Showing papers by "Icahn School of Medicine at Mount Sinai published in 2022"

Post-acute COVID-19 syndrome (PCS) and health-related quality of life (HRQoL)-A systematic review and meta-analysis.

Abstract: There is an established literature on the symptoms and complications of COVID-19 but the after-effects of COVID-19 are not well understood with few studies reporting persistent symptoms and quality of life. We aim to evaluate the pooled prevalence of poor quality of life in post-acute COVID-19 syndrome (PCS) and conducted meta-regression to evaluate the effects of persistent symptoms and intensive care unit (ICU) admission on the poor quality of life. We extracted data from observational studies describing persistent symptoms and quality of life in post-COVID-19 patients from March 10, 2020, to March 10, 2021, following PRISMA guidelines with a consensus of two independent reviewers. We calculated the pooled prevalence with 95% confidence interval (CI) and created forest plots using random-effects models. A total of 12 studies with 4828 PCS patients were included. We found that amongst PCS patients, the pooled prevalence of poor quality of life (EQ-VAS) was (59%; 95% CI: 42%-75%). Based on individual factors in the EQ-5D-5L questionnaire, the prevalence of mobility was (36, 10-67), personal care (8, 1-21), usual quality (28, 2-65), pain/discomfort (42, 28-55), and anxiety/depression (38, 19-58). The prevalence of persistent symptoms was fatigue (64, 54-73), dyspnea (39.5, 20-60), anosmia (20, 15-24), arthralgia (24.3, 14-36), headache (21, 3-47), sleep disturbances (47, 7-89), and mental health (14.5, 4-29). Meta-regression analysis showed the poor quality of life was significantly higher among post-COVID-19 patients with ICU admission (p = 0.004) and fatigue (p = 0.0015). Our study concludes that PCS is associated with poor quality of life, persistent symptoms including fatigue, dyspnea, anosmia, sleep disturbances, and worse mental health. This suggests that we need more research on PCS patients to understand the risk factors causing it and eventually leading to poor quality of life.

Post-acute COVID-19 Syndrome Negatively Impacts Physical Function, Cognitive Function, Health-Related Quality of Life, and Participation

Abstract: This report describes persistent symptoms associated with post-acute COVID-19 syndrome (PACS) and the impact of these symptoms on physical function, cognitive function, health-related quality of life, and participation.This study used a cross-sectional observational study design. Patients attending Mount Sinai's post-acute COVID-19 syndrome clinic completed surveys containing patient-reported outcomes.A total of 156 patients completed the survey, at a median (range) time of 351 days (82-457 days) after COVID-19 infection. All patients were prevaccination. The most common persistent symptoms reported were fatigue (n = 128, 82%), brain fog (n = 105, 67%), and headache (n = 94, 60%). The most common triggers of symptom exacerbation were physical exertion (n = 134, 86%), stress (n = 107, 69%), and dehydration (n = 77, 49%). Increased levels of fatigue (Fatigue Severity Scale) and dyspnea (Medical Research Council) were reported, alongside reductions in levels of regularly completed physical activity. Ninety-eight patients (63%) scored for at least mild cognitive impairment (Neuro-Qol), and the domain of the EuroQol: 5 dimension, 5 level most impacted was Self-care, Anxiety/Depression and Usual Activities.Persistent symptoms associated with post-acute COVID-19 syndrome seem to impact physical and cognitive function, health-related quality of life, and participation in society. More research is needed to further clarify the relationship between COVID-19 infection and post-acute COVID-19 syndrome symptoms, the underlying mechanisms, and treatment options.

Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

Abstract: •The S:655Y mutation in SARS-CoV-2 enhances spike protein cleavage and fusogenicity•The S:655Y mutation increases transmissibility in vivo•S:655Y was able to outcompete ancestral S:655H in vivo•SARS-CoV-2 VOCs evolve to acquire an increased spike cleavage and fusogenic ability SARS-CoV-2 lineages have diverged into highly prevalent variants termed “variants of concern” (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission. SARS-CoV-2 lineages have diverged into highly prevalent variants termed “variants of concern” (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission. SARS-CoV-2 has been spreading worldwide and causing millions of infections and deaths since its emergence in Wuhan, China in late 2019. Apart from humans, ferrets, cats, dogs, Syrian golden hamsters, and nonhuman primates are also susceptible to SARS-CoV-2 infection and transmission (Fenollar et al., 2021Fenollar F. Mediannikov O. Maurin M. Devaux C. Colson P. Levasseur A. Fournier P.E. Raoult D. Mink, SARS-CoV-2, and the Human-Animal Interface.Front. Microbiol. 2021; 12: 663815Crossref PubMed Scopus (39) Google Scholar; Shi et al., 2020Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (873) Google Scholar). In addition, cases of viral spread in mink farms and mink-to-human cross-species transmission have been reported (Hammer et al., 2021Hammer A.S. Quaade M.L. Rasmussen T.B. Fonager J. Rasmussen M. Mundbjerg K. Lohse L. Strandbygaard B. Jørgensen C.S. Alfaro-Núñez A. et al.SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.Emerg. Infect. Dis. 2021; 27: 547-551Crossref PubMed Scopus (91) Google Scholar; Oude Munnink et al., 2021Oude Munnink B.B. Sikkema R.S. Nieuwenhuijse D.F. Molenaar R.J. Munger E. Molenkamp R. van der Spek A. Tolsma P. Rietveld A. Brouwer M. et al.Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.Science. 2021; 371: 172-177Crossref PubMed Scopus (376) Google Scholar). The spike (S) glycoprotein of SARS-CoV-2 is the main determinant of host tropism and susceptibility and the main target of antibody responses (Tong et al., 2021Tong P. Gautam A. Windsor I. Travers M. Chen Y. Garcia N. Whiteman N.B. McKay L.G.A. Lelis F.J.N. Habibi S. et al.Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.03.10.434840Crossref Scopus (0) Google Scholar). Therefore, the emergence of adaptive mutations present in the S protein can strongly affect host tropism and viral transmission (Johnson et al., 2020Johnson B.A. Xie X. Kalveram B. Lokugamage K.G. Muruato A. Zou J. Zhang X. Juelich T. Smith J.K. Zhang L. et al.Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis.Preprint at bioRxiv. 2020; https://doi.org/10.1101/2020.08.26.268854Crossref Scopus (0) Google Scholar; Starr et al., 2020Starr T.N. Greaney A.J. Hilton S.K. Ellis D. Crawford K.H.D. Dingens A.S. Navarro M.J. Bowen J.E. Tortorici M.A. Walls A.C. et al.Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.Cell. 2020; 182: 1295-1310.e20Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). The S protein is composed of 2 subunits: S1, which contains the receptor-binding domain (RBD) that initiates infection by binding to the angiotensin converting enzyme 2 (ACE2) receptor present in the host cell surface, and the S2 subunit that mediates fusion between viral and cellular membranes (Duan et al., 2020Duan L. Zheng Q. Zhang H. Niu Y. Lou Y. Wang H. The SARS-CoV-2 Spike Glycoprotein Biosynthesis, Structure, Function, and Antigenicity: Implications for the Design of Spike-Based Vaccine Immunogens.Front. Immunol. 2020; 11: 576622Crossref PubMed Scopus (98) Google Scholar; Huang et al., 2020Huang Y. Yang C. Xu X.F. Xu W. Liu S.W. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19.Acta Pharmacol. Sin. 2020; 41: 1141-1149Crossref PubMed Scopus (602) Google Scholar). To fuse with the host cell, the S protein needs to be cleaved by cellular proteases at the S1/S2 and S2′ sites. Importantly, the S1/S2 site of SARS-CoV-2 viruses contains a multibasic furin motif (681PRRXR685) absent in other beta coronaviruses (Coutard et al., 2020Coutard B. Valle C. de Lamballerie X. Canard B. Seidah N.G. Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade.Antiviral Res. 2020; 176: 104742Crossref PubMed Scopus (885) Google Scholar; Hoffmann et al., 2020Hoffmann M. Kleine-Weber H. Pohlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.Mol. Cell. 2020; 78: 779-784.e5Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar) that can be processed by furin proteases but also by transmembrane serine proteases, such as TMPRSS2, or by cathepsins present in the endosomes (Bestle et al., 2020Bestle D. Heindl M.R. Limburg H. Van Lam van T. Pilgram O. Moulton H. Stein D.A. Hardes K. Eickmann M. Dolnik O. et al.TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.Life Sci Alliance. 2020; 3: e202000786Crossref PubMed Google Scholar; Hoffmann et al., 2020Hoffmann M. Kleine-Weber H. Pohlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.Mol. Cell. 2020; 78: 779-784.e5Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar; Matsuyama et al., 2010Matsuyama S. Nagata N. Shirato K. Kawase M. Takeda M. Taguchi F. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2.J. Virol. 2010; 84: 12658-12664Crossref PubMed Scopus (444) Google Scholar; Örd et al., 2020Örd M. Faustova I. Loog M. The sequence at Spike S1/S2 site enables cleavage by furin and phospho-regulation in SARS-CoV2 but not in SARS-CoV1 or MERS-CoV.Sci. Rep. 2020; 10: 16944Crossref PubMed Scopus (43) Google Scholar; Tang et al., 2021Tang T. Jaimes J.A. Bidon M.K. Straus M.R. Daniel S. Whittaker G.R. Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin.ACS Infect. Dis. 2021; 7: 264-272Crossref PubMed Scopus (52) Google Scholar). The S1/S2 cleavage exposes the S2′ site, and a second cleavage of the S2′ is needed to release an internal fusion peptide that mediates membrane fusion (Xia et al., 2020Xia S. Zhu Y. Liu M. Lan Q. Xu W. Wu Y. Ying T. Liu S. Shi Z. Jiang S. Lu L. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein.Cell. Mol. Immunol. 2020; 17: 765-767Crossref PubMed Scopus (340) Google Scholar). Since 2019, several SARS-CoV-2 lineages have emerged, leading to the divergence of an extensive subset of SARS-CoV-2 variants termed “variants of concern” (VOCs). This has led to the natural selection of several mutations in the S protein with different functional consequences, some of them unknown. As SARS-CoV-2 variants are arising, more research is needed to understand what the drivers of evolutionary changes are over time and the potential impact on epidemiology, antigenicity, escape from neutralizing antibodies induced by previous infection, and vaccination and virus fitness. The first widely adaptative substitution described was the S protein amino acid change S:D614G, which became dominant in March 2020 and is present in most of the variants currently circulating worldwide. This substitution is known to enhance viral replication in the upper respiratory tract as well as in vivo transmission (Korber et al., 2020Korber B. Fischer W.M. Gnanakaran S. Yoon H. Theiler J. Abfalterer W. Hengartner N. Giorgi E.E. Bhattacharya T. Foley B. et al.Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.Cell. 2020; 182: 812-827.e19Abstract Full Text Full Text PDF PubMed Scopus (1817) Google Scholar; Plante et al., 2021Plante J.A. Liu Y. Liu J. Xia H. Johnson B.A. Lokugamage K.G. Zhang X. Muruato A.E. Zou J. Fontes-Garfias C.R. et al.Spike mutation D614G alters SARS-CoV-2 fitness.Nature. 2021; 592: 116-121Crossref PubMed Scopus (604) Google Scholar). Several other polymorphisms became dominant in late 2020. The N501Y substitution convergently evolved in emerging VOCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and omicron (B.1.1.529) variants and has been associated with an enhanced spike affinity for the cellular ACE2 receptor (Liu et al., 2021Liu Y. Liu J. Plante K.S. Plante J.A. Xie X. Zhang X. Ku Z. An Z. Scharton D. Schindewolf C. et al.The N501Y spike substitution enhances SARS-CoV-2 transmission.Nature. 2021; (Published online November 24, 2021)https://doi.org/10.1038/s41586-021-04245-0Crossref Scopus (36) Google Scholar; Yi et al., 2020Yi C. Sun X. Ye J. Ding L. Liu M. Yang Z. Lu X. Zhang Y. Ma L. Gu W. et al.Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies.Cell. Mol. Immunol. 2020; 17: 621-630Crossref PubMed Scopus (208) Google Scholar). This mutation is located in the receptor-binding motif of the RBD, the primary target of many neutralizing antibodies. Importantly, accumulation of mutations in the RBD can decrease neutralizing antibody responses elicited by infection or vaccination against ancestral SARS-CoV-2 variants (Aydillo et al., 2021Aydillo T. Rombauts A. Stadlbauer D. Aslam S. Abelenda-Alonso G. Escalera A. Amanat F. Jiang K. Krammer F. Carratala J. García-Sastre A. Immunological imprinting of the antibody response in COVID-19 patients.Nat. Commun. 2021; 12: 3781Crossref PubMed Scopus (34) Google Scholar). Similarly, the later SARS-CoV-2 kappa (B.1.617.1) and delta (B.1.617.2) variants have also shown a significantly reduced sensitivity to convalescent and immune sera (Edara et al., 2021Edara V.V. Lai L. Sahoo M.K. Floyd K. Sibai M. Solis D. Flowers M.W. Hussaini L. Ciric C.R. Bechnack S. et al.Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant.Prepint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.09.443299Crossref Google Scholar; Mlcochova et al., 2021aMlcochova P. Kemp S. Dhar M.S. Papa G. Meng B. Mishra S. Whittaker C. Mellan T. Ferreira I. Datir R. et al.SARS-CoV-2 B.1.617.2 Delta variant emergence, replication and sensitivity to neutralising antibodies.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.08.443253Crossref Scopus (0) Google Scholar; Planas et al., 2021bPlanas D. Veyer D. Baidaliuk A. Staropoli I. Guivel-Benhassine F. Rajah M.M. Planchais C. Porrot F. Robillard N. Puech J. et al.Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.Nature. 2021; 596: 276-280Crossref PubMed Scopus (648) Google Scholar). Other mutations outside the RBD have also become prevalent. A clear example is the polymorphism found at position S:681 in the furin cleavage site, which includes P681H and P681R in the alpha/omicron and kappa/delta variants, respectively. Some preliminary reports have pointed to an enhancement in virus transmissibility associated with this polymorphism, perhaps due to an increase of spike cleavage (Peacock et al., 2021Peacock T.P. Sheppard C.M. Brown J.C. Goonawardane N. Zhou J. Whiteley M. Consortium P.V. de Silva T.I. Barclay W.S. The SARS-CoV-2 variants associated with infections in India, B.1.617, show enhanced spike cleavage by furin.Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.05.28.446163Crossref Scopus (0) Google Scholar). Additionally, several other mutations have been identified at the edge of the furin cleavage site. This is the case of the H655Y substitution found in the gamma (P.1) and omicron variants (B.1.1.529). This mutation was associated with changes in antigenicity by conferring escape from human monoclonal antibodies (Baum et al., 2020Baum A. Fulton B.O. Wloga E. Copin R. Pascal K.E. Russo V. Giordano S. Lanza K. Negron N. Ni M. et al.Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies.Science. 2020; 369: 1014-1018Crossref PubMed Scopus (577) Google Scholar). Moreover, it has also been found to be selected in animal models after experimental infection in vivo (Braun et al., 2021Braun K.M. Moreno G.K. Halfmann P.J. Hodcroft E.B. Baker D.A. Boehm E.C. Weiler A.M. Haj A.K. Hatta M. Chiba S. et al.Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.PLoS Pathog. 2021; 17: e1009373Crossref PubMed Google Scholar; Rathnasinghe et al., 2021Rathnasinghe R. Jangra S. Cupic A. Martinez-Romero C. Mulder L.C.F. Kehrer T. Yildiz S. Choi A. Mena I. De Vrieze J. et al.The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera.Preprint at medRxiv. 2021; https://doi.org/10.1101/2021.01.19.21249592Crossref Scopus (0) Google Scholar), indicating a potential role in host replication, transmissibility, and pathogenicity. Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. Using the mink model of COVID-19, we found that the S:H655Y substitution was acquired in vivo after infection with the WA1 isolate (USA-WA1/2020). To investigate the advantage conferred by S:H655Y, we analyzed the kinetics, spike processing by cellular proteases, and syncytium formation ability of a panel of SARS-CoV-2 variants harboring 655Y, including human isolates derived from patients seeking care at the Mount Sinai Health System in New York (NY) City, which was one of the major early epicenters of the COVID-19 pandemic. Our results demonstrate that the 655Y polymorphism enhances spike cleavage and viral growth. Furthermore, the S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to overcome S:655H in the human airway epithelial system. Finally, and in the context of the current epidemiological situation, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. We demonstrate that novel circulating VOCs that have become more prevalent have independently acquired mutations associated with a gain in spike cleavage and syncytia formation. Taken together, our study shows a link between an increased spike processing and increased virus transmission due to the spike mutations present in SARS-CoV-2 variants that have become epidemiologically more prevalent in humans. Minks have been suggested to play a role in the initial local spread and evolution of SARS-CoV-2 variants in different countries in Europe (Hammer et al., 2021Hammer A.S. Quaade M.L. Rasmussen T.B. Fonager J. Rasmussen M. Mundbjerg K. Lohse L. Strandbygaard B. Jørgensen C.S. Alfaro-Núñez A. et al.SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.Emerg. Infect. Dis. 2021; 27: 547-551Crossref PubMed Scopus (91) Google Scholar; Oude Munnink et al., 2021Oude Munnink B.B. Sikkema R.S. Nieuwenhuijse D.F. Molenaar R.J. Munger E. Molenkamp R. van der Spek A. Tolsma P. Rietveld A. Brouwer M. et al.Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans.Science. 2021; 371: 172-177Crossref PubMed Scopus (376) Google Scholar). While minks are susceptible to SARS-CoV-2, they are also capable for zoonotic transmission of SARS-CoV-2 because of the similarity of the ACE2 receptor between minks and humans. We used the mink model to investigate the replication and pathogenicity of the WA1 (USA-WA1/2020) isolate of SARS-CoV-2 as a representative of the first original human viruses that initiated the SARS-CoV-2 pandemic. This variant corresponds to one of the first isolates of the United States of America (USA) and does not contain any changes on the S protein when compared to the initial isolates from Wuhan, such as the Wuhan-1 virus. For this purpose, 6 minks were intranasally infected with 106 pfu of WA1 isolate, resulting in productive viral replication in the upper respiratory tract with infectious virus recovered from nasal washes at days 1, 3, and 5 post-infection (p.i.) (Figures S1A and S1B). At day 4 post-inoculation, infectious virus was detected by plaque assays from left cranial lung and nasal turbinates, but not from any of the other tissues analyzed (Figure S1C). We then selected small and large viral plaques in the Vero E6 cell-based plaque assays from infected mink lung specimens and performed next generation sequencing of the genome from the plaque-isolated viruses. As compared to the Wuhan-1 and WA1 reference sequences, all mink-derived viral isolates encoded the H655Y amino acid substitution within S (Figure S1D). Additionally, the 3 viral isolates with the small plaque phenotype encoded the T259K amino acid substitution while the 3 viral isolates with the large plaque phenotype encoded the R682W amino acid substitution. It is known that S:682W/Q substitution in the furin cleavage site region may emerge after subsequent passages in Vero E6 cells (Lamers et al., 2021Lamers M.M. Mykytyn A.Z. Breugem T.I. Wang Y. Wu D.C. Riesebosch S. van den Doel P.B. Schipper D. Bestebroer T. Wu N.C. Haagmans B.L. Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.eLife. 2021; 10: e66815Crossref PubMed Google Scholar; Liu et al., 2020Liu Z. Zheng H. Lin H. Li M. Yuan R. Peng J. Xiong Q. Sun J. Li B. Wu J. et al.Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.J. Virol. 2020; 94 (e00790-20)Crossref Scopus (69) Google Scholar). Next generation sequencing analysis of the viral quasispecies population in mink lung specimens and WA1 stock demonstrated the presence of around 30% of the S:682W/Q substitution, indicating that this mutation was selected during the course of the Vero E6 infections and stock generation and not during the infection in minks. On the other hand, S:655Y was present in less than 10% of the WA1 original stock, but it was dominant in all the mink isolates in 99% of the viral RNA obtained from the lungs, suggesting that this mutation was selected and may confer an advantage in the mink host. To understand the magnitude and the spread of the 655Y polymorphism over time, we investigated the frequency of S:655Y in sequences sampled worldwide since the initial outbreak to the end of the first wave of SARS-CoV-2 (Figure 1A). For this, 7,059 sequences sampled from GISAID up to September 2020 were used. Human variants harboring the 655Y mutation were spread throughout the phylogenetic tree and distributed in all clades with no differences according to temporal distribution, suggesting that the 655Y mutation arose independently multiple times. Remarkably, the S:H655Y polymorphism was also found among the initial variants introduced in NY City in March 2020. To determine the replication phenotype, we decided to investigate this NY 655Y variant (NY7) together with some of its contemporaneous SARS-CoV-2 isolates circulating in NY during the early pandemic outbreak (Gonzalez-Reiche et al., 2020Gonzalez-Reiche A.S. Hernandez M.M. Sullivan M.J. Ciferri B. Alshammary H. Obla A. Fabre S. Kleiner G. Polanco J. Khan Z. et al.Introductions and early spread of SARS-CoV-2 in the New York City area.Science. 2020; 369: 297-301Crossref PubMed Scopus (157) Google Scholar). To this end, we isolated 12 viruses based on their genotypes (Gonzalez-Reiche et al., 2020Gonzalez-Reiche A.S. Hernandez M.M. Sullivan M.J. Ciferri B. Alshammary H. Obla A. Fabre S. Kleiner G. Polanco J. Khan Z. et al.Introductions and early spread of SARS-CoV-2 in the New York City area.Science. 2020; 369: 297-301Crossref PubMed Scopus (157) Google Scholar), including NY7, which carries the S:655Y mutation for culture directly from nasopharyngeal specimens obtained from COVID-19-infected patients. Of note, the dominant 614G spike polymorphism was present in seven (NY1, NY2, NY3, NY4, NY9, NY10, and NY12; 58%) of the selected human SARS-CoV-2 (hCoV) NY isolates, which is consistent with its early emergence and rapid spread worldwide (Korber et al., 2020Korber B. Fischer W.M. Gnanakaran S. Yoon H. Theiler J. Abfalterer W. Hengartner N. Giorgi E.E. Bhattacharya T. Foley B. et al.Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.Cell. 2020; 182: 812-827.e19Abstract Full Text Full Text PDF PubMed Scopus (1817) Google Scholar; Plante et al., 2021Plante J.A. Liu Y. Liu J. Xia H. Johnson B.A. Lokugamage K.G. Zhang X. Muruato A.E. Zou J. Fontes-Garfias C.R. et al.Spike mutation D614G alters SARS-CoV-2 fitness.Nature. 2021; 592: 116-121Crossref PubMed Scopus (604) Google Scholar). Confirmation sequencing of the isolates showed that 682W/Q substitutions appeared in 4 (33%) viruses after initial isolation and culturing in Vero E6 cells. However, this mutation was absent in the original nasal swabs collected from the corresponding COVID-19-infected patients. This is consistent with in vitro adaptative mutations previously described (Lamers et al., 2021Lamers M.M. Mykytyn A.Z. Breugem T.I. Wang Y. Wu D.C. Riesebosch S. van den Doel P.B. Schipper D. Bestebroer T. Wu N.C. Haagmans B.L. Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.eLife. 2021; 10: e66815Crossref PubMed Google Scholar). Moreover, a 5-amino-acid sequence (Δ675–679) flanking the furin cleavage site was deleted in five (42%) of the isolates as compared to the sequence from the original specimen. This deletion has been previously reported to be a common in vitro mutation selected in Vero cells (Liu et al., 2020Liu Z. Zheng H. Lin H. Li M. Yuan R. Peng J. Xiong Q. Sun J. Li B. Wu J. et al.Identification of Common Deletions in the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus 2.J. Virol. 2020; 94 (e00790-20)Crossref Scopus (69) Google Scholar). Amino acids substitutions of the S protein of these initial human isolates compared to the Wuhan-1 reference are shown in Figure S2A and Table S2. We next studied the replication kinetics of the NY SARS-CoV-2 isolates by comparing their multicycle growth curves at an MOI of 0.01 in Vero E6 and human Caco-2 cells. As expected, NY2, NY4, and NY9 containing the 682Q/W showed an advantage in growth at 48 h p.i. in Vero E6 cells, while no differences could be found in Caco-2 cells (Figures S2B–S2E). Remarkably, NY7 (S:655Y) showed higher growth at 48 h p.i in Caco-2 cells (Figures S2C and S2E) when compared to the rest of these early SARS-CoV-2 isolates. These results support our conclusion that the 655Y polymorphism conferred a viral advantage. To investigate the spike cleavage efficiency of the 655Y versus other human isolates, we performed infections in Vero E6, and supernatants were analyzed by western blot for the S2 domain of the S protein. Importantly, 2 bands were clearly visible for the NY7 (S:655Y) (Figure 1B), corresponding to both the cleaved (95 kDa) and uncleaved (180 kDa) form of the S protein. In contrast, subtle bands for S2 were detected in NY6, NY10, and NY12, and only the uncleaved S form was detected in the other early human isolates. Consistently, quantification of full-length and cleaved S protein showed that around 80% of the NY7 (S:655Y) S was in its S2 cleaved form (Figure 1C), indicating that the 655Y polymorphism may facilitate S protein processing. To confirm these results in a primary human cell model, we assessed the S protein processing of the twelve early NY human isolates in pneumocyte-like cells. This system has been previously described as a robust model to study SARS-CoV-2 infection (Riva et al., 2020Riva L. Yuan S. Yin X. Martin-Sancho L. Matsunaga N. Pache L. Burgstaller-Muehlbacher S. De Jesus P.D. Teriete P. Hull M.V. et al.Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.Nature. 2020; 586: 113-119Crossref PubMed Scopus (343) Google Scholar; White et al., 2021White K.M. Rosales R. Yildiz S. Kehrer T. Miorin L. Moreno E. Jangra S. Uccellini M.B. Rathnasinghe R. Coughlan L. et al.Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.Science. 2021; 371: 926-931Crossref PubMed Scopus (96) Google Scholar). Human pneumocyte-like cells were differentiated from human induced pluripotent stem cell and infected with the NY SARS-CoV-2 isolates. Cell extracts were collected at 48 h p.i and analyzed by western blot (Figure 1D). As expected, NY7 (S:655Y) showed an increase cleavage efficiency with 70% of cleaved S protein (Figure 1E). Additionally, we observed S2 cleaved protein in NY12 consistent with the S2 subtle bands detected in Vero E6 cells. This isolate only contains the S:D614G mutation. Although other NY variants used in this study also harbor the D614G, no enhancement in spike cleavage was observed. This can be explained by the acquisition of additional S mutations during in vitro culture (Δ675–679, 682Q/W) in the isolates containing the D614G, which could be masking the effect of D614G in spike processing. To confirm whether the 655Y mutation was solely responsible for the increased S cleavage, we analyzed the replication and cleavage efficiency of a panel of SARS-CoV-2 viruses, all bearing the 655Y substitution but containing additional substitutions across the genome. We included two of the isolated mink variants (MiA1 and MiA2), NY7 and another human isolate derived from a COVID-19 patient infected in February 2021 (NY13, S:614G, 655Y), and a WA1-655Y variant isolated after wild-type WA1 infection in Vero E6 cells and subsequent plaque purification. Additionally, the WA1 reference and NY6 were used as controls since they lack the 655Y substitution. It should be noted that NY6 has a 5-amino-acid deletion before the furin cleavage site (Figures 2A and S2A). We assessed differences in replication and S processing of this panel of viruses by comparing growth in both Vero E6 and Vero-TMPRSS2 cells. As shown in Figure 2B, WA1-655Y infection yielded higher titers in both Vero E6 and Vero-TMPRSS2 cells as compared to infection by WA1 (statistically significant in Vero E6 at 24 h p.i.; Table S4). These isolates only differ in the position 655Y while the rest of the genome is isogenic, supporting that 655Y spike polymorphism enhances viral replication and growth. Next, viral supernatants were used to analyze the plaque phenotype in Vero E6 and Vero-TMPRSS2 and to compare S protein expression levels after infection. In general, all isolates showed higher plaque size in the presence of TMPRSS2, consistent with enhancement of cell entry (Figure S3A). However, differences were found by western blot, and only the isolates bearing 655Y showed enhanced spike cleavage in both Vero E6 and Vero-TMPRSS2 (Figure 2C). More than 90% of the total S protein from these 655Y variants corresponded to the S2 cleaved form of the spike. In contrast, NY6 and WA1 controls showed poor cleavage efficiency (Figures 2D and 2E). Finally, we performed infections with a representative panel of viruses containing the 655Y (NY7, NY13, WA1-655Y, NY6, and WA1) in human pneumocyte-like cells to assess viral growth and S protein processing. Consistent with our previous findings, WA1-655Y demonstrated higher replication efficiency in our human airway epithelial system compared to WA1 wild type (Figure 2F; Table S4). Moreover, all isolates encoding the 655Y spike mutation exhibited enhanced spike cleavage as shown by western blot (Figures 2G and 2H). This demonstrates that the S:655Y polymorphism plays a crucial role in SARS-CoV-2 S protein processing and cell entry in human pneumocyte-like cells. Syncytia formation has been described as one of the hallmarks of SARS-CoV-2 infection and pathogenesis in the lungs. It is mediated by the interaction

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures

Abstract: Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors

Abstract: Abstract It is currently accepted that cancer-associated fibroblasts (CAF) participate in T-cell exclusion from tumor nests. To unbiasedly test this, we used single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors. We identified four main CAF populations, two of which are associated with T-cell exclusion: (i) MYH11+αSMA+ CAF, which are present in early-stage tumors and form a single cell layer lining cancer aggregates, and (ii) FAP+αSMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both populations orchestrate a particular structural tissue organization through dense and aligned fiber deposition compared with T cell–permissive CAF. Yet they produce distinct matrix molecules, including collagen IV (MYH11+αSMA+ CAF) and collagen XI/XII (FAP+αSMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T-cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell–excluded tumors. Significance: The cellular and molecular programs driving T-cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T-cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to developing new strategies targeting T cell–excluding CAF. See related commentary by Sherman, p. 2501. This article is highlighted in the In This Issue feature, p. 2483

Efficacy, immunogenicity, and safety of a quadrivalent HPV vaccine in men: results of an open-label, long-term extension of a randomised, placebo-controlled, phase 3 trial

Abstract: The quadrivalent human papillomavirus (HPV) vaccine was shown to prevent infections and lesions related to HPV6, 11, 16, and 18 in a randomised, placebo-controlled study in men aged 16-26 years. We assessed the incidences of external genital warts related to HPV6 or 11, and external genital lesions and anal dysplasia related to HPV6, 11, 16, or 18, over 10 years of follow-up.The 3-year base study was an international, multicentre, double-blind, randomised, placebo-controlled trial done at 71 sites in 18 countries. Eligible participants were heterosexual men (aged 16-23 years) or men who have sex with men (MSM; aged 16-26 years). Men who had clinically detectable anogenital warts or genital lesions at screening that were suggestive of infection with non-HPV sexually transmitted diseases, or who had a history of such findings, were excluded. Eligible participants were randomly assigned (1:1) to receive three doses of either quadrivalent HPV vaccine or placebo on day 1, month 2, and month 6, administered as a 0·5-mL injection into the deltoid muscle. The 7-year, open-label, long-term follow-up extension study was done at 46 centres in 16 countries. Participants who received one or more doses of the quadrivalent HPV vaccine in the base study were eligible for enrolment into the long-term follow-up study (early vaccination group). Placebo recipients were offered the three-dose quadrivalent HPV vaccine at the end of the base study; those who received one or more quadrivalent HPV vaccine doses were eligible for enrolment into the long-term follow-up study (catch-up vaccination group). The primary efficacy endpoints were the incidence of external genital warts related to HPV6 or 11 and the incidence of external genital lesions related to HPV6, 11, 16, or 18 in all participants and the incidence of anal intraepithelial neoplasia (including anal warts and flat lesions) or anal cancer related to HPV6, 11, 16, or 18 in MSM only. The primary efficacy analysis was done in the per-protocol population for the early vaccination group, which included participants who received all three vaccine doses, were seronegative at day 1 and PCR-negative from day 1 through month 7 of the base study for the HPV type being analysed, had no protocol violations that could affect evaluation of vaccine efficacy, and had attended at least one visit during the long-term follow-up study. For the catch-up vaccination group, efficacy was assessed in the modified intention-to-treat population, which included participants who had received at least one vaccine dose, were seronegative and PCR-negative for HPV types analysed from day 1 of the base study to the final follow-up visit before receiving the quadrivalent HPV vaccine, and had at least one long-term follow-up visit. Safety was assessed in all randomised participants who received at least one vaccine dose. This study is registered with ClinicalTrials.gov, NCT00090285.Between Aug 10, 2010, and April 3, 2017, 1803 participants were enrolled in the long-term follow-up study, of whom 936 (827 heterosexual men and 109 MSM) were included in the early vaccination group and 867 (739 heterosexual men and 128 MSM) were included in the catch-up vaccination group. Participants in the early vaccination group were followed up for a median of 9·5 years (range 0·1-11·5) after receiving the third dose of the quadrivalent HPV vaccine, and participants in the catch-up vaccination group were followed up for a median of 4·7 years (0·0-6·6) after receiving the third dose. In early vaccine group participants during long-term follow-up compared with the placebo group in the base study, the incidence per 10 000 person-years of external genital warts related to HPV6 or 11 was 0·0 (95% CI 0·0-8·7) versus 137·3 (83·9-212·1), of external genital lesions related to HPV6, 11, 16, or 18 was 0·0 (0·0-7·7) versus 140·4 (89·0-210·7), and of anal intraepithelial neoplasia or anal cancer related to HPV6, 11, 16, or 18 in MSM only was 20·5 (0·5-114·4) versus 906·2 (553·5-1399·5). Compared with during the base study (ie, before quadrivalent HPV vaccine administration), during the long-term follow-up period, participants in the catch-up vaccination group had no new reported cases of external genital warts related to HPV6 or 11 (149·6 cases per 10 000 person-years [95% CI 101·6-212·3] vs 0 cases per 10 000 person-years [0·0-13·5]) or external genital lesions related to HPV6, 11, 16, or 18 (155·1 cases per 10 000 person-years [108·0-215·7] vs 0 cases per 10 000 person-years [0·0-10·2]), and a lower incidence of anal intraepithelial neoplasia or anal cancer related to HPV6, 11, 16, or 18 (886·0 cases per 10 000 person-years [583·9-1289·1] vs 101·3 cases per 10 000 person-years [32·9-236·3]). No vaccine-related serious adverse events were reported.The quadrivalent HPV vaccine provides durable protection against anogenital disease related to HPV6, 11, 16, and 18. The results support quadrivalent HPV vaccination in men, including catch-up vaccination.Merck Sharp & Dohme.

Opportunistic experiments to constrain aerosol effective radiative forcing

Abstract: Aerosol-cloud interactions (ACIs) are considered to be the most uncertain driver of present-day radiative forcing due to human activities. The nonlinearity of cloud-state changes to aerosol perturbations make it challenging to attribute causality in observed relationships of aerosol radiative forcing. Using correlations to infer causality can be challenging when meteorological variability also drives both aerosol and cloud changes independently. Natural and anthropogenic aerosol perturbations from well-defined sources provide "opportunistic experiments" (also known as natural experiments) to investigate ACI in cases where causality may be more confidently inferred. These perturbations cover a wide range of locations and spatiotemporal scales, including point sources such as volcanic eruptions or industrial sources, plumes from biomass burning or forest fires, and tracks from individual ships or shipping corridors. We review the different experimental conditions and conduct a synthesis of the available satellite datasets and field campaigns to place these opportunistic experiments on a common footing, facilitating new insights and a clearer understanding of key uncertainties in aerosol radiative forcing. Cloud albedo perturbations are strongly sensitive to background meteorological conditions. Strong liquid water path increases due to aerosol perturbations are largely ruled out by averaging across experiments. Opportunistic experiments have significantly improved process-level understanding of ACI, but it remains unclear how reliably the relationships found can be scaled to the global level, thus demonstrating a need for deeper investigation in order to improve assessments of aerosol radiative forcing and climate change.

An NR2F1-specific agonist suppresses metastasis by inducing cancer cell dormancy

Abstract: We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 agonists to induce dormancy as a therapeutic strategy to prevent metastasis.

Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4

Abstract: Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Long-term analysis of antibodies elicited by SPUTNIK V: A prospective cohort study in Tucumán, Argentina.

Abstract: Background Gam-COVID-Vac (SPUTNIK V) has been granted emergency use authorization in 70 nations and has been administered to millions worldwide. However, there are very few peer-reviewed studies describing its effects. Independent reports regarding safety and effectiveness could accelerate the final approval by the WHO. We aimed to study the long-term humoral immune response in naive and previously infected volunteers who received SPUTNIK V. Methods Humoral immune responses, assayed by anti-SARS-CoV-2-spike-RBD IgG ELISA and neutralization assays, were measured in 602 healthcare workers at 0, 14, 28, 60 and 180 days after receiving SPUTNIK V between December 2020 and July 2021 in Tucuman, Argentina. Findings Seroconversion was detected in 97% of individuals after 28 days post-vaccination (dpv) (N = 405). Anti-RBD titers began to decrease after 60 dpv (N = 328), but remained detectable in 94% at 90 dpv (N = 224). At 180 dpv, anti-RDB titers persisted in 31% (N = 146). Previous infection triggered an increased immune response to the first dose and increased neutralization activity against variants of concern (VOC). Second doses in previously infected individuals further increased titers, even 90 dpv (N = 75). Basal antibody titers had more influence on post-vaccination anti-RBD responses than the time elapsed between diagnosis and vaccination (N = 274). Interpretation Data presented herein provides essential knowledge regarding the kinetics of antibodies induced by SPUTNIK V up to six months after immunization, and suggests that when considering one-dose vaccination policies for individuals with previous SARS-CoV-2 infection, serological studies to determine basal titers may be important, independent of when diagnosis occurred. Funding Tucuman Public Health System (SIPROSA), Argentinean National Research Council (CONICET), National University of Tucuman (UNT).

Ritlecitinib and brepocitinib demonstrate significant improvement in scalp alopecia areata biomarkers

Abstract: Janus kinase (JAK) inhibitors have shown encouraging results in the treatment of alopecia areata (AA), an autoimmune form of hair loss, in small, uncontrolled studies and case reports.We conducted a biopsy substudy during the randomized, double-blind, placebo-controlled first 24 weeks of a phase 2a clinical trial that evaluated the efficacy and safety of ritlecitinib, an inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, and brepocitinib, an inhibitor of tyrosine kinase 2 (TYK2)/JAK1 in the treatment of AA.Change in biomarkers in lesional scalp biopsy samples between baseline and weeks 12 and 24 was an exploratory end point, and 46 patients participated from the ritlecitinib (n = 18), brepocitinib (n = 16), and placebo (n = 12) groups. Correlations of biomarkers with hair regrowth, measured using the Severity of Alopecia Tool (SALT) score, were also evaluated.NCT02974868.At week 24, both ritlecitinib and brepocitinib demonstrated improvement exceeding 100% in the lesional scalp transcriptome toward a nonlesional profile. At week 12, the improvements in scalp tissue were greater with brepocitinib than ritlecitinib; however, at week 24, the improvements were greater with ritlecitinib.For both ritlecitinib and brepocitinib, improvement in the SALT scores was positively associated with expression of TH1 markers and negatively associated with expression of hair keratins. Larger, long-term clinical trials are warranted.