SPECIFIC DETECTION OF SARS-COV-2 B.1.1.529 (OMICRON) VARIANT BY FOUR RT-qPCR DIFFERENTIAL ASSAYS (preprint)

In this report, we describe four RT-qPCR assays that enable rapid identification of the newly emerging SARS-COV-2 Omicron (B.1.1.529) variant of concern. The assays target Omicron characteristic mutations in the nsp6 (Orf1a), spike and nucleocapsid genes. We demonstrate that the assays are straightforward to assemble and perform, are amendable for multiplexing, and may be used as a reliable first-line tool to identify B.1.1.529 suspected samples. Importantly, this is a preliminary development report. Further validation and optimization of the assays described herein will be published hereafter.

Covid-19 Vaccine Effectiveness in Healthcare Personnel in six Israeli Hospitals (CoVEHPI) (preprint)

BackgroundMethodologically rigorous studies on Covid-19 vaccine effectiveness (VE) in preventing SARS-CoV-2 infection are critically needed to inform national and global policy on Covid-19 vaccine use. In Israel, healthcare personnel (HCP) were initially prioritized for Covid-19 vaccination, creating an ideal setting to evaluate real-world VE in a closely monitored population. MethodsWe conducted a prospective study among HCP in 6 hospitals to estimate the effectiveness of the BNT162b2 mRNA Covid-19 vaccine in preventing SARS-CoV-2 infection. Participants filled out weekly symptom questionnaires, provided weekly nasal specimens, and three serology samples - at enrollment, 30 days and 90 days. We estimated VE against PCR-confirmed SARS-CoV-2 infection using the Cox Proportional Hazards model and against a combined PCR/serology endpoint using Fishers exact test. FindingsOf the 1,567 HCP enrolled between December 27, 2020 and February 15, 2021, 1,250 previously uninfected participants were included in the primary analysis; 998 (79.8%) were vaccinated with their first dose prior to or at enrollment, all with Pfizer BNT162b2 mRNA vaccine. There were four PCR-positive events among vaccinated participants, and nine among unvaccinated participants. Adjusted two-dose VE against any PCR- confirmed infection was 94.5% (95% CI: 82.6%-98.2%); adjusted two-dose VE against a combined endpoint of PCR and seroconversion for a 60-day follow-up period was 94.5% (95% CI: 63.0%-99.0%). Five PCR-positive samples from study participants were sequenced; all were alpha variant. InterpretationOur prospective VE study of HCP in Israel with rigorous weekly surveillance found very high VE for two doses of Pfizer BNT162b2 mRNA vaccine against SARS-CoV-2 during a period of predominant alpha variant circulation. FundingClalit Health Services

Global disparities in SARS-CoV-2 genomic surveillance (preprint)

Genomic sequencing provides critical information to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments and vaccines, and guide public health responses. To investigate the spatiotemporal heterogeneity in the global SARS-CoV-2 genomic surveillance, we estimated the impact of sequencing intensity and turnaround times (TAT) on variant detection in 167 countries. Most countries submit genomes >21 days after sample collection, and 77% of low and middle income countries sequenced <0.5% of their cases. We found that sequencing at least 0.5% of the cases, with a TAT <21 days, could be a benchmark for SARS-CoV-2 genomic surveillance efforts. Socioeconomic inequalities substantially impact our ability to quickly detect SARS-CoV-2 variants, and undermine the global pandemic preparedness. One-Sentence SummarySocioeconomic inequalities impacted the SARS-CoV-2 genomic surveillance, and undermined the global pandemic preparedness.

The unique evolutionary dynamics of the SARS-CoV-2 Delta variant (preprint)

The SARS-Coronavirus-2 (SARS-CoV-2) driven pandemic was first recognized in late 2019, and the first few months of its evolution were relatively clock-like, dominated mostly by neutral substitutions. In contrast, the second year of the pandemic was punctuated by the emergence of several variants that bore evidence of dramatic evolution. Here, we compare and contrast evolutionary patterns of various variants, with a focus on the recent Delta variant. Most variants are characterized by long branches leading to their emergence, with an excess of non-synonymous substitutions occurring particularly in the Spike and Nucleocapsid proteins. In contrast, the Delta variant that is now becoming globally dominant, lacks the signature long branch, and is characterized by a step-wise evolutionary process that is ongoing. Contrary to the "star-like" topologies of other variants, we note the formation of several distinct clades within Delta that we denote as clades A-E. We find that sequences from the Delta D clade are dramatically increasing in frequency across different regions of the globe. Delta D is characterized by an excess of non-synonymous mutations, mostly occurring in ORF1a/b, some of which occurred in parallel in other notable variants. We conclude that the Delta surge these days is composed almost exclusively of Delta D, and discuss whether selection or random genetic drift has driven the emergence of Delta D.

BNT162b2 Vaccination efficacy is marginally affected by the SARS-CoV-2 B.1.351 variant in fully vaccinated individuals (preprint)

ABSTRACT Background Israeli has vaccinated over 80% of its adult population, with two doses of the Pfizer BNT162b2 vaccine. This intervention has been highly successful in curtailing the coronavirus 2 outbreak. One major concern is the ability of the virus to mutate which potentially can cause SARS-CoV-2 to partially escape from the immune system. Here we evaluate the efficacy of the Pfizer vaccine against the B.1.351 variant. Methods The Ministry of Health, initiated sequencing of selected positive swab samples identified as being of interest. We used logistic regression, with variant type as the dependent variable, vaccination status as the main explanatory variable, controlling for age, sex, subpopulation, place of residence and time of sample, to estimate the odds ratio for a vaccinated case to have the B. 1.351 versus the B.1.1.7 variant, within vaccinated and unvaccinated persons who tested positive. Findings There were 19 cases of B.1.351 variant (3.2%) among those vaccinated more than 14 days before the positive sample and 88 (3.5%) among the unvaccinated. The estimated odds ratio was 1.29 [95% CI: 0.66-2.50]. From this result, assuming the efficacy against the B.1.1.7 variant to be 95%, the estimated efficacy against the B.1.351 variant was 94% [95% CI: 87-97%]. Interpretation Despite the concerns caused by the B.1.351 variant, the BNT162b2 vaccine seems to provide substantial immunity against both that variant and the B.1.1.7. Our results suggest that from 14 days following the second vaccine dose the efficacy of BNT162b2 vaccine is at most marginally affected by the B.1.351 variant. Funding No funding

BNT162b2 Vaccination Efficacy is Marginally Affected by the SARS-CoV-2 B.1.351 Variant in Fully Vaccinated Individuals (preprint)

Background: Israeli has vaccinated over 80% of its adult population, with two doses of the Pfizer BNT162b2 vaccine. This intervention has been highly successful in curtailing the coronavirus 2 outbreak. One major concern is the ability of the virus to mutate which potentially can cause SARS-CoV-2 to partially escape from the immune system. Here we evaluate the efficacy of the Pfizer vaccine against the B.1.351 variant.Methods: The Ministry of Health, initiated sequencing of selected positive swab samples identified as being of interest. We used logistic regression, with variant type as the dependent variable, vaccination status as the main explanatory variable, controlling for age, sex, subpopulation, place of residence and time of sample, to estimate the odds ratio for a vaccinated case to have the B. 1.351 versus the B.1.1.7 variant, within vaccinated and unvaccinated persons who tested positive.Findings: There were 19 cases of B.1.351 variant (3.2%) among those vaccinated more than 14 days before the positive sample and 88 (3.5%) among the unvaccinated. The estimated odds ratio was 1.29 [95% CI: 0.66-2.50]. From this result, assuming the efficacy against the B.1.1.7 variant to be 95%, the estimated efficacy against the B.1.351 variant was 94% [95% CI: 87-97%].Interpretation: Despite the concerns caused by the B.1.351 variant, the BNT162b2 vaccine seems to provide substantial immunity against both that variant and the B.1.1.7. Our results suggest that from 14 days following the second vaccine dose the efficacy of BNT162b2 vaccine is at most marginally affected by the B.1.351 variant.Funding Information: No funding.Declaration of Interests: I confirm that none of the authors has related work submitted to another journal and that none have any conflict of interest regarding the content of the manuscript.

The neutralization potency of anti-SARS-CoV-2 therapeutic human monoclonal antibodies is retained against novel viral variants (preprint)

Summary A wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority. Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains. Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.

HiSpike: A high-throughput cost effective sequencing method for the SARS-CoV-2 spike gene (preprint)

The changing nature of the corona virus of the SARS-CoV-2 pandemic poses unprecedented challenges to the world’s health systems. New and virulent emerging spike gene variants, such as the UK 20I/501Y.V1 and South African 20H/501Y.V2, could jeopardize global efforts to produce immunity and reduce mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and therefore, it is most susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is also the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Therefore, tight monitoring of the spike protein gene variants is key to mitigating COVID-19 spread and vaccine escape mutants. Currently, the ARTIC method for SARS-CoV-2 whole genome sequencing is applied worldwide. However, this method commonly requires more than 96 hours (4–5 days) from start to finish and at present high sample sequence demands, sequencing resources are quickly exhausted. In this work, we present HiSpike, a method for high-throughput targeted next generation sequencing (NGS) of the spike gene. This simple three-step method can be completed in less than 30 hours and can sequence 10-fold more samples compared to the conventional ARTIC method and at a fraction of the cost. HiSpike was proven valid, and has identified, at high quality, multiple spike variants from real-time field samples, such as the UK and the South African variants. This method will certainly be effective in discovering future spike mutations. Therefore, running HiSpike for full sequencing of the spike gene of all positive SARS-CoV-2 samples could be considered for near real-time detection of known and emerging spike mutations as they evolve. HiSpike provides affordable sequencing options to help laboratories conserve resources, hence it provides a tool for widespread monitoring, that can support critical knowledge-based decisions.

Initial real world evidence for lower viral load of individuals who have been vaccinated by BNT162b2 (preprint)

One of the key questions regarding COVID19 vaccines is whether they can reduce viral shedding. To date, Israel vaccinated substantial parts of the adult population, which enables extracting real world signals. The vaccination rollout started on Dec 20th 2020, utilized mainly the BNT162b2 vaccine, and focused on individuals who are 60 years or older. By now, more than 75% of the individuals of this age group have been at least 14 days after the first dose, compared to 25% of the individuals between ages 40-60 years old. Here, we traced the Ct value distribution of 16,297 positive qPCR tests in our lab between Dec 1st to Jan 31st that came from these two age groups. As we do not have access to the vaccine status of each test, our hypothesis was that if vaccines reduce viral load, we should see a difference in the Ct values between these two age groups in late January but not before. Consistent with this hypothesis, until Jan 15th, we did not find any statistically significant differences in the average Ct value between the groups. In stark contrast, our results in the last two weeks of January show a significant weakening in the average Ct value of 60+ individuals to the 40-60 group. To further corroborate these results, we also used a series nested linear models to explain the Ct values of the positive tests. This analysis favored a model that included an interaction between age and the late January time period, consistent with the effect of vaccination. We then used demographic data and the daily vaccination rates to estimate the effect of vaccination on viral load reduction. Our estimate suggests that vaccination reduces the viral load by 1.6x to 20x in individuals who are positive for SARS-CoV-2. This estimate might improve after more individuals receive the second dose. Taken together, our findings indicate vaccination is not only important for individual’s protection but can reduce transmission.