Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification (preprint)

The ability to distinguish between SARS-CoV-2 variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, response to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in Reaction 1, and del69-70, K417N, and T478K in Reaction 2. This assay had 95-100% agreement with WGS in 502 upper respiratory swabs collected between April 26 and August 1, 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

Direct Comparison of Antibody Responses to Four SARS-CoV-2 Vaccines in Mongolia (preprint)

Different vaccines for SARS-CoV-2 are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four Covid vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant RBD proteins reveal marked differences in the vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control the COVID-19 pandemic in Mongolia and worldwide.

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury (preprint)

Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinib's cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and Venezuelan equine encephalitis virus infection and is a molecular target mediating lapatinib's antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in acute and chronic lung injury downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and tissue injury via ErbBs and propose approved candidates to counteract these effects with implications for coronaviruses and unrelated viruses.

Multiplex SARS-CoV-2 Genotyping PCR for Population-Level Variant Screening and Epidemiologic Surveillance (preprint)

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a spike genotyping nucleic acid amplification test (NAAT) could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population. Between December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had near-perfect (98-100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.