Tracing the origin of SARS-CoV-2 Omicron-like Spike sequences detected in wastewater

ImportanceThe origin of highly divergent "cryptic" SARS-CoV-2 Spike sequences, which appear in wastewater but not clinical samples, is unknown. These wastewater sequences have harbored many of the same mutations that later emerged in Omicron variants. If these enigmatic sequences are human-derived and transmissible, they could both be a source of future variants and a valuable tool for forecasting sequences that should be incorporated into vaccines and therapeutics. ObjectiveTo determine whether enigmatic SARS-CoV-2 lineages detected in wastewater have a human or non-human (i.e., animal) source. DesignOn January 11, 2022, an unusual Spike sequence was detected in municipal wastewater from a metropolitan area. Over the next four months, more focused wastewater sampling resolved the source of this variant. SettingThis study was performed in Wisconsin, United States, which has a comprehensive program for detecting SARS-CoV-2 in wastewater. ParticipantsComposite wastewater samples were used for this study; therefore, no individuals participated. Main Outcome(s) and Measure(s)The primary outcome was to determine the host(s) responsible for shedding this variant in wastewater. Both human and non-human hosts were plausible candidates at the studys outset. ResultsThe presence of the cryptic virus was narrowed from a municipal wastewater sample (catchment area >100,000 people) to an indoor wastewater sample from a single facility (catchment area [~]30 people), indicating the human origin of this virus. Extraordinarily high concentrations of viral RNA ([~]520,000,000 genome copies / L and [~]1,600,000,000 genome copies / L in June and August 2022, respectively) were detected in the indoor wastewater sample. The virus sequence harbored a combination of fixed nucleotide substitutions previously observed only in Pango lineage B.1.234, a variant that circulated at low levels in Wisconsin from October 2020 to February 2021. Conclusions and RelevanceHigh levels of persistent SARS-CoV-2 shedding from the gastrointestinal tract of an infected individual likely explain the presence of evolutionarily advanced "cryptic variants" observed in some wastewater samples. Key points QuestionWhat is the source of unusual SARS-CoV-2 Omicron-like Spike variants detected in wastewater but not in clinical samples? FindingsWe identified a cryptic SARS-CoV-2 lineage in wastewater collected at a central wastewater treatment facility and traced its source to a single wastewater outlet serving six restrooms. The virus in this sample resembled a 2020-2021 lineage except for the Spike protein, in which Omicron-like variants were observed. MeaningProlonged shedding from the human gastrointestinal tract is the most likely source for evolutionarily advanced SARS-CoV-2 variant sequences found in wastewater.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel SARS-CoV-2 variants, particularly when both the immune system and antiviral therapy fail to clear the infection, thereby promoting adaptation. Here we describe an approximately 16-month case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individuals virus was resistant to this antibody via a globally unique Spike amino acid variant (E484T) that evolved from E484A earlier in infection. With the emergence and spread of the Omicron Variant of Concern, which also contains Spike E484A, E484T may arise again as an antibody-resistant derivative of E484A.

Reduced Pathogenicity of the SARS-CoV-2 Omicron Variant in Hamsters

The SARS-CoV-2 Omicron (B.1.1.529) variant has proven highly transmissible and has outcompeted the Delta variant in many regions of the world1. Early reports have also suggested that Omicron may result in less severe clinical disease in humans. Here we show that Omicron is less pathogenic than prior SARS-CoV-2 variants in Syrian golden hamsters. Infection of hamsters with the SARS-CoV-2 WA1/2020, Alpha, Beta, or Delta strains led to 4-10% weight loss by day 4 and 10-17% weight loss by day 6, as expected2,3. In contrast, infection of hamsters with two different Omicron challenge stocks did not result in any detectable weight loss, even at high challenge doses. Omicron infection still led to substantial viral replication in both the upper and lower respiratory tracts and pulmonary pathology, but with a trend towards higher viral loads in nasal turbinates and lower viral loads in lung parenchyma compared with WA1/2020 infection. These data suggest that the SARS-CoV-2 Omicron variant may result in more robust upper respiratory tract infection but less severe lower respiratory tract clinical disease compared with prior SARS-CoV-2 variants.

mRNA-1273 and Ad26.COV2.S vaccines protect against the B.1.621 variant of SARS-CoV-2

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, viral variants with greater transmissibility or immune evasion properties have arisen, which could jeopardize recently deployed vaccine and antibody-based countermeasures. Here, we evaluated in mice and hamsters the efficacy of preclinical non-GMP Moderna mRNA vaccine (mRNA-1273) and the Johnson & Johnson recombinant adenoviral-vectored vaccine (Ad26.COV2.S) against the B.1.621 (Mu) South American variant of SARS-CoV-2, which contains spike mutations T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, and D950N. Immunization of 129S2 and K18-human ACE2 transgenic mice with mRNA-1273 vaccine protected against weight loss, lung infection, and lung pathology after challenge with B.1.621 or WA1/2020 N501Y/D614G SARS-CoV-2 strain. Similarly, immunization of 129S2 mice and Syrian hamsters with a high dose of Ad26.COV2.S reduced lung infection after B.1.621 virus challenge. Thus, immunity induced by mRNA-1273 or Ad26.COV2.S vaccines can protect against the B.1.621 variant of SARS-CoV-2 in multiple animal models.

An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by several therapeutic monoclonal antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic resulting in millions of deaths worldwide. Despite the development and deployment of highly effective antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. Indeed, the recent emergence of the highly-transmissible B.1.1.529 Omicron variant is especially concerning because of the number of mutations, deletions, and insertions in the spike protein. Here, using a panel of anti-receptor binding domain (RBD) monoclonal antibodies (mAbs) corresponding to those with emergency use authorization (EUA) or in advanced clinical development by Vir Biotechnology (S309, the parent mAbs of VIR-7381), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Lilly (LY-CoV555 and LY-CoV016), and Celltrion (CT-P59), we report the impact on neutralization of a prevailing, infectious B.1.1.529 Omicron isolate compared to a historical WA1/2020 D614G strain. Several highly neutralizing mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987, and CT-P59) completely lost inhibitory activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced ([~]12-fold decrease, COV2-2196 and COV2-2130 combination) or minimally affected (S309). Our results suggest that several, but not all, of the antibody products in clinical use will lose efficacy against the B.1.1.529 Omicron variant and related strains.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Durability of immune responses to the BNT162b2 mRNA vaccine

The development of the highly efficacious mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology. However, reports of waning vaccine efficacy, coupled with the emergence of variants of concern that are resistant to antibody neutralization, have raised concerns about the potential lack of durability of immunity to vaccination. We recently reported findings from a comprehensive analysis of innate and adaptive immune responses in 56 healthy volunteers who received two doses of the BNT162b2 vaccination. Here, we analyzed antibody responses to the homologous Wu strain as well as several variants of concern, including the emerging Mu (B.1.621) variant, and T cell responses in a subset of these volunteers at six months (day 210 post-primary vaccination) after the second dose. Our data demonstrate a substantial waning of antibody responses and T cell immunity to SARS-CoV-2 and its variants, at 6 months following the second immunization with the BNT162b2 vaccine. Notably, a significant proportion of vaccinees have neutralizing titers below the detection limit, and suggest a 3rd booster immunization might be warranted to enhance the antibody titers and T cell responses.

A method to reduce ELISA serial dilution assay workload applied to SARS-CoV-2 and seasonal HCoVs

ObjectivesAssays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method to reduce the workload of these assays, and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. MethodsWe apply Bayesian hierarchical modelling to ELISA measurements of human serum samples against SARS-CoV-2 and HCoV antigens. ResultsInflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from three spread-out dilutions correlated with those inferred from eight consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. ConclusionsIn serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

Highly efficient SARS-CoV-2 infection of human cardiomyocytes: spike protein-mediated cell fusion and its inhibition

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to a bystander effect: the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) that beat spontaneously. These cardiomyocytes express the angiotensin I converting-enzyme 2 (ACE2) receptor and a subset of the proteases that mediate spike protein cleavage in the lungs, but not transmembrane protease serine 2 (TMPRSS2). Nevertheless, SARS-CoV-2 infection was productive: viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CM, smooth walled exocytic vesicles contained numerous 65-90 nm particles with typical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand the mechanisms of SARS-CoV-2 spread in hiPSC-CM we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm signal from cell to cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin abolished cell fusion. A spike mutant with the single amino acid change R682S that inactivates the furin cleavage site was fusion inactive. Thus, SARS-CoV-2 can replicate efficiently in hiPSC-CM and furin activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform provides an opportunity for target-based drug discovery in cardiac COVID-19. Author SummaryIt is unclear whether the cardiac complications frequently observed in COVID-19 patients are due exclusively to systemic inflammation and thrombosis. Viral replication has occasionally been confirmed in cardiac tissue, but rigorous analyses are restricted to rare autopsy materials. Moreover, there are few animal models to study cardiovascular complications of coronavirus infections. To overcome these limitations, we developed an in vitro model of SARS-CoV-2 spread in induced pluripotent stem cell-derived cardiomyocytes. In these cells, infection is highly productive: viral transcription levels exceed those documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread we expressed a fluorescent version of its spike protein that allowed to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin cleavage site lost cytopathic function. The spike protein of the Middle East Respiratory Syndrome (MERS) coronavirus drove cardiomyocyte fusion with slow kinetics, whereas the spike proteins of SARS-CoV and the respiratory coronavirus 229E were inactive. These fusion activities correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 has the potential to cause cardiac damage by fusing cardiomyocytes.

Vaccinated and unvaccinated individuals have similar viral loads in communities with a high prevalence of the SARS-CoV-2 delta variant

The SARS-CoV-2 Delta Variant of Concern is highly transmissible and contains mutations that confer partial immune escape. The emergence of Delta in North America caused the first surge in COVID-19 cases after SARS-CoV-2 vaccines became widely available. To determine whether individuals infected despite vaccination might be capable of transmitting SARS-CoV-2, we compared RT-PCR cycle threshold (Ct) data from 20,431 test-positive anterior nasal swab specimens from fully vaccinated (n = 9,347) or unvaccinated (n=11,084) individuals tested at a single commercial laboratory during the interval 28 June - 1 December 2021 when Delta variants were predominant. We observed no significant effect of vaccine status alone on Ct value, nor when controlling for vaccine product or sex. Testing a subset of low-Ct (<25) samples, we detected infectious virus at similar rates, and at similar titers, in specimens from vaccinated and unvaccinated individuals. These data indicate that vaccinated individuals infected with Delta variants are capable of shedding infectious SARS-CoV-2 and could play a role in spreading COVID-19.

SARS-CoV-2 D614G Variant Exhibits Enhanced Replication ex vivo and Earlier Transmission in vivo

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.