SARS-CoV-2 BA.4/5 infection triggers more cross-reactive FcγRIIIa signaling and neutralization than BA.1, in the context of hybrid immunity.

SARS-CoV-2 variants of concern (VOCs) differentially trigger neutralizing and antibody-dependent cellular cytotoxic (ADCC) antibodies with variable cross-reactivity. Omicron BA.4/5 was approved for inclusion in bivalent vaccination boosters, and therefore the antigenic profile of antibodies elicited by this variant is critical to understand. Here, we investigate the ability of BA.4/5-elicited antibodies following the first documented (primary) infection (n = 13) or breakthrough infection after vaccination (n = 9) to mediate neutralization and FcγRIIIa signaling across multiple SARS-CoV-2 variants including XBB.1.5 and BQ.1. Using a pseudovirus neutralization assay and a FcγRIIIa crosslinking assay to measure ADCC potential, we show that unlike SARS-CoV-2 Omicron BA.1, BA.4/5 infection triggers highly cross-reactive functional antibodies. Cross-reactivity was observed both in the absence of prior vaccination and in breakthrough infections following vaccination. However, BQ.1 and XBB.1.5 neutralization and FcγRIIIa signaling were significantly compromised compared to other VOCs, regardless of prior vaccination status. BA.4/5 triggered FcγRIIIa signaling was significantly more resilient against VOCs (<10-fold decrease in magnitude) compared to neutralization (10- to 100-fold decrease). Overall, this study shows that BA.4/5 triggered antibodies are highly cross-reactive compared to those triggered by other variants. Although this is consistent with enhanced neutralization and FcγRIIIa signaling breadth of BA.4/5 vaccine boosters, the reduced activity against XBB.1.5 supports the need to update vaccines with XBB sublineage immunogens to provide adequate coverage of these highly antibody evasive variants. IMPORTANCE: The continued evolution of SARS-CoV-2 has resulted in a number of variants of concern. Of these, the Omicron sublineage is the most immune evasive. Within Omicron, the BA.4/5 sublineage drove the fifth wave of infection in South Africa prior to becoming the dominant variant globally. As a result this spike sequence was approved as part of a bivalent vaccine booster, and rolled out worldwide. We aimed to understand the cross-reactivity of neutralizing and Fc mediated cytotoxic functions elicited by BA.4/5 infection following infection or breakthrough infection. We find that, in contrast to BA.1 which triggered fairly strain-specific antibodies, BA.4/5 triggered antibodies that are highly cross-reactive for neutralization and antibody-dependent cellular cytotoxicity potential. Despite this cross-reactivity, these antibodies are compromised against highly resistant variants such as XBB.1.5 and BQ.1. This suggests that next-generation vaccines will require XBB sublineage immunogens in order to protect against these evasive variants.

Hybrid Immunity and the Incidence of SARS-CoV-2 Reinfections during the Omicron Era in Frontline Healthcare Workers.

During the coronavirus disease (COVID-19) pandemic healthcare workers (HCWs) acquired immunity by vaccination or exposure to multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our study is a comparative analysis between subgroups of HCWs constructed based on the number of SARS-CoV-2 infections, vaccination, and the dominant variant of SARS-CoV-2 in the population. We collected and analyzed data using the χ2 test and density incidence of reinfections in Microsoft Excel for Mac, Version 16.84, and MedCalc®, 22.026. Of the 829 HCWs, 70.1% (581) had only one SARS-CoV-2 infection and 29.9% (248) had two infections. Of the subjects with two infections, 77.4% (192) worked in high-risk departments and 93.2% (231) of the second infections were registered during Omicron dominance. The density incidence of reinfections was higher in HCWs vaccinated with the primary schedule than those vaccinated with the first booster, and the incidence ratio was 2.8 (95% CI: 1.2; 6.7). The probability of reinfection was five times lower (95% CI: 2.9; 9.2) in HCWs vaccinated with the primary schedule if the first infection was acquired during Omicron dominance. The subjects vaccinated with the first booster had a density incidence of reinfection three times lower (95% CI: 1.9; 5.8) if the first infection was during Omicron. The incidence ratio in subgroups constructed based on characteristics such as gender, age group, job category, and department also registered significant differences in density incidence. The history of SARS-CoV-2 infection by variant is important when interpreting and understanding public health data and the results of studies related to vaccine efficacy for hybrid immunity subgroup populations.

Serum cytokine dysregulation signatures associated with COVID-19 outcomes in high mortality intensive care unit cohorts across pandemic waves and variants.

The aim of this study was to characterize the systemic cytokine signature of critically ill COVID-19 patients in a high mortality setting aiming to identify biomarkers of severity, and to explore their associations with viral loads and clinical characteristics. We studied two COVID-19 critically ill patient cohorts from a referral centre located in Central Europe. The cohorts were recruited during the pre-alpha/alpha (November 2020 to April 2021) and delta (end of 2021) period respectively. We determined both the serum and bronchoalveolar SARS-CoV-2 viral load and identified the variant of concern (VoC) involved. Using a cytokine multiplex assay, we quantified systemic cytokine concentrations and analyzed their relationship with clinical findings, routine laboratory workup and pulmonary function data obtained during the ICU stay. Patients who did not survive had a significantly higher systemic and pulmonary viral load. Patients infected with the pre-alpha VoC showed a significantly lower viral load in comparison to those infected with the alpha- and delta-variants. Levels of systemic CTACK, M-CSF and IL-18 were significantly higher in non-survivors in comparison to survivors. CTACK correlated directly with APACHE II scores. We observed differences in lung compliance and the association between cytokine levels and pulmonary function, dependent on the VoC identified. An intra-cytokine analysis revealed a loss of correlation in the non-survival group in comparison to survivors in both cohorts. Critically ill COVID-19 patients exhibited a distinct systemic cytokine profile based on their survival outcomes. CTACK, M-CSF and IL-18 were identified as mortality-associated analytes independently of the VoC involved. The Intra-cytokine correlation analysis suggested the potential role of a dysregulated systemic network of inflammatory mediators in severe COVID-19 mortality.

VOE: automated analysis of variant epitopes of SARS-CoV-2 for the development of diagnostic tests or vaccines for COVID-19.

Background: The development of serodiagnostic tests and vaccines for COVID-19 depends on the identification of epitopes from the SARS-CoV-2 genome. An epitope is the specific part of an antigen that is recognized by the immune system and can elicit an immune response. However, when the genetic variants contained in epitopes are used to develop rapid antigen tests (Ag-RDTs) and DNA or RNA vaccines, test sensitivity and vaccine efficacy can be low. Methods: Here, we developed a "variant on epitope (VOE)" software, a new Python script for identifying variants located on an epitope. Variant analysis and sensitivity calculation for seven recommended epitopes were processed by VOE. Variants in 1,011 Omicron SRA reads from two variant databases (BCFtools and SARS-CoV-2-Freebayes) were processed by VOE. Results: A variant with HIGH or MODERATE impact was found on all epitopes from both variant databases except the epitopes KLNDLCFTNV, RVQPTES, LKPFERD, and ITLCFTLKRK on the S gene and ORF7a gene. All epitope variants from the BCFtools and SARS-CoV-2 Freebayes variant databases showed about 100% sensitivity except epitopes APGQTGK and DSKVGGNYN on the S gene, which showed respective sensitivities of 28.4866% and 6.8249%, and 87.7349% and 71.1177%. Conclusions: Therefore, the epitopes KLNDLCFTNV, RVQPTES, LKPFERD, and ITLCFTLKRK may be useful for the development of an epitope-based peptide vaccine and GGDGKMKD on the N gene may be useful for the development of serodiagnostic tests. Moreover, VOE can also be used to analyze other epitopes, and a new variant database for VOE may be further established when a new variant of SARS-CoV-2 emerges.

Comparative Analysis of Vaccine-Induced Neutralizing Antibodies against the Alpha, Beta, Delta, and Omicron Variants of SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 660 million people and caused nearly seven million deaths worldwide. During the pandemic, a number of SARS-CoV-2 vaccines were rapidly developed, and several are currently licensed for use in Europe. However, the optimization of vaccination regimens is still ongoing, particularly with regard to booster vaccinations. At the same time, the emergence of new virus variants poses an ongoing challenge to vaccine efficacy. In this study, we focused on a comparative analysis of the neutralization capacity of vaccine-induced antibodies against four different variants of concern (i.e., Alpha, Beta, Delta, and Omicron) after two and three doses of COVID-19 vaccine. We were able to show that both two (prime/boost) and three (prime/boost/boost) vaccinations elicit highly variable levels of neutralizing antibodies. In addition, we did not observe a significant difference in antibody levels after two and three vaccinations. We also observed a significant decrease in the neutralization susceptibility of all but one SARS-CoV-2 variants to vaccine-induced antibodies. In contrast, a SARS-CoV-2 breakthrough infection between the second and third vaccination results in overall higher levels of neutralizing antibodies with a concomitant improved neutralization of all virus variants. Titer levels remained highly variable across the cohort but a common trend was observed. This may be due to the fact that at the time of this study, all licensed vaccines were still based exclusively on wild-type SARS-CoV-2, whereas infections were caused by virus variants. Overall, our data demonstrate the importance of (booster) vaccinations, but at the same time emphasize the need for the continued adaptation of vaccines to induce a protective immune response against virus variants in order to be prepared for future (seasonal) SARS-CoV-2 outbreaks.

Wide Real-Life Data Support Reduced Sensitivity of Antigen Tests for Omicron SARS-CoV-2 Infections.

With the continuous spread of new SARS-CoV-2 variants of concern (VOCs), the monitoring of diagnostic test performances is mandatory. We evaluated the changes in antigen diagnostic tests' (ADTs) accuracy along the Delta to Omicron VOCs transition, exploring the N protein mutations possibly affecting ADT sensitivity and assessing the best sampling site for the diagnosis of Omicron infections. In total, 5175 subjects were enrolled from 1 October 2021 to 15 July 2022. The inclusion criteria were SARS-CoV-2 ADT combined with a same-day RT-PCR swab test. For the sampling site analysis, 61 patients were prospectively recruited during the Omicron period for nasal and oral swab analyses by RT-PCR. Next-Generation Sequencing data were obtained to evaluate the different sublineages. Using RT-PCR as a reference, 387 subjects resulted in becoming infected and the overall sensitivity of the ADT decreased from 63% in the Delta period to 33% in the Omicron period. This decrease was highly statistically significant (p < 0.001), and no decrease in viral load was detected at the RNA level. The nasal site presented a significantly higher viral load than the oral site during the Omicron wave. The reduced detection rate of Omicron infections by ADT should be considered in the global testing strategy to preserve accurate diagnoses across the changing SARS-CoV-2 variants.

Incursion of SARS-CoV-2 BA.2.86.1 variant into Israel: National-scale wastewater surveillance using a novel quantitative real-time PCR assay.

The emergence of the SARS-CoV-2 variant BA.2.86.1 raised a considerable concern, due to the large number of potentially virulent mutations. In this study, we developed a novel assay that specifically detects variant BA.2.86.1, and used it to screen environmental samples from wastewater treatment sites across Israel. By using a multiplex assay that included a general SARS-CoV-2 reaction, together with the BA.2.86.1-specific reaction and a control reaction, we quantified the absolute number of viral copies in each sample and its relative abundance, compared with the total copy number of circulating SARS-CoV-2. Evaluation of the new reactions showed that they are both sensitive and specific, detecting down to four copies per reaction, and maintain specificity in the presence of Omicron variants BA.1, 2 and 4 RNA. Examination of 279 samples from 30 wastewater collection sites during August-September 2023 showed that 35 samples (12.5 %) were positive, from 18 sites. Quantitative analysis of the samples showed that the relative abundance of variant BA.2.86.1 with respect to the total viral load of SARS-CoV-2 was very low and consisted between 0.01 % and 0.6 % of the total SARS-CoV-2 circulation. This study demonstrates the importance of combining wastewater surveillance with the development of specialized diagnostic assays, when clinical testing is insufficient. This approach may be useful for timely response by public health authorities in future outbreaks.

Mutational dynamics of SARS-CoV-2: Impact on future COVID-19 vaccine strategies.

The rapid emergence of multiple strains of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has sparked profound concerns regarding the ongoing evolution of the virus and its potential impact on global health. Classified by the World Health Organization (WHO) as variants of concern (VOC), these strains exhibit heightened transmissibility and pathogenicity, posing significant challenges to existing vaccine strategies. Despite widespread vaccination efforts, the continual evolution of SARS-CoV-2 variants presents a formidable obstacle to achieving herd immunity. Of particular concern is the coronavirus spike (S) protein, a pivotal viral surface protein crucial for host cell entry and infectivity. Mutations within the S protein have been shown to enhance transmissibility and confer resistance to antibody-mediated neutralization, undermining the efficacy of traditional vaccine platforms. Moreover, the S protein undergoes rapid molecular evolution under selective immune pressure, leading to the emergence of diverse variants with distinct mutation profiles. This review underscores the urgent need for vigilance and adaptation in vaccine development efforts to combat the evolving landscape of SARS-CoV-2 mutations and ensure the long-term effectiveness of global immunization campaigns.

Characteristics and clinical manifestations of patients, including organ transplant patients, during the surge of JN.1: Insights from Saudi Arabia.

BACKGROUND: Amidst the persistent global health threat posed by the evolving SARS-CoV-2 virus throughout the four-year-long COVID-19 pandemic, the focus has now turned to the Omicron variant and its subvariant, JN.1, which has rapidly disseminated worldwide. This study reports on the characteristics and clinical manifestations of patients during the surge of the JN.1 variant in Saudi Arabia; it also investigates the evolution of SARS-CoV-2 variants in organ transplant patients and identifies patient risk factors. METHODS: A total of 151 nasopharyngeal samples from patients with PCR-confirmed SARS-CoV-2 infection were collected between September 2023 and January 2024. Demographic and clinical data of the patients were obtained from electronic health records. All confirmed positive samples underwent sequencing using Ion GeneStudio and the Ion AmpliSeq™ SARS-CoV-2 panel. RESULTS: During the surge of the JN.1 variant, the average age of the patients was 40 years, ranging from 3 to 93 years, and nearly 50% of the patients were male. Our investigation revealed that the J.N variant predominantly infected patients with comorbidities or organ transplant recipients (57.6%). Moreover, patients with comorbidities or organ transplants exhibited a higher number of mutations. In our organ transplant cohort, an increased total number of spike mutations was associated with a lower risk of developing severe disease (OR = 0.96, 95% CI: 0.93-0.98). CONCLUSIONS: Although JN.1 may not prove to be particularly harmful, it is crucial to recognize the persistent emergence of concerning variants, which create new pathways for the virus to evolve. The ongoing evolution of SARS-CoV-2 is evident in the continuous divergence of these variants from the original strain that marked the onset of the pandemic nearly four years ago.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

Developing variant-adapted COVID-19 vaccines to improve protection against Omicron and other recent variants: a plain language summary.

SummaryWhat are variant-adapted COVID-19 vaccines?The COVID-19 vaccine developed by BioNTech and Pfizer is known as BNT162b2 (Comirnaty). BNT162b2 contains messenger RNA, or mRNA, from SARS-CoV-2. SARS-CoV-2 is the virus responsible for COVID-19. mRNA is a type of genetic material that contains the instructions that tell cells in the body how to make a protein. The mRNA in BNT162b2 tells the body to make one of the proteins from SARS-CoV-2 known as the spike protein.This teaches the body's defense system, known as the immune system, to recognize and respond to a SARS-CoV-2 infection.The BNT162b2 vaccine contains mRNA from the first SARS-CoV-2 virus, which was detected in December 2019. Since this original vaccine was developed, the SARS-CoV-2 virus has evolved, resulting in the appearance of new versions of the virus, known as variants. Certain variants that might be more concerning for public health are labeled as either 'variants of concern' or 'variants of interest' by the World Health Organization (WHO). Variants have differences in their proteins compared with the original virus that can affect how well the original vaccine works against them. Therefore, BioNTech and Pfizer developed updated versions of the BNT162b2 vaccine that contain mRNA from certain variants. These new vaccines are called variant-adapted COVID-19 mRNA vaccines.Another company, Moderna, has also developed their own variant-adapted versions of their COVID-19 mRNA vaccine, mRNA-1273 (SpikeVax).Variant-adapted vaccines can contain parts of the variant mRNA either in addition to, or instead of, that from the original virus. Vaccines that contain mRNA from two different viruses are known as bivalent, whereas vaccines that contain mRNA from a single virus are called monovalent.

Measuring disease burden of dominant variants of COVID-19 in Taiwan.

Objectives: The disability-adjusted life years (DALYs) of COVID-19 have been applied as a time-based measurement to estimate years of life lost due to premature mortality or healthy life lost in different countries. Limited information was found for DALYs among different variants of concern (VOC). Methods: Disease severities based on categories of asymptomatic, mild, moderate, severe, and critical cases were explored among different VOC by analyzing the proportions in confirmed cases. DALY or years of healthy life lost due to disability (YLD)-based annual burdens of COVID-19 on different ages, genders as well as trend analysis were also evaluated for VOC in Taiwan. Results: Different trends were observed in years of life lost due to premature mortality (YLLs) or YLD for various age or gender categories. Disease severity at critical stage had the highest percentage for overall YLDs encompassed from 2020 to 2022. Also, critical-grade cases were found to be predominantly caused by Wild-type, Alpha, and Omicron variants in 2020, 2021, and 2022, respectively. Conclusion: Precautionary measures are also suggested for policy makers to take in specific seasons, age or gender groups based on YLL and YLD analyses.

Gastrointestinal microbiota and metabolites possibly contribute to distinct pathogenicity of SARS-CoV-2 proto or its variants in rhesus monkeys.

Gastrointestinal (GI) infection is evidenced with involvement in COVID-19 pathogenesis caused by SARS-CoV-2. However, the correlation between GI microbiota and the distinct pathogenicity of SARS-CoV-2 Proto and its emerging variants remains unclear. In this study, we aimed to determine if GI microbiota impacted COVID-19 pathogenesis and if the effect varied between SARS-CoV-2 Proto and its variants. We performed an integrative analysis of histopathology, microbiomics, and transcriptomics on the GI tract fragments from rhesus monkeys infected with SARS-CoV-2 proto or its variants. Based on the degree of pathological damage and microbiota profile in the GI tract, five of SARS-CoV-2 strains were classified into two distinct clusters, namely, the clusters of Alpha, Beta and Delta (ABD), and Proto and Omicron (PO). Notably, the abundance of potentially pathogenic microorganisms increased in ABD but not in the PO-infected rhesus monkeys. Specifically, the high abundance of UCG-002, UCG-005, and Treponema in ABD virus-infected animals positively correlated with interleukin, integrins, and antiviral genes. Overall, this study revealed that infection-induced alteration of GI microbiota and metabolites could increase the systemic burdens of inflammation or pathological injury in infected animals, especially in those infected with ABD viruses. Distinct GI microbiota and metabolite profiles may be responsible for the differential pathological phenotypes of PO and ABD virus-infected animals. These findings improve our understanding the roles of the GI microbiota in SARS-CoV-2 infection and provide important information for the precise prevention, control, and treatment of COVID-19.

Mutations in the SARS-CoV-2 spike receptor binding domain and their delicate balance between ACE2 affinity and antibody evasion.

Intensive selection pressure constrains the evolutionary trajectory of SARS-CoV-2 genomes and results in various novel variants with distinct mutation profiles. Point mutations, particularly those within the receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein, lead to the functional alteration in both receptor engagement and monoclonal antibody (mAb) recognition. Here, we review the data of the RBD point mutations possessed by major SARS-CoV-2 variants and discuss their individual effects on ACE2 affinity and immune evasion. Many single amino acid substitutions within RBD epitopes crucial for the antibody evasion capacity may conversely weaken ACE2 binding affinity. However, this weakened effect could be largely compensated by specific epistatic mutations, such as N501Y, thus maintaining the overall ACE2 affinity for the spike protein of all major variants. The predominant direction of SARS-CoV-2 evolution lies neither in promoting ACE2 affinity nor evading mAb neutralization but in maintaining a delicate balance between these two dimensions. Together, this review interprets how RBD mutations efficiently resist antibody neutralization and meanwhile how the affinity between ACE2 and spike protein is maintained, emphasizing the significance of comprehensive assessment of spike mutations.

AlphaFold2 Reveals Structural Patterns of Seasonal Haplotype Diversification in SARS-CoV-2 Spike Protein Variants.

The slow experimental acquisition of high-quality atomic structures of the rapidly changing proteins of the COVID-19 virus challenges vaccine and therapeutic drug development efforts. Fortunately, deep learning tools such as AlphaFold2 can quickly generate reliable models of atomic structure at experimental resolution. Current modeling studies have focused solely on definitions of mutant constellations of Variants of Concern (VOCs), leaving out the impact of haplotypes on protein structure. Here, we conduct a thorough comparative structural analysis of S-proteins belonging to major VOCs and corresponding latitude-delimited haplotypes that affect viral seasonal behavior. Our approach identified molecular regions of importance as well as patterns of structural recruitment. The S1 subunit hosted the majority of structural changes, especially those involving the N-terminal domain (NTD) and the receptor-binding domain (RBD). In particular, structural changes in the NTD were much greater than just translations in three-dimensional space, altering the sub-structures to greater extents. We also revealed a notable pattern of structural recruitment with the early VOCs Alpha and Delta behaving antagonistically by suppressing regions of structural change introduced by their corresponding haplotypes, and the current VOC Omicron behaving synergistically by amplifying or collecting structural change. Remarkably, haplotypes altering the galectin-like structure of the NTD were major contributors to seasonal behavior, supporting its putative environmental-sensing role. Our results provide an extensive view of the evolutionary landscape of the S-protein across the COVID-19 pandemic. This view will help predict important regions of structural change in future variants and haplotypes for more efficient vaccine and drug development.

Previous SARS-CoV-2 infections and their impact on the protection from reinfection during the Omicron BA.5 wave - a nested case-control study among vaccinated adults in Sweden.

Objectives: We evaluated the protection afforded by SARS-CoV-2 infection-induced immunity against reinfection among working-age vaccinated individuals during a calendar period from June to December 2022 when Omicron BA.5 was the dominating subvariant in Scania County, Sweden. Methods: The study cohort (n = 71,592) mainly consisted of health care workers. We analyzed 4144 infected cases during the Omicron BA.5 dominance and 41,440 sex- and age-matched controls with conditional logistic regression. Results: The average protection against reinfection was marginal (16%, 95% confidence interval [CI] 7-23%) during the study period but substantially higher for recent infections. Recent infection (3-6 months) with Omicron BA.2 and BA.5 offered strong protection (86%, 95% CI 68-94% and 78%, 95% CI 69-84%), whereas more distant infection (6-12 months) with Omicron BA.1, BA.2, and the variants before Omicron offered marginal or no protection. Conclusions: These findings suggest that infection-induced immunity contributes to short-term population protection against infection with the subvariant BA.5 among working-age vaccinated individuals but wanes considerably with time, independent of the virus variant.

Design of human ACE2 mimic miniprotein binders that interact with RBD of SARS-CoV-2 variants of concerns.

The world of medicine demands from the research community solutions to the emerging problem of SARS-CoV-2 variants and other such potential global pandemics. With advantages of specificity over small molecule drugs and designability over antibodies, miniprotein therapeutics offers a unique solution to the threats of rapidly emerging SARS-CoV-2 variants. Unfortunately, most of the promising miniprotein binders are de novo designed and it is not viable to generate molecules for each new variant. Therefore in this study, we demonstrate a method for design of miniprotein mimics from the interaction interphase of human angiotensin converting enzyme 2 (ACE2). ACE2 is the natural interacting partner for the SARS-CoV-2 spike receptor binding domain (RBD) and acts as a recognition molecule for viral entry into the host cells. Starting with ACE2 N-terminal triple helix interaction interphase, we generated more than 70 miniprotein sequences. Employing Rosetta folding and docking scores we selected 10 promising miniprotein candidates amongst which 3 were found to be soluble in lab studies. Further, using molecular mechanics (MM) calculations on molecular dynamics (MD) trajectories we test interaction of miniproteins with RBD from various variants of concern (VOC). Presently, we report two key findings; miniproteins in this study are generated using less than 10 lab testing experiments, yet when tested through in-vitro experiments, they show submicro to nanomolar affinities towards SARS-CoV-2 RBD. Also in simulation studies, when compared with previously developed therapeutics, our miniproteins display remarkable ability to mimic ACE2 interphase; making them an ideal solution to the ever evolving problem of VOCs.Communicated by Ramaswamy H. Sarma.

Dynamic clade transitions and the influence of vaccine rollout on the spatiotemporal circulation of SARS-CoV-2 variants in São Paulo, Brazil.

Since 2021, the emergence of variants of concern (VOC) has led Brazil to experience record numbers of in COVID-19 cases and deaths. The expanded spread of the SARS-CoV-2 combined with a low vaccination rate has contributed to the emergence of new mutations that may enhance viral fitness, leading to the persistence of the disease. Due to limitations in the real-time genomic monitoring of new variants in some Brazilian states, we aimed to investigate whether genomic surveillance, coupled with epidemiological data and SARS-CoV-2 variants spatiotemporal spread in a smaller region, can reflect the pandemic progression at a national level. Our findings revealed three SARS-CoV-2 variant replacements from 2021 to early 2022, corresponding to the introduction and increase in the frequency of Gamma, Delta, and Omicron variants, as indicated by peaks of the Effective Reproductive Number (Reff). These distinct clade replacements triggered two waves of COVID-19 cases, influenced by the increasing vaccine uptake over time. Our results indicated that the effectiveness of vaccination in preventing new cases during the Delta and Omicron circulations was six and eleven times higher, respectively, than during the period when Gamma was predominant, and it was highly efficient in reducing the number of deaths. Furthermore, we demonstrated that genomic monitoring at a local level can reflect the national trends in the spread and evolution of SARS-CoV-2.

Emergence and spread of SARS-CoV-2 variants of concern in Canada: a retrospective analysis from clinical and wastewater data.

BACKGROUND: The spread of SARS-CoV-2 has been studied at unprecedented levels worldwide. In jurisdictions where molecular analysis was performed on large scales, the emergence and competition of numerous SARS-CoV-2lineages have been observed in near real-time. Lineage identification, traditionally performed from clinical samples, can also be determined by sampling wastewater from sewersheds serving populations of interest. Variants of concern (VOCs) and SARS-CoV-2 lineages associated with increased transmissibility and/or severity are of particular interest. METHOD: Here, we consider clinical and wastewater data sources to assess the emergence and spread of VOCs in Canada retrospectively. RESULTS: We show that, overall, wastewater-based VOC identification provides similar insights to the surveillance based on clinical samples. Based on clinical data, we observed synchrony in VOC introduction as well as similar emergence speeds across most Canadian provinces despite the large geographical size of the country and differences in provincial public health measures. CONCLUSION: In particular, it took approximately four months for VOC Alpha and Delta to contribute to half of the incidence. In contrast, VOC Omicron achieved the same contribution in less than one month. This study provides significant benchmarks to enhance planning for future VOCs, and to some extent for future pandemics caused by other pathogens, by quantifying the rate of SARS-CoV-2 VOCs invasion in Canada.

Successful treatment of SARS-CoV-2 in an immunocompromised patient with persistent infection for 245 days: A case report.

Background: Immunocompromised patients receiving B-cell-depleting therapies are at increased risk of persistent SARS-CoV-2 infection, with many experiencing fatal outcomes. We report a successful outcome in a patient with rheumatoid arthritis (RA) on rituximab diagnosed with COVID-19 in July 2020 with persistent infection for over 245 days. Results: The patient received numerous treatment courses for persistent COVID-19 infection, including remdesivir, baricitinib, immunoglobulin and high doses of corticosteroids followed by a prolonged taper due to persistent respiratory symptoms and cryptogenic organizing pneumonia. Her clinical course was complicated by Pseudomonas aeruginosa sinusitis with secondary bacteremia, and cytomegalovirus (CMV) viremia and pneumonitis. SARS-CoV-2 positive RNA samples were extracted from two nasopharyngeal swabs and sequenced using targeted amplicon Next-Generation Sequencing which were analyzed for virus evolution over time. Viral sequencing indicated lineage B.1.585.3 SARS-CoV-2 accumulated Spike protein mutations associated with immune evasion and resistance to therapeutics. Upon slowly decreasing the patient's steroids, she had resolution of her symptoms and had a negative nasopharyngeal SARS-CoV-2 PCR and serum CMV PCR in March 2021. Conclusion: A patient with RA on B-cell depleting therapy developed persistent SARS-CoV-2 infection allowing for virus evolution and had numerous complications, including viral and bacterial co-infections with opportunistic pathogens. Despite intra-host evolution with a more immune evasive SARS-CoV-2 lineage, it was cleared after 245 days with reconstitution of the patient's immune system.