Using Genome Sequence Data to Predict SARS-CoV-2 Detection Cycle Threshold Values (preprint)

The continuing emergence of SARS-CoV-2 variants of concern (VOCs) presents a serious public health threat, exacerbating the effects of the COVID19 pandemic. Although millions of genomes have been deposited in public archives since the start of the pandemic, predicting SARS-CoV-2 clinical characteristics from the genome sequence remains challenging. In this study, we used a collection of over 29,000 high quality SARS-CoV-2 genomes to build machine learning models for predicting clinical detection cycle threshold (Ct) values, which correspond with viral load. After evaluating several machine learning methods and parameters, our best model was a random forest regressor that used 10-mer oligonucleotides as features and achieved an R2 score of 0.521 +/- 0.010 (95% confidence interval over 5 folds) and an RMSE of 5.7 +/- 0.034, demonstrating the ability of the models to detect the presence of a signal in the genomic data. In an attempt to predict Ct values for newly emerging variants, we predicted Ct values for Omicron variants using models trained on previous variants. We found that approximately 5% of the data in the model needed to be from the new variant in order to learn its Ct values. Finally, to understand how the model is working, we evaluated the top features and found that the model is using a multitude of k-mers from across the genome to make the predictions. However, when we looked at the top k-mers that occurred most frequently across the set of genomes, we observed a clustering of k-mers that span spike protein regions corresponding with key variations that are hallmarks of the VOCs including G339, K417, L452, N501, and P681, indicating that these sites are informative in the model and may impact the Ct values that are observed in clinical samples.

Antibody escape and cryptic cross-domain stabilization in the SARS CoV-2 Omicron spike protein (preprint)

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.

Detection of SARS-CoV-2 Omicron variant (B.1.1.529) infection of white-tailed deer (preprint)

White-tailed deer (Odocoileus virginianus) are highly susceptible to infection by SARS-CoV-2, with multiple reports of widespread spillover of virus from humans to free-living deer. While the recently emerged SARS-CoV-2 B.1.1.529 Omicron variant of concern (VoC) has been shown to be notably more transmissible amongst humans, its ability to cause infection and spillover to non-human animals remains a challenge of concern. We found that 19 of the 131 (14.5%; 95% CI: 0.10-0.22) white-tailed deer opportunistically sampled on Staten Island, New York, between December 12, 2021, and January 31, 2022, were positive for SARS-CoV-2 specific serum antibodies using a surrogate virus neutralization assay, indicating prior exposure. The results also revealed strong evidence of age-dependence in antibody prevalence. A significantly ({chi}2, p < 0.001) greater proportion of yearling deer possessed neutralizing antibodies as compared with fawns (OR=12.7; 95% CI 4-37.5). Importantly, SARS-CoV-2 nucleic acid was detected in nasal swabs from seven of 68 (10.29%; 95% CI: 0.0-0.20) of the sampled deer, and whole-genome sequencing identified the SARS-CoV-2 Omicron VoC (B.1.1.529) is circulating amongst the white-tailed deer on Staten Island. Phylogenetic analyses revealed the deer Omicron sequences clustered closely with other, recently reported Omicron sequences recovered from infected humans in New York City and elsewhere, consistent with human to deer spillover. Interestingly, one individual deer was positive for viral RNA and had a high level of neutralizing antibodies, suggesting either rapid serological conversion during an ongoing infection or a breakthrough infection in a previously exposed animal. Together, our findings show that the SARS-CoV-2 B.1.1.529 Omicron VoC can infect white-tailed deer and highlights an urgent need for comprehensive surveillance of susceptible animal species to identify ecological transmission networks and better assess the potential risks of spillback to humans.

Multiple spillovers and onward transmission of SARS-Cov-2 in free-living and captive White-tailed deer (Odocoileus virginianus) (preprint)

Many animal species are susceptible to SARS-CoV-2 and could potentially act as reservoirs, yet transmission in non-human free-living animals has not been documented. White-tailed deer (Odocoileus virginianus), the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns transmit the virus to other captive deer. To test the hypothesis that SARS-CoV-2 may be circulating in deer, we evaluated 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through December of 2020 for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 deer (33.2%; 95% CI: 28, 38.9) samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, between Nov 23, 2020, and January 10, 2021, 80 of 97 (82.5%; 95% CI 73.7, 88.8) RPLN samples had detectable SARS-CoV-2 RNA by RT-PCR. Whole genome sequencing of the 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%), and B.1.311 (n = 19; 20%) accounting for ~75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple zooanthroponotic spillover events and deer-to-deer transmission. The discovery of sylvatic and enzootic SARS-CoV-2 transmission in deer has important implications for the ecology and long-term persistence, as well as the potential for spillover to other animals and spillback into humans. These findings highlight an urgent need for a robust and proactive One Health approach to obtaining a better understanding of the ecology and evolution of SARS-CoV-2.

Analysis of the ARTIC version 3 and version 4 SARS-CoV-2 primers and their impact on the detection of the G142D amino acid substitution in the spike protein (preprint)

The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. ImportanceARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.

Delta variants of SARS-CoV-2 cause significantly increased vaccine breakthrough COVID-19 cases in Houston, Texas (preprint)

Genetic variants of SARS-CoV-2 have repeatedly altered the course of the COVID-19 pandemic, and disease in individual patients. Delta variants (B.1.617.2, AY.2, and AY.3) are now the focus of international concern because they are causing widespread COVID-19 disease globally. Vaccine breakthrough cases caused by SARS-CoV-2 variants also are of considerable public health and medical concern worldwide. As part of a comprehensive project, we sequenced the genomes of 3,913 SARS-CoV-2 from patient samples acquired March 15, 2021 through July 3, 2021 in the Houston Methodist hospital system and studied vaccine breakthrough cases. During the study period Delta variants increased to cause 58% of all COVID-19 cases and spread throughout the metropolitan Houston area. In addition, Delta variants caused a significantly higher rate of vaccine breakthrough cases (19.7% compared to 5.8% for all other variants). Importantly, only 6.5% of all COVID-19 cases occurred in fully immunized individuals, and relatively few of these patients required hospitalization. Our genomic and epidemiologic data emphasize that vaccines used in the United States are highly effective in decreasing severe COVID-19 disease, hospitalizations, and deaths.

Sequence Analysis of 20,453 SARS-CoV-2 Genomes from the Houston Metropolitan Area Identifies the Emergence and Widespread Distribution of Multiple Isolates of All Major Variants of Concern (preprint)

[Abstract]Since the beginning of the SARS-CoV-2 pandemic, there has been international concern about the emergence of virus variants with mutations that increase transmissibility, enhance escape from the human immune response, or otherwise alter biologically important phenotypes. In late 2020, several "variants of concern" emerged globally, including the UK variant (B.1.1.7), South Africa variant (B.1.351), Brazil variants (P.1 and P.2), and two related California "variants of interest" (B.1.429 and B.1.427). These variants are believed to have enhanced transmissibility capacity. For the South Africa and Brazil variants, there is evidence that mutations in spike protein permit it to escape from some vaccines and therapeutic monoclonal antibodies. Based on our extensive genome sequencing program involving 20,453 virus specimens from COVID-19 patients dating from March 2020, we report identification of all important SARS-CoV-2 variants among Houston Methodist Hospital patients residing in the greater metropolitan area. Although these variants are currently at relatively low frequency in the population, they are geographically widespread. Houston is the first city in the United States to have all variants documented by genome sequencing. As vaccine deployment accelerates worldwide, increased genomic surveillance of SARS-CoV-2 is essential to understanding the presence and frequency of consequential variants and their patterns and trajectory of dissemination. This information is critical for medical and public health efforts to effectively address and mitigate this global crisis.

Early transfusion of a large cohort of COVID-19 patients with high titer anti-SARS-CoV-2 spike protein IgG convalescent plasma confirms a signal of significantly decreased mortality (preprint)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 remains a global threat with few proven efficacious treatments. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 disease has emerged as a promising therapy and has been granted emergency use authorization by the U.S. Food and Drug Administration (FDA). We recently reported results from interim analysis of a propensity-score matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We here present results from 60-day follow up of our cohort of 351 transfused hospitalized patients. Prospective determination of ELISA anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff (S/C) ratio of 24.0 for transfused units, a value far exceeding the recently FDA-required cutoff of 12.0 for designation of high titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours post-hospitalization for transfusing COVID-19 patients with high titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.

Molecular Architecture of Early Disseminationand Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area (preprint)

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. IMPORTANCEThere is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, Texas, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture, evolution, and relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our study provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Limited window for donation of convalescent plasma with high live-virus neutralizing antibodies for COVID-19 immunotherapy (preprint)

The optimal timeframe for donating convalescent plasma to be used for COVID-19 immunotherapy is unknown. To address this important knowledge deficit, we determined in vitro live-virus neutralizing capacity and persistence of IgM and IgG antibody responses against the receptor-binding domain and S1 ectodomain of the SARS-CoV-2 spike glycoprotein in 540 convalescent plasma samples obtained from 175 COVID-19 plasma donors for up to 142 days post-symptom onset. Robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 persist, in the aggregate, for at least 100 days post-symptom onset. However, a notable acceleration in decline in virus neutralization titers [≥]160, a value suitable for convalescent plasma therapy, was observed starting 60 days after first symptom onset. Together, these findings better define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor, including age and COVID-19 disease severity score. One Sentence SummaryEvaluation of SARS-CoV-2 anti-spike protein IgM, IgG, and live-virus neutralizing titer profiles reveals that the optimal window for donating convalescent plasma for use in immunotherapy is within the first 60 days of symptom onset.

Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma (preprint)

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer [≥]160 was 80% or greater with anti-RBD or anti-ECD titers of [≥]1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers [≥]160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of [≥]1:1350, and evidence of VN [≥]1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of [≥]1:1350 may provide critical information about protection against COVID-19 disease.

Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas (preprint)

Background: COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. Methods: Patients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 to April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. Results: At baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. Conclusions: The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

Molecular Architecture of Early Dissemination and Evolution of the SARS-CoV-2 Virus in Metropolitan Houston, Texas (preprint)

We sequenced the genomes of 320 SARS-CoV-2 strains from COVID-19 patients in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. These genomes were from the viruses causing infections in the earliest recognized phase of the pandemic affecting Houston. Substantial viral genomic diversity was identified, which we interpret to mean that the virus was introduced into Houston many times independently by individuals who had traveled from different parts of the country and the world. The majority of viruses are apparent progeny of strains derived from Europe and Asia. We found no significant evidence of more virulent viral types, stressing the linkage between severe disease, underlying medical conditions, and perhaps host genetics. We discovered a signal of selection acting on the spike protein, the primary target of massive vaccine efforts worldwide. The data provide a critical resource for assessing virus evolution, the origin of new outbreaks, and the effect of host immune response. SignificanceCOVID-19, the disease caused by the SARS-CoV-2 virus, is a global pandemic. To better understand the first phase of virus spread in metropolitan Houston, Texas, we sequenced the genomes of 320 SARS-CoV-2 strains recovered from COVID-19 patients early in the Houston viral arc. We identified no evidence that a particular strain or its progeny causes more severe disease, underscoring the connection between severe disease, underlying health conditions, and host genetics. Some amino acid replacements in the spike protein suggest positive immune selection is at work in shaping variation in this protein. Our analysis traces the early molecular architecture of SARS-CoV-2 in Houston, and will help us to understand the origin and trajectory of future infection spikes.