Yan, Jinyu Huang, Li Zhao, Siqi Xu, Shibo Jiang, Chungen Pan, Ming Liao

Several outbreaks of COVID-19 were associated with seafood markets, raising concerns that fish-attached SARS-CoV-2 may exhibit prolonged survival in low-temperature environments. Here we showed that salmon-attached SARS-CoV-2 at 4°C could remain infectious for more than one week, suggesting that fish-attached SARS-CoV-2 may be a source of transmission. ### Competing Interest Statement The authors have declared no competing interest.

Elon Musk, Neuralink

Brain-machine interfaces (BMIs) hold promise for the restoration of sensory and motor function and the treatment of neurological disorders, but clinical BMIs have not yet been widely adopted, in part because modest channel counts have limited their potential. In this white paper, we describe Neuralink’s first steps toward a scalable high-bandwidth BMI system. We have built arrays of small and flexible electrode “threads”, with as many as 3,072 electrodes per array distributed across 96 threads. We have also built a neurosurgical robot capable of inserting six threads (192 electrodes) per minute. Each thread can be individually inserted into the brain with micron precision for avoidance of surface vasculature and targeting specific brain regions. The electrode array is packaged into a small implantable device that contains custom chips for low-power on-board amplification and digitization: the package for 3,072 channels occupies less than (23 × 18.5 × 2) mm3. A single USB-C cable provides full-bandwidth data streaming from the device, recording from all channels simultaneously. This system has achieved a spiking yield of up to 70% in chronically implanted electrodes. Neuralink’s approach to BMI has unprecedented packaging density and scalability in a clinically relevant package.

Bas B. Oude Munnink, Reina S. Sikkema, David F. Nieuwenhuijse, Robert Jan Molenaar, Emmanuelle Munger, Richard Molenkamp, Arco van der Spek, Paulin Tolsma, Ariene Rietveld, Miranda Brouwer, Noortje Bouwmeester-Vincken, Frank Harders, Renate Hakze-van der Honing, Marjolein C.A. Wegdam-Blans, Ruth J. Bouwstra, Corine GeurtsvanKessel, Annemiek A. van der Eijk, Francisca C. Velkers, Lidwien A.M. Smit, Arjan Stegeman, Wim H.M. van der Poel, Marion P.G. Koopmans

The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms. One sentence summary SARS-CoV-2 transmission on mink farms. ### Competing Interest Statement The authors have declared no competing interest.

Juan A Pérez-Bermejo, Serah Kang, Sarah J. Rockwood, Camille R. Simoneau, David A. Joy, Gokul N. Ramadoss, Ana C. Silva, Will R. Flanigan, Huihui Li, Ken Nakamura, Jeffrey D. Whitman, Melanie Ott, Bruce R. Conklin, Todd C. McDevitt

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19. ### Competing Interest Statement B.R.C. is a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including genetic cardiomyopathies. B.R.C. and T.C.M. hold equity in Tenaya.

Neeltje van Doremalen, Teresa Lambe, Alexandra Spencer, Sandra Belij-Rammerstorfer, Jyothi N. Purushotham, Julia R. Port, Victoria Avanzato, Trenton Bushmaker, Amy Flaxman, Marta Ulaszewska, Friederike Feldmann, Elizabeth R. Allen, Hannah Sharpe, Jonathan Schulz, Myndi Holbrook, Atsushi Okumura, Kimberly Meade-White, Lizzette Pérez-Pérez, Cameron Bissett, Ciaran Gilbride, Brandi N. Williamson, Rebecca Rosenke, Dan Long, Alka Ishwarbhai, Reshma Kailath, Louisa Rose, Susan Morris, Claire Powers, Jamie Lovaglio, Patrick W. Hanley, Dana Scott, Greg Saturday, Emmie de Wit, Sarah C. Gilbert, Vincent Munster

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the COVID-19 pandemic3. Vaccines are an essential countermeasure urgently needed to control the pandemic4. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials. ### Competing Interest Statement SCG is a board member of Vaccitech and named as an inventor on a patent covering use of ChAdOx1-vectored vaccines and a patent application covering a SARS-CoV-2 (nCoV-19) vaccine. Teresa Lambe is named as an inventor on a patent application covering a SARS-CoV-2 (nCoV-19) vaccine. The remaining authors declare no competing interests.

Takuya Sekine, André Perez-Potti, Olga Rivera-Ballesteros, Kristoffer Strålin, Jean-Baptiste Gorin, Annika Olsson, Sian Llewellyn-Lacey, Habiba Kamal, Gordana Bogdanovic, Sandra Muschiol, David J. Wullimann, Tobias Kammann, Johanna Emgård, Tiphaine Parrot, Elin Folkesson, Olav Rooyackers, Lars I Eriksson, Anders Sönnerborg, Tobias Allander, Jan Albert, Morten Nielsen, Jonas Klingström, Sara Gredmark-Russ, Niklas K Björkström, Johan K. Sandberg, David A. Price, Hans-Gustaf Ljunggren, Soo Aleman, Marcus Buggert, Karolinska COVID-19 Study Group

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in a large cohort of unexposed individuals as well as exposed family members and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative family members and individuals with a history of asymptomatic or mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust memory T cell responses akin to those observed in the context of successful vaccines, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19 also in seronegative individuals. ### Competing Interest Statement The authors have declared no competing interest.

Kenneth S Plante, Jessica A. Plante, Diana Fernandez, Divya Mirchandani, Nathen Bopp, Patricia V Aguilar, K Jagannadha Sastry, Robert A Newman, Scott C. Weaver

With continued expansion of the COVID-19 pandemic, antiviral drugs are desperately needed to treat patients at high risk of life-threatening disease and even to limit spread if administered early during infection. Typically, the fastest route to identifying and licensing a safe and effective antiviral drug is to test those already shown safe in early clinical trials for other infections or diseases. Here, we tested in vitro oleandrin, derived from the Nerium oleander plant and shown previously to have inhibitory activity against several viruses. Using Vero cells, we found that prophylactic oleandrin administration at concentrations down to 0.05 μg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 μg/ml dose resulted in a greater than 3,000-fold reduction in infectious virus production. The EC50 values were 11.98 ng/ml when virus output was measured at 24 hours post-infection, and 7.07 ng/ml measured at 48 hours post-infection. Therapeutic (post-infection) treatment up to 24 hours after infection of Vero cells also reduced viral titers, with the 0.1 μg/ml dose causing greater than 100-fold reductions as measured at 48 hours, and the 0.05 μg/ml dose resulting in a 78-fold reduction. The potent prophylactic and therapeutic antiviral activities demonstrated here strongly support the further development of oleandrin to reduce the severity of COVID-19 and potentially also to reduce spread by persons diagnosed early after infection. ### Competing Interest Statement RAN is Chief Science Officer of Phoenix Biotechnology Inc. KJS is a paid consultant with Phoenix Biotechnology Inc.

Hugo Zeberg, Svante Pääbo

A recent genetic association study (Ellinghaus et al. 2020) identified a gene cluster on chromosome 3 as a risk locus for respiratory failure in SARS-CoV-2. Recent data comprising 3,199 hospitalized COVID-19 patients and controls reproduce this and find that it is the major genetic risk factor for severe SARS-CoV-2 infection and hospitalization (COVID-19 Host Genetics Initiative). Here, we show that the risk is conferred by a genomic segment of ~50 kb that is inherited from Neandertals and occurs at a frequency of ~30% in south Asia and ~8% in Europe. ### Competing Interest Statement The authors have declared no competing interest.

Alexander E. Gorbalenya, Susan C. Baker, Ralph S. Baric, Raoul J. de Groot, Christian Drosten, Anastasia A. Gulyaeva, Bart L. Haagmans, Chris Lauber, Andrey M Leontovich, Benjamin W Neuman, Dmitry Penzar, Stanley Perlman, Leo L.M. Poon, Dmitry Samborskiy, Igor A. Sidorov, Isabel Sola, John Ziebuhr

The present outbreak of lower respiratory tract infections, including respiratory distress syndrome, is the third spillover, in only two decades, of an animal coronavirus to humans resulting in a major epidemic. Here, the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the official classification of viruses and taxa naming (taxonomy) of the Coronaviridae family, assessed the novelty of the human pathogen tentatively named 2019-nCoV. Based on phylogeny, taxonomy and established practice, the CSG formally recognizes this virus as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus and designates it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate communication, the CSG further proposes to use the following naming convention for individual isolates: SARS-CoV-2/Isolate/Host/Date/Location. The spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined. The independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying the entire (virus) species to complement research focused on individual pathogenic viruses of immediate significance. This research will improve our understanding of virus-host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

Graham K. Shunk, Xavier R. Gomez, Nils J. H. Averesch

The greatest hazard for humans on deep-space exploration missions is radiation. To protect astronauts venturing out beyond Earth’s protective magnetosphere and sustain a permanent presence on Moon and/or Mars, advanced passive radiation protection is highly sought after. Due to the complex nature of space radiation, there is likely no one-size-fits-all solution to this problem, which is further aggravated by up-mass restrictions. In search of innovative radiation-shields, biotechnology holds unique advantages such as suitability for in-situ resource utilization (ISRU), self-regeneration, and adaptability. Certain fungi thrive in high-radiation environments on Earth, such as the contamination radius of the Chernobyl Nuclear Power Plant. Analogous to photosynthesis, these organisms appear to perform radiosynthesis, using pigments known as melanin to convert gamma-radiation into chemical energy. It is hypothesized that these organisms can be employed as a radiation shield to protect other lifeforms. Here, growth of Cladosporium sphaerospermum and its capability to attenuate ionizing radiation, was studied aboard the International Space Station (ISS) over a time of 30 days, as an analog to habitation on the surface of Mars. At full maturity, radiation beneath a ≈ 1.7 mm thick lawn of the melanized radiotrophic fungus (180° protection radius) was 2.17±0.35% lower as compared to the negative control. Estimations based on linear attenuation coefficients indicated that a ∼ 21 cm thick layer of this fungus could largely negate the annual dose-equivalent of the radiation environment on the surface of Mars, whereas only ∼ 9 cm would be required with an equimolar mixture of melanin and Martian regolith. Compatible with ISRU, such composites are promising as a means to increase radiation shielding while reducing overall up-mass, as is compulsory for future Mars-missions. ### Competing Interest Statement The authors have declared no competing interest.

Annette B. Vogel, Isis Kanevsky, Ye Che, Kena A. Swanson, Alexander Muik, Mathias Vormehr, Lena M. Kranz, Kerstin C. Walzer, Stephanie Hein, Alptekin Güler, Jakob Loschko, Mohan S. Maddur, Kristin Tompkins, Journey Cole, Bonny G. Lui, Thomas Ziegenhals, Arianne Plaschke, David Eisel, Sarah C. Dany, Stephanie Fesser, Stephanie Erbar, Ferdia Bates, Diana Schneider, Bernadette Jesionek, Bianca Sänger, Ann-Kathrin Wallisch, Yvonne Feuchter, Hanna Junginger, Stefanie A. Krumm, André P. Heinen, Petra Adams-Quack, Julia Schlereth, Christoph Kröner, Shannan Hall-Ursone, Kathleen Brasky, Matthew C. Griffor, Seungil Han, Joshua A. Lees, Ellene H. Mashalidis, Parag V. Sahasrabudhe, Charles Y. Tan, Danka Pavliakova, Guy Singh, Camila Fontes-Garfias, Michael Pride, Ingrid L. Scully, Tara Ciolino, Jennifer Obregon, Michal Gazi, Ricardo Carrion, Kendra J. Alfson, Warren V. Kalina, Deepak Kaushal, Pei-Yong Shi, Thorsten Klamp, Corinna Rosenbaum, Andreas N. Kuhn, Özlem Türeci, Philip R. Dormitzer, Kathrin U. Jansen, Ugur Sahin

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial ([NCT04368728][1]). ### Competing Interest Statement U.S. and O.T. are management board members and employees at BioNTech SE (Mainz, Germany); K.C.W., B.G.L., D.S., B.J., T.K. and C.R. are employees at BioNTech SE; A.B.V., A.M., M.V., L.M.K., S.He., A.G., T.Z., A.P., D.E., S.C.D., S.F., S.E., F.B., B.S., A.W., Y.F., H.J., S.A.K., A.P.H., P.A., J.S., C.K., and A.N.K. are employees at BioNTech RNA Pharmaceuticals GmbH (Mainz, Germany); A.B.V., A.M., K.C.W., A.G., S.F., A.N.K and U.S. are inventors on patents and patent applications related to RNA technology and COVID-19 vaccine; A.B.V., A.M., M.V., L.M.K., K.C.W., S.He., B.G.L., A.P., D.E., S.C.D., S.F., S.E., D.S., B.J., B.S., A.P.H., P.A., J.S., C.K., T.K., C.R., A.N.K., O.T. and U.S. have securities from BioNTech SE; I.K., Y.C., K.A.S., J.L., M.M., K.T., M.C.G., S.H., J.A.L.,E.H.M., P.V.S., C.Y.T., D.P., G.S., M.P., I.L.S., T.C., J.O., W.V.K., P.R.D. and K.U.J. are employees of Pfizer and may hold stock options; C.F.-G. and P.-Y.S. received compensation from Pfizer to perform neutralisation assays; J.C., S.H.-U, K.B., R.C., jr., K.J.A. and D.K., are employees of Southwest National Primate Research Center, which received compensation from Pfizer to conduct the animal challenge work; no other relationships or activities that could appear to have influenced the submitted work. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04368728&atom=%2Fbiorxiv%2Fearly%2F2020%2F09%2F08%2F2020.09.08.280818.atom

Pamela C. Proud, Daphne Tsitoura, Robert J Watson, Brendon Y. Chua, Marilyn J Aram, Kevin R Bewley, Breeze E Cavell, Rebecca Cobb, Stuart Dowall, Susan A Fotheringham, Catherine M K Ho, Vanessa Lucas, Didier Ngabo, Emma Rayner, Kathryn A Ryan, Gillian S Slack, Stephen Thomas, Nadina I Wand, Paul Yeates, Christophe Demaison, David C. Jackson, Nathan W Bartlett, Francesca Mercuri, Miles W Carroll

Respiratory viruses such as coronaviruses represent major ongoing global threats, causing epidemics and pandemics with huge economic burden. Rapid spread of virus through populations poses an enormous challenge for outbreak control. Like all respiratory viruses, the most recent novel human coronavirus SARS-CoV-2, initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT to reduce SARS-CoV-2 transmission and provide protection against COVID-19. ### Competing Interest Statement The authors have declared no competing interest.

Yuhan Hao, Stephanie Hao, Erica Andersen-Nissen, William M. Mauck, Shiwei Zheng, Andrew Butler, Maddie J. Lee, Aaron J. Wilk, Charlotte Darby, Michael Zagar, Paul Hoffman, Marlon Stoeckius, Efthymia Papalexi, Eleni P. Mimitou, Jaison Jain, Avi Srivastava, Tim Stuart, Lamar B. Fleming, Bertrand Yeung, Angela J. Rogers, M. Juliana McElrath, Catherine A. Blish, Raphael Gottardo, Peter Smibert, Rahul Satija

The simultaneous measurement of multiple modalities, known as multimodal analysis, represents an exciting frontier for single-cell genomics and necessitates new computational methods that can define cellular states based on multiple data types. Here, we introduce "weighted-nearest neighbor analysis", an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of hundreds of thousands of human white blood cells alongside a panel of 228 antibodies to construct a multimodal reference atlas of the circulating immune system. We demonstrate that integrative analysis substantially improves our ability to resolve cell states and validate the presence of previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets, and to interpret immune responses to vaccination and COVID-19. Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets, including paired measurements of RNA and chromatin state, and to look beyond the transcriptome towards a unified and multimodal definition of cellular identity. Availability: Installation instructions, documentation, tutorials, and CITE-seq datasets are available at http://www.satijalab.org/seurat ### Competing Interest Statement In the past three years, RS has worked as a consultant for Bristol-Myers Squibb, Regeneron, and Kallyope, and served as an SAB member for ImmunAI, Apollo Life Sciences GmbH, Nanostring and the NYC Pandemic Response Lab. R.G. has received consulting income from Juno Therapeutics, Takeda, Infotech Soft, Celgene, Merck and has received research support from Janssen Pharmaceuticals and Juno Therapeutics, and declares ownership in CellSpace Biosciences. PS is a co-inventor of a patent related to this work. BZY is an employee at BioLegend Inc., which is the exclusive licensee of the New York Genome Center patent application related to this work.