In December 2020 an unexpected rise in SARS-CoV-2 infections in the UK was attributed to the emergence of a new SARS-CoV-2 variant of concern (VOC), lineage B.1.1.7 (20I/501.V1, VOC 202012/01), first detected by an efficient national scale genomic surveillance network. B.1.1.7 shows a 40–70% higher transmission rate 1,2, mainly explained by the N501Y amino acid substitution in the Spike-Receptor Binding Domain (S-RBD) that increases the affinity of S-RBD for the human ACE2 receptor 7-to 19-fold 3,4. B.1.1.7 rapidly became the dominant strain in the UK and many European countries 5. Its spread could be accurately monitored by an accidental interference by one of its defining mutations, the Spike 69/70 deletion (69.70del) in the widely used TaqPath PCR resulting in a characteristic signature with preserved amplification of N-gene and ORF1ab gene targets but S gene target failure (SGTF or S-dropout). Though recent data indicate that B.1.1.7 is overall 60% more deadly 6,7, it does not appear to display immune escape. B.1.1.7 appears refractory to N-terminal domain (NTD) targeting antibodies, but not to the S-RBD neutralizing antibodies that are induced by all currently approved vaccines 4,8, with generally preserved in vitro neutralization convalescent sera after wild type virus infection and by immune sera after the BNT162b2 (Pfizer/BioNTech) and AZD1222 (AstraZeneca/Oxford) 4,9,10 vaccines.
In an example of convergent evolution, the N501Y mutation independently arose in several other SARS-CoV-2 lineages, such as the B.1.351 (20H/501.V2, variant of concern 2) in South-Africa 11 and the P.1 (20J/501Y.V3, variant of concern 3) in Brazil 12. Both lineages had higher infectivity than the wild type virus and rapidly achieved regional dominance. In another, more concerning, example of convergent evolution, the B.1.351 and P.1 lineages share the additional E484K Spike mutation that was independently confirmed as powerful driver of clinical immune escape. This partial immune escape explains the resurgence of COVID-19 in Manaus, Brazil by P.1 in a population with 76% seroprevalence 13 and the absence of protective immunity of prior wild type infection to B.1.351 in the placebo arms of the NVX-CoV2373 vaccine trial in South-Africa.
The impact of various SARS-CoV-2 mutations/variants on immune escape was mainly derived from several small-scale in vitro pseudovirus neutralization studies using polyclonal convalescent sera or monoclonal antibodies after wild type (non-variant of concern, non-VOC) infections and polyclonal sera after vaccination with the current first generation S-RBD targeted vaccines. Introduction of the E484K in a B.1.1.7 background leads to a 6-fold lower neutralization by convalescent sera 3,4. Also, vaccine efficacy is reduced: P.1 shows a moderately (2- to 3-fold) lower neutralization by mRNA-1273 and BNT162b2 vaccine sera 8. In case of B.1.351 the effect is more pronounced, likely due to additional effects of the NTD-mutations in this lineage, leading to 6 to 12-fold lower neutralization by both vaccines 8,14,15. These effects are consistently confirmed in vivo. Recent data collected in South-Africa showed a drop of vaccine efficacy to prevent moderate to severe COVID-19 by the B.1.351 lineage as compared to non-VOC strains: from 95.6–50% for the NVX-CoV2373 (Novavax) vaccine, from 72–57% for Ad26.COV2.S (Janssen) and a complete loss of efficacy of the AZD1222 (AstraZeneca/Oxford) (66.7% for all variants to 10.6% for B.1.351) 16.
All data thus indicate that aggressively containing B.1.351 and P.1 variants in populations with low prevalence of these strains is crucial to safeguard the global vaccination strategy using the first-generation vaccines. This requires a combination of several test strategies: intensified genome surveillance by whole genome sequencing (WGS), the flexible introduction of targeted reflex PCR assays for lineage defining mutations in the spike protein (i.e. at position N501, D253, Q677) or mutations associated with potential immune escape (i.e. at position E484, L452, S477,…) depending on regional strain prevalences but also the intelligent use of subtle variations in standard SARS-CoV-2 PCR amplification curves. The latter could allow fast, high-throughput and low-cost screening of variants of concern, as illustrated by the impact of the SGFT/S-gene dropout for B.1.1.7 surveillance 1,17,18, rendering variant screening also available for health care systems with limited resources.
Here we describe a delayed amplification of the N gene-target, characteristic for the B.1.351 variant, in the Allplex Sars-CoV-2 Assay (Seegene, Korea) that is currently used in high volumes in over 70 countries worldwide. The N-gene delay was investigated in a study cohort of SARS-CoV-2 isolates containing a representative number of wild type lineages, B.1.1.7 and B.1.351. A probability score was calculated for presence of B.1.351 variant of concern 2 (VOC.V2 score) based on E/N/S-RdRP cycle threshold (Ct) values. We subsequently studied the diagnostic power of this new VOC.V2 probability score as separate test result in an independent validation cohort, as stand-alone test and in conjunction with N501Y and 69.70del mutation-specific PCRs.