Emergence of N antigen SARS-CoV-2 genetic variants escaping detection of antigenic tests

SARS-CoV-2 genetic variants are emerging as a major threat to vaccination efforts worldwide as they may increase virus transmission rate and/or confer the ability to escape vaccine induced immunity with knock on effects on the level of herd immunity and vaccine efficacy respectively. These variants concern the Spike protein, which is encoded by the S gene, involved in virus entry into host cells and the major target of vaccine development. We report here that genetic variants of the N gene can impair our ability to utilize antigenic tests for both diagnosis and mass testing efforts aimed at controlling virus transmission. While conducting a large validation study on the Abbott Panbio COVID-19 Ag test, we noticed that some swab samples failed to generate a positive result in spite of a high viral load in Rt-PCR assays. Sequencing analysis of viruses showing discordant results in the RT-PCR and antigen assays revealed the presence of multiple disruptive amino-acid substitutions in the N antigen (the viral protein detected in the antigen test) clustered from position 229 to 374 a region known to contain an immunodominant epitope. A relevant fraction of the variants, undetected by the antigen test, contained the mutations A376T coupled to M241I. Intriguingly we found that virus sequences with this mutation were over-represented in the antigen-test-negative and PCR-positive samples and progressively increased in frequency over time in Veneto, a region of Italy that has aggressively scaled up the utilization of antigen tests, which reached nearly 68% of all the SARS-CoV-2 swab assays performed there. We speculate that mass utilization of antigen assays could create a selection pressure on the target that may favor the spread of undetectable virus variants.