Evolution of SARS-CoV-2 variants of concern over a period of Delta and Omicron cocirculation, among patients hospitalized for COVID-19 in an Italian reference hospital: Impact on clinical outcomes.

Despite the higher transmissibility of Omicron Variant of Concern (VOC), several reports have suggested lower risk for hospitalization and severe outcomes compared to previous variants of SARS-CoV-2. This study, enrolling all COVID-19 adults admitted to a reference hospital who underwent both the S-gene-target-failure test and VOC identification by Sanger sequencing, aimed to describe the evolving prevalence of Delta and Omicron variants and to compare the main in-hospital outcomes of severity, during a trimester (December 2021 to March 2022) of VOCs' cocirculation. Factors associated with clinical progression to noninvasive ventilation (NIV)/mechanical ventilation (MV)/death within 10 days and to MV/admission to intensive care unit (ICU)/death within 28 days, were investigated through multivariable logistic regressions. Overall, VOCs were: Delta n = 130/428, Omicron n = 298/428 (sublineages BA.1 n = 275 and BA.2 n = 23). Until mid-February, Delta predominance shifted to BA.1, which was gradually displaced by BA.2 until mid-March. Participants with Omicron VOC were more likely to be older, fully vaccinated, with multiple comorbidities and to have a shorter time from symptoms' onset, and less likely to have systemic symptoms and respiratory complications. Although the need of NIV within 10 days and MV within 28 days from hospitalization and the admission to ICU were less frequent for patients with Omicron compared to those with Delta infections, mortality was similar between the two VOCs. In the adjusted analysis, multiple comorbidities and a longer time from symptoms' onset predicted 10-day clinical progression, while complete vaccination halved the risk. Multimorbidity was the only risk factor associated with 28-day clinical progression. In our population, in the first trimester of 2022, Omicron rapidly displaced Delta in COVID-19 hospitalized adults. Clinical profile and presentation differed between the two VOCs and, although Omicron infections showed a less severe clinical picture, no substantial differences for clinical progression were found. This finding suggests that any hospitalization, especially in more vulnerable individuals, may be at risk for severe progression, which is more related to the underlying frailty of patients than to the intrinsic severity of the viral variant.

Multisystem inflammatory syndrome in children during the first two years of the COVID-19 pandemic in Luxembourg.

Objective: Estimate the incidence of multisystem inflammatory syndrome (MIS-C) in children (0-15 years), the role of SARS-CoV-2 variants during the first two years of COVID-19 pandemic in Luxembourg; and describe the demographic, biological and clinical characteristics of the patients. Method: Observational retrospective cohort study. Cases between March 2020 and February 2022 were ascertained from the national registry of MIS-C cases by a retrospective review of medical records. Reported SARS-CoV-2 infections were obtained from the national COVID-19 surveillance system. We calculated monthly MIS-C incidence, the ratio between MIS-C and SARS-CoV-2 infections and associated rate ratios by the periods corresponding to the circulation of different variants. Results: 18 children were diagnosed with MIS-C among 35,200 reported infections. The incidence rate of MIS-C was 7.2 [95% confidence interval (CI) 4.5-11.4] per 1,000,000 person-months. A higher incidence of MIS-C was observed between September and December 2021, corresponding to the circulation of the Delta variant than during the first year of the pandemic (RR 3.6, 95% CI, 1.1-12.3). The lowest rate of MIS-C per infection was observed during the Omicron (RR 0.17, 95% CI, 0.03-0.82). Median age at diagnosis was 6.5 years. Previously healthy children made up 88% of MIS-C cases, none were vaccinated against SARS-CoV-2. 33% required intensive care. All patients recovered fully. Conclusions: MIS-C incidence and MIS-C risk per infection changed significantly over time during the first two years of COVID-19 pandemic. Monitoring of MIS-C incidence in future SARS-CoV-2 waves will be essential to guide public health interventions and vaccination policies for children.

CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/µL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

Intrinsic D614G and P681R/H mutations in SARS-CoV-2 VoCs Alpha, Delta, Omicron and viruses with D614G plus key signature mutations in spike protein alters fusogenicity and infectivity.

The SARS-CoV-2 virus has been rapidly evolving over the time and the genetic variation has led to the generation of Variants of Concerns (VoC), which have shown increased fitness. These VoC viruses contain the key mutations in the spike protein which have allowed better survival and evasion of host defense mechanisms. The D614G mutation in the spike domain is found in the majority of VoC; additionally, the P681R/H mutation at the S1/S2 furin cleavage site junction is also found to be highly conserved in major VoCs; Alpha, Delta, Omicron, and its' current variants. The impact of these genetic alterations of the SARS-CoV-2 VoCs on the host cell entry, transmissibility, and infectivity has not been clearly identified. In our study, Delta and D614G + P681R synthetic double mutant pseudoviruses showed a significant increase in the cell entry, cell-to-cell fusion and infectivity. In contrast, the Omicron and P681H synthetic single mutant pseudoviruses showed TMPRSS2 independent cell entry, less fusion and infectivity as compared to Delta and D614G + P681R double mutants. Addition of exogenous trypsin further enhanced fusion in Delta viruses as compared to Omicron. Furthermore, Delta viruses showed susceptibility to both E64d and Camostat mesylate inhibitors suggesting, that the Delta virus could exploit both endosomal and TMPRSS2 dependent entry pathways as compared to the Omicron virus. Taken together, these results indicate that the D614G and P681R/H mutations in the spike protein are pivotal which might be favoring the VoC replication in different host compartments, and thus allowing a balance of mutation vs selection for better long-term adaptation.

The spread of the omicron variant: Identification of knowledge gaps, virus diffusion modelling, and future research needs.

The World Health Organization (WHO) recognised variant B.1.1.529 of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a variant of concern, termed "Omicron", on November 26, 2021. Its diffusion was attributed to its several mutations, which allow promoting its ability to diffuse worldwide and its capability in immune evasion. As a consequence, some additional serious threats to public health posed the risk to undermine the global efforts made in the last two years to control the pandemic. In the past, several works were devoted to discussing a possible contribution of air pollution to the SARS-CoV-2 spread. However, to the best of the authors' knowledge, there are still no works dealing with the Omicron variant diffusion mechanisms. This work represents a snapshot of what we know right now, in the frame of an analysis of the Omicron variant spread. The paper proposes the use of a single indicator, commercial trade data, to model the virus spread. It is proposed as a surrogate of the interactions occurring between humans (the virus transmission mechanism due to human-to-human contacts) and could be considered for other diseases. It allows also to explain the unexpected increase in infection cases in China, detected at beginning of 2023. The air quality data are also analyzed to evaluate for the first time the role of air particulate matter (PM) as a carrier of the Omicron variant diffusion. Due to emerging concerns associated with other viruses (such as smallpox-like virus diffusion in Europe and America), the proposed approach seems to be promising to model the virus spreading.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Discovery of lead natural products for developing pan-SARS-CoV-2 therapeutics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC50) below 50 µM against mNG-SARS-CoV-2; 16 of these had EC50 values below 10 µM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants.

Preclinical assessment and randomized Phase I study of CT-P63, a broadly neutralizing antibody targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant morbidity and mortality worldwide. Despite a successful vaccination programme, the emergence of mutated variants that can escape current levels of immunity mean infections continue. Herein, we report the development of CT-P63, a broad-spectrum neutralizing monoclonal antibody. In vitro studies demonstrated potent neutralizing activity against the most prevalent variants, including Delta and the BA.1 and BA.2 sub-lineages of Omicron. In a transgenic mouse model, prophylactic CT-P63 significantly reduced wild-type viral titres in the respiratory tract and CT-P63 treatment proved efficacious against infection with Beta, Delta, and Omicron variants of SARS-CoV-2 with no detectable infectious virus in the lungs of treated animals. A randomized, double-blind, parallel-group, placebo-controlled, Phase I, single ascending dose study in healthy volunteers (NCT05017168) confirmed the safety, tolerability, and pharmacokinetics of CT-P63. Twenty-four participants were randomized and received the planned dose of CT-P63 or placebo. The safety and tolerability of CT-P63 were evaluated as primary objectives. Eight participants (33.3%) experienced a treatment-emergent adverse event (TEAE), including one grade ≥3 (blood creatine phosphokinase increased). There were no deaths, treatment-emergent serious adverse events, TEAEs of special interest, or TEAEs leading to study drug discontinuation in the CT-P63 groups. Serum CT-P63 concentrations rapidly peaked before declining in a biphasic manner and systemic exposure was dose proportional. Overall, CT-P63 was clinically safe and showed broad-spectrum neutralizing activity against SARS-CoV-2 variants in vitro and in vivo.

Cross-variant protection against SARS-CoV-2 infection in hamsters immunized with monovalent and bivalent inactivated vaccines.

Rapid development and successful use of vaccines against SARS-CoV-2 might hold the key to curb the ongoing pandemic of COVID-19. Emergence of vaccine-evasive SARS-CoV-2 variants of concern (VOCs) has posed a new challenge to vaccine design and development. One urgent need is to determine what types of variant-specific and bivalent vaccines should be developed. Here, we compared homotypic and heterotypic protection against SARS-CoV-2 infection of hamsters with monovalent and bivalent whole-virion inactivated vaccines derived from representative VOCs. In addition to the ancestral SARS-CoV-2 Wuhan strain, Delta (B.1.617.2; δ) and Theta (P.3; θ) variants were used in vaccine preparation. Additional VOCs including Omicron (B.1.1.529) and Alpha (B.1.1.7) variants were employed in the challenge experiment. Consistent with previous findings, Omicron variant exhibited the highest degree of immune evasion, rendering all different forms of inactivated vaccines substantially less efficacious. Notably, monovalent and bivalent Delta variant-specific inactivated vaccines provided optimal protection against challenge with Delta variant. Yet, some cross-variant protection against Omicron and Alpha variants was seen with all monovalent and bivalent inactivated vaccines tested. Taken together, our findings support the notion that an optimal next-generation inactivated vaccine against SARS-CoV-2 should contain the predominant VOC in circulation. Further investigations are underway to test whether a bivalent vaccine for Delta and Omicron variants can serve this purpose.

Heterogeneous Infectivity and Pathogenesis of SARS-CoV-2 Variants Beta, Delta and Omicron in Transgenic K18-hACE2 and Wildtype Mice.

The emerging SARS-CoV-2 variants of concern (VOCs) may display enhanced transmissibility, more severity and/or immune evasion; however, the pathogenesis of these new VOCs in experimental SARS-CoV-2 models or the potential infection of other animal species is not completely understood. Here we infected K18-hACE2 transgenic mice with B.1, B.1.351/Beta, B.1.617.2/Delta and BA.1.1/Omicron isolates and demonstrated heterogeneous infectivity and pathogenesis. B.1.351/Beta variant was the most pathogenic, while BA.1.1/Omicron led to lower viral RNA in the absence of major visible clinical signs. In parallel, we infected wildtype (WT) mice and confirmed that, contrary to B.1 and B.1.617.2/Delta, B.1.351/Beta and BA.1.1/Omicron can infect them. Infection in WT mice coursed without major clinical signs and viral RNA was transient and undetectable in the lungs by day 7 post-infection. In silico modeling supported these findings by predicting B.1.351/Beta receptor binding domain (RBD) mutations result in an increased affinity for both human and murine ACE2 receptors, while BA.1/Omicron RBD mutations only show increased affinity for murine ACE2.

Optimizing the Illumina COVIDSeq laboratorial and bioinformatics pipeline on thousands of samples for SARS-CoV-2 Variants of Concern tracking.

The SARS-CoV-2 Variants of Concern tracking via Whole Genome Sequencing represents a pillar of public health measures for the containment of the pandemic. The ability to track down the lineage distribution on a local and global scale leads to a better understanding of immune escape and to adopting interventions to contain novel outbreaks. This scenario poses a challenge for NGS laboratories worldwide that are pressed to have both a faster turnaround time and a high-throughput processing of swabs for sequencing and analysis. In this study, we present an optimization of the Illumina COVID-seq protocol carried out on thousands of SARS-CoV-2 samples at the wet and dry level. We discuss the unique challenges related to processing hundreds of swabs per week such as the tradeoff between ultra-high sensitivity and negative contamination levels, cost efficiency and bioinformatics quality metrics.

Molecular Cross-Talk between Integrins and Cadherins Leads to a Loss of Vascular Barrier Integrity during SARS-CoV-2 Infection.

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins are emerging as possible receptor candidates for SARS-CoV-2, and their complex intracellular signaling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation by hijacking integrin αVß3, expressed on human endothelial cells. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin αVß3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein-through its RGD motif-binds to αVß3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.

Resilience of Spike-Specific Immunity Induced by COVID-19 Vaccines against SARS-CoV-2 Variants.

The outbreak of SARS-CoV-2 leading to the declaration of the COVID-19 global pandemic has led to the urgent development and deployment of several COVID-19 vaccines. Many of these new vaccines, including those based on mRNA and adenoviruses, are aimed to generate neutralizing antibodies against the spike glycoprotein, which is known to bind to the receptor angiotensin converting enzyme 2 (ACE2) in host cells via the receptor-binding domain (RBD). Antibodies binding to this domain can block the interaction with the receptor and prevent viral entry into the cells. Additionally, these vaccines can also induce spike-specific T cells which could contribute to providing protection against the virus. However, the emergence of new SARS-CoV-2 variants can impair the immunity generated by COVID-19 vaccines if mutations occur in cognate epitopes, precluding immune recognition. Here, we evaluated the chance of five SARS-CoV-2 variants of concern (VOCs), Alpha, Beta, Gamma, Delta and Omicron, to escape spike-specific immunity induced by vaccines. To that end, we examined the impact of the SARS-CoV-2 variant mutations on residues located on experimentally verified spike-specific epitopes, deposited at the Immune Epitope Database, that are targeted by neutralizing antibodies or recognized by T cells. We found about 300 of such B cell epitopes, which were largely overlapping, and could be grouped into 54 B cell epitope clusters sharing ≥ 7 residues. Most of the B cell epitope clusters map in the RBD domain (39 out of 54) and 20%, 50%, 37%, 44% and 57% of the total are mutated in SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron variants, respectively. We also found 234 experimentally verified CD8 and CD4 T cell epitopes that were distributed evenly throughout the spike protein. Interestingly, in each SARS-CoV-2 VOC, over 87% and 79% of CD8 and CD4 T cell epitopes, respectively, are not mutated. These observations suggest that SARS-CoV-2 VOCs-particularly the Omicron variant-may be prone to escape spike-specific antibody immunity, but not cellular immunity, elicited by COVID-19 vaccines.

Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the ß-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern.

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.

SARS-CoV-2 mRNA vaccine BNT162b2 triggers a consistent cross-variant humoral and cellular response.

As the SARS-CoV-2 pandemic continues to rage worldwide, the emergence of numerous variants of concern (VOC) represents a challenge for the vaccinal protective efficacy and the reliability of commercially available high-throughput immunoassays. Our study demonstrates the administration of two doses of the BNT162b2 vaccine that elicited a robust SARS-CoV-2-specific immune response which was assessed up to 3 months after full vaccination in a cohort of 37 health care workers (HCWs). SARS-CoV-2-specific antibody response, evaluated by four commercially available chemiluminescence immunoassays (CLIA), was qualitatively consistent with the results provided by the gold-standard in vitro neutralization assay (NTA). However, we could not observe a correlation between the quantity of the antibody detected by CLIA assays and their neutralizing activity tested by NTA. Almost all subjects developed a SARS-CoV-2-specific T-cell response. Moreover, vaccinated HCWs developed a similar protective neutralizing antibodies response against the EU (B.1), Alpha (B.1.1.7), Gamma (P.1), and Eta (B.1.525) SARS-CoV-2 variants, while Beta (B.1.351) and Delta (B.1.617.2) strains displayed a consistent partial immune evasion. These results underline the importance of a solid vaccine-elicited immune response and a robust antibody titre. We believe that these relevant results should be taken into consideration in the definition of future vaccinal strategies.

Serum Neutralizing Activity of mRNA-1273 against SARS-CoV-2 Variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to growing concerns over increased transmissibility and the ability of some variants to partially escape immunity. Sera from participants immunized on a prime-boost schedule with the mRNA-1273 COVID-19 vaccine were tested for neutralizing activity against several SARS-CoV-2 variants, including variants of concern (VOCs) and variants of interest (VOIs), compared to neutralization of the wild-type SARS-CoV-2 virus (designated D614G). Results showed minimal, statistically nonsignificant effects on neutralization titers against the B.1.1.7 (Alpha) variant (1.2-fold reduction compared with D614G); other VOCs, such as B.1.351 (Beta, including B.1.351-v1, B.1.351-v2, and B.1.351-v3), P.1 (Gamma), and B.1.617.2 (Delta), showed significantly decreased neutralization titers ranging from 2.1-fold to 8.4-fold reductions compared with D614G, although all remained susceptible to mRNA-1273-elicited serum neutralization. IMPORTANCE In light of multiple variants of SARS-CoV-2 that have been documented globally during the COVID-19 pandemic, it remains important to continually assess the ability of currently available vaccines to confer protection against newly emerging variants. Data presented herein indicate that immunization with the mRNA-1273 COVID-19 vaccine produces neutralizing antibodies against key emerging variants tested, including variants of concern and variants of interest. While the serum neutralization elicited by mRNA-1273 against most variants tested was reduced compared with that against the wild-type virus, the level of neutralization is still expected to be protective. Such data are crucial to inform ongoing and future vaccination strategies to combat COVID-19.