Molecular therapy : the journal of the American Society of Gene Therapy
Yu, D;Han, HJ;Yu, J;Kim, J;Lee, GH;Yang, JH;Song, BM;Tark, D;Choi, BS;Kang, SM;Heo, WD;
PMID: 36945774 | DOI: 10.1016/j.ymthe.2023.03.018
CRISPR-Cas13-mediated viral genome targeting is a novel strategy for defending against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we generated mRNA-encoded Cas13b targeting the open reading frame 1b (ORF1b) region to effectively degrade the RNA-dependent RNA polymerase gene. Of the 12 designed CRISPR RNAs (crRNAs), those targeting the pseudoknot site upstream of ORF1b were found to be the most effective in suppressing SARS-CoV-2 propagation. Pseudoknot-targeting Cas13b reduced expression of the spike protein and attenuated viral replication by 99%. It also inhibited the replication of multiple SARS-CoV-2 variants, exhibiting broad potency. We validated the therapeutic efficacy of this system in SARS-CoV-2-infected hACE2 transgenic mice, demonstrating that crRNA treatment significantly reduced viral titers. Our findings suggest that the pseudoknot region is a strategic site for targeted genomic degradation of SARS-CoV-2. Hence, pseudoknot-targeting Cas13b could be a breakthrough therapy for overcoming infections by SARS-CoV-2 or other RNA viruses.
Barnett, KC;Xie, Y;Asakura, T;Song, D;Liang, K;Taft-Benz, SA;Guo, H;Yang, S;Okuda, K;Gilmore, RC;Loome, JF;Oguin Iii, TH;Sempowski, GD;Randell, SH;Heise, MT;Lei, YL;Boucher, RC;Ting, JP;
PMID: 36563691 | DOI: 10.1016/j.chom.2022.12.005
Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.
Berry, N;Ferguson, D;Kempster, S;Hall, J;Ham, C;Jenkins, A;Rannow, V;Giles, E;Leahy, R;Goulding, S;Fernandez, A;Adedeji, Y;Vessillier, S;Rajagopal, D;Prior, S;Le Duff, Y;Hurley, M;Gilbert, S;Fritzsche, M;Mate, R;Rose, N;Francis, RJ;MacLellan-Gibson, K;Suarez-Bonnet, A;Priestnall, S;Almond, N;
PMID: 36333445 | DOI: 10.1038/s41598-022-23339-x
SARS-CoV-2 exhibits a diverse host species range with variable outcomes, enabling differential host susceptibility studies to assess suitability for pre-clinical countermeasure and pathogenesis studies. Baseline virological, molecular and pathological outcomes were determined among multiple species-one Old World non-human primate (NHP) species (cynomolgus macaques), two New World NHP species (red-bellied tamarins; common marmosets) and Syrian hamsters-following single-dose, atraumatic intranasal administration of SARS-CoV-2/Victoria-01. After serial sacrifice 2, 10 and 28-days post-infection (dpi), hamsters and cynomolgus macaques displayed differential virus biodistribution across respiratory, gastrointestinal and cardiovascular systems. Uniquely, New World tamarins, unlike marmosets, exhibited high levels of acute upper airway infection, infectious virus recovery associated with mild lung pathology representing a host previously unrecognized as susceptible to SARS-CoV-2. Across all species, lung pathology was identified post-clearance of virus shedding (antigen/RNA), with an association of virus particles within replication organelles in lung sections analysed by electron microscopy. Disrupted cell ultrastructure and lung architecture, including abnormal morphology of mitochondria 10-28 dpi, represented on-going pathophysiological consequences of SARS-CoV-2 in predominantly asymptomatic hosts. Infection kinetics and host pathology comparators using standardized methodologies enables model selection to bridge differential outcomes within upper and lower respiratory tracts and elucidate longer-term consequences of asymptomatic SARS-CoV-2 infection.
Proceedings of the National Academy of Sciences of the United States of America
Morrison, CB;Edwards, CE;Shaffer, KM;Araba, KC;Wykoff, JA;Williams, DR;Asakura, T;Dang, H;Morton, LC;Gilmore, RC;O'Neal, WK;Boucher, RC;Baric, RS;Ehre, C;
PMID: 35353667 | DOI: 10.1073/pnas.2119680119
Significance Gaining insights into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) high transmissibility and the role played by inflammatory mediators in viral proliferation are critical to investigating new therapeutic targets against COVID-19. Electron microscopy reveals important SARS-CoV-2 features, including the combination of large, rapidly released viral clusters and the massive shedding of epithelial cells packed with virions. Interleukin-13 (IL-13), a Th2 cytokine up-regulated in allergic asthma and associated with less severe COVID-19, protects against SARS-CoV-2 viral and cell shedding. Using gene expression analyses and biochemical assays, IL-13 is shown to affect viral entry, replication, and cell-to-cell transmission. Given the broad spectrum of COVID-19 clinical symptoms, it is important to elucidate intrinsic factors that modulate viral load and spreading mechanisms.
Chang, YC;Yang, CF;Chen, YF;Yang, CC;Chou, YL;Chou, HW;Chang, TY;Chao, TL;Hsu, SC;Ieong, SM;Tsai, YM;Liu, PC;Chin, YF;Fang, JT;Kao, HC;Lu, HY;Chang, JY;Weng, RS;Tu, QW;Chang, FY;Huang, KY;Lee, TY;Chang, SY;Yang, PC;
PMID: 35138028 | DOI: 10.15252/emmm.202115298
The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants has altered the trajectory of the COVID-19 pandemic and raised some uncertainty on the long-term efficiency of vaccine strategy. The development of new therapeutics against a wide range of SARS-CoV-2 variants is imperative. We, here, have designed an inhalable siRNA, C6G25S, which covers 99.8% of current SARS-CoV-2 variants and is capable of inhibiting dominant strains, including Alpha, Delta, Gamma, and Epsilon, at picomolar ranges of IC50 in vitro. Moreover, C6G25S could completely inhibit the production of infectious virions in lungs by prophylactic treatment, and decrease 96.2% of virions by cotreatment in K18-hACE2-transgenic mice, accompanied by a significant prevention of virus-associated extensive pulmonary alveolar damage, vascular thrombi, and immune cell infiltrations. Our data suggest that C6G25S provides an alternative and effective approach to combating the COVID-19 pandemic.
Kaufmann, E;Khan, N;Tran, KA;Ulndreaj, A;Pernet, E;Fontes, G;Lupien, A;Desmeules, P;McIntosh, F;Abow, A;Moorlag, SJCFM;Debisarun, P;Mossman, K;Banerjee, A;Karo-Atar, D;Sadeghi, M;Mubareka, S;Vinh, DC;King, IL;Robbins, CS;Behr, MA;Netea, MG;Joubert, P;Divangahi, M;
PMID: 35235831 | DOI: 10.1016/j.celrep.2022.110502
Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.
Lee, JY;Wing, PA;Gala, DS;Noerenberg, M;Järvelin, AI;Titlow, J;Zhuang, X;Palmalux, N;Iselin, L;Thompson, MK;Parton, RM;Prange-Barczynska, M;Wainman, A;Salguero, FJ;Bishop, T;Agranoff, D;James, W;Castello, A;McKeating, JA;Davis, I;
PMID: 35049501 | DOI: 10.7554/eLife.74153
Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.
Luu, R;Valdebenito, S;Scemes, E;Cibelli, A;Spray, D;Rovegno, M;Tichauer, J;Cottignies-Calamarte, A;Rosenberg, A;Capron, C;Belouzard, S;Dubuisson, J;Annane, D;Lorin de la Grandmaison, G;Cramer-Bordé, E;Bomsel, M;Eugenin, E;
PMID: 34841222 | DOI: 10.1016/j.isci.2021.103478
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly rampaged worldwide, causing a pandemic of coronavirus disease (COVID -19), but the biology of SARS-CoV-2 is under investigation. We demonstrate that both SARS-CoV-2 spike protein and human coronavirus 229E (hCoV-229E) or its purified S protein, one of the main viruses responsible for the common cold, induce the transient opening of Pannexin-1 (Panx-1) channels in human lung epithelial cells. However, the Panx-1 channel opening induced by SARS-CoV-2 is greater and more prolonged than hCoV-229E/S protein, resulting in ATP, PGE2, and IL-1β release. Analysis of lung lavage and tissues indicate that Panx-1 mRNA expression is associated with increased ATP, PGE2, and IL-1β levels. Panx-1 channel opening induced by SARS-CoV-2 spike protein is angiotensin-converting enzyme 2 (ACE-2), endocytosis, and furin dependent. Overall, we demonstrated that Panx-1 is a critical contributor to SARS-CoV-2 infection and should be considered an alternative therapy.
Gajewski, T;Rouhani, S;Trujillo, J;Pyzer, A;Yu, J;Fessler, J;Cabanov, A;Higgs, E;Cron, K;Zha, Y;Lu, Y;Bloodworth, J;Abasiyanik, M;Okrah, S;Flood, B;Hatogai, K;Leung, M;Pezeshk, A;Kozloff, L;Reschke, R;Strohbehn, G;Chervin, CS;Kumar, M;Schrantz, S;Madariaga, ML;Beavis, K;Yeo, KT;Sweis, R;Segal, J;Tay, S;Izumchenko, E;Mueller, J;Chen, L;
PMID: 34845442 | DOI: 10.21203/rs.3.rs-1083825/v1
The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.
Molecular Analysis of the Kidney From a Patient With COVID-19-Associated Collapsing Glomerulopathy
Meliambro, K;Li, X;Salem, F;Yi, Z;Sun, Z;Chan, L;Chung, M;Chancay, J;Vy, HMT;Nadkarni, G;Wong, JS;Fu, J;Lee, K;Zhang, W;He, JC;Campbell, KN;
PMID: 33942030 | DOI: 10.1016/j.xkme.2021.02.012
Recent Case reports suggest COVID-19 is associated with collapsing glomerulopathy in African Americans with APOL1 risk alleles, however, it is unclear if disease pathogenesis is similar to HIVAN. Here RNA sequencing analysis of a kidney biopsy specimen from a patient with COVID-19-associated collapsing glomerulopathy and APOL1 risk alleles (G1/G1) revealed similar levels of APOL1 and ACE2 mRNA transcripts as compared to 12 control kidney samples downloaded from the GTEx Portal. Whole genome sequencing of the COVID-19-associated collapsing glomerulopathy kidney sample identified four indel gene variants, three of which are of unknown significance with respect to chronic kidney disease and/or FSGS. Molecular profiling of the kidney demonstrated activation of COVID-19-associated cell injury pathways such as inflammation and coagulation. Evidence for direct SARS-CoV-2 infection of kidney cells was lacking, which is consistent with the findings of several recent studies. Interestingly, immunostaining of kidney biopsy sections revealed increased expression of phospho-STAT3 in both COVID-19-associated collapsing glomerulopathy and HIVAN as compared to control kidney tissue. Importantly, IL-6-induced activation of STAT3 may be a targetable mechanism driving COVID-19-associated acute kidney injury.
Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages
Lv, J;Wang, Z;Qu, Y;Zhu, H;Zhu, Q;Tong, W;Bao, L;Lv, Q;Cong, J;Li, D;Deng, W;Yu, P;Song, J;Tong, WM;Liu, J;Liu, Y;Qin, C;Huang, B;
PMID: 33850112 | DOI: 10.1038/s41421-021-00258-1
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.
Journal of the American Society of Nephrology : JASN
Kudose, S;Santoriello, D;Bomback, AS;Sekulic, M;Batal, I;Stokes, MB;Ghavami, IA;Kim, JS;Marasa, M;Xu, K;Peleg, Y;Barasch, J;Canetta, P;Rasouly, HM;Gharavi, AG;Markowitz, GS;D'Agati, VD;
PMID: 34670811 | DOI: 10.1681/ASN.2021070931
The long-term outcome of COVID-19-associated collapsing glomerulopathy is unknown.We retrospectively identified 76 native kidney biopsies from patients with history of COVID-19 between March 2020 and April 2021. Presenting and outcome data were obtained for all 23 patients with collapsing glomerulopathy and for seven patients with noncollapsing podocytopathies. We performed APOL1 genotyping by Sanger sequencing, immunostaining for spike and nucleocapsid proteins, and in situ hybridization for SARS-CoV-2.The 23 patients with COVID-19-associated collapsing glomerulopathy were median age 57 years (range, 35-72), included 16 men, and were predominantly (91%) Black. Severity of COVID-19 was mild or moderate in most (77%) patients. All but one patient presented with AKI, 17 had nephrotic-range proteinuria, and six had nephrotic syndrome. Fourteen (61%) patients required dialysis at presentation. Among 17 patients genotyped, 16 (94%) were high-risk APOL1. Among 22 (96%) patients with median follow-up at 155 days (range, 30-412), 11 (50%) received treatment for COVID-19, and eight (36%) received glucocorticoid therapy for podocytopathy. At follow-up, 19 (86%) patients were alive, and 15 (68%) were dialysis free, including seven of 14 who initially required dialysis. The dialysis-free patients included 64% (seven of 11) of those treated for COVID-19 and 75% (six of eight) of those treated with glucocorticoids for podocytopathy. Overall, 36% achieved partial remission of proteinuria, 32% had no remission, and 32% reached combined end points of ESKD or death. Viral infection of the kidney was not detected.Half of 14 patients with COVID-19-associated collapsing glomerulopathy requiring dialysis achieved dialysis independence, but the long-term prognosis of residual proteinuric CKD remains guarded, indicating a need for more effective therapy.
