Study: Pre-Omicron vaccine breakthrough infection induces superior cross-neutralization against SARS-CoV-2 Omicron BA.1 than primo infection. Image Credit: Lightspring/Shutterstock

Breakthrough infection with pre-Omicron variant induces cross-neutralizing activity against Omicron

In a recent study posted to the medRxiv* preprint server, researchers evaluated the neutralizing ability of sera from patients with coronavirus disease 2019 (COVID-19).

Study: Breakthrough Infection of Pre-Omicron Vaccine Induces Superior Cross Neutralization Against SARS-CoV-2 Omicron BA.1 Than Primo Infection† Image Credit: Lightspring/Shutterstock

The causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), evolved during the pandemic and emerges as mutant variants with enhanced characteristics. SARS-CoV-2 variants that are highly transmissible or immune-evasive easily outperform previously dominant strains. Vaccines such as BNT162b2 from Pfizer, mRNA-1273 from Moderna, Ad.26COV2.S from Janssen and ChAdOx1 from AstraZeneca are effective in protecting against serious diseases.

Still, protection against infection is poor, especially for SARS-CoV-2 Delta and Omicron variants. The protection conferred by prior infection with pre-Omicron variants of care (VOCs) against the Omicron variant is debatable. Some reports indicated that breakthrough infection with the Omicron BA.1 variant leads to: neutralizing antibodies (nAbs) against BA.1 and earlier VOCs; yet these nAbs exhibit poor cross-neutralizing activity against Omicron sublines.

Omicron infection in unvaccinated humans causes nAbs with insufficient neutralization against all VOCs. The extensive distribution of SARS-CoV-2 Omicron warrants a better understanding of its susceptibility to neutralization by vaccination or infection-induced antibodies.

About the study

In the current study, researchers compared the neutralization of Omicron BA.1 by sera from unvaccinated recoveries and those with vaccine breakthrough infections. Serum samples were obtained from 70 convalescent individuals (henceforth convalescent sera) infected with ancestral B.1 strain and from 16 vaccinated individuals (BTI sera) experiencing breakthrough infection with Gamma or Delta VOC. The severity of COVID-19 was divided into mild/asymptomatic, moderate and severe illness.

The research team isolated the D614G strain and Gamma, Delta, or Omicron VOCs from patients’ leftover nasopharyngeal swabs. They added residual smear preservation medium to Vero E6 cells and visually monitored cytopathic effect (CPE). The viral supernatant was then used to reinfect Vero E6 cells (passage 2) to establish a viral stock. SARS-CoV-2 variants in the remaining smears were sequenced using next-generation sequencing (NGS), and the spike gene (S) was resequenced after passage 2 to verify sequence conservation. Finally, they performed a neutralization test for live viruses.


Most recovering sera samples were collected from patients with moderate or severe disease. The half-maximal inhibitory concentration (IC50) values ​​of restorative sera showed a wide range of neutralizing activity against B.1, Gamma and Delta variants by similar geometric means.

All but one recovering samples did not neutralize Omicron at the highest serum concentration tested.

Samples with low/no neutralization activity against one variant often had a higher neutralization against another variant. Recovering sera from patients with moderate COVID-19 had the highest neutralizing activity against B.1 and VOCs. They then measured antibodies directed against the S protein, its receptor binding (RBD) and N-terminal (NTD) domains, and the nucleocapsid (N) protein. All patients had levels of anti-N, anti-S and anti-RBD antibodies detected.

Anti-NTD antibodies were lower/undetectable in some patients with severe disease. The authors noted a moderate correlation between neutralizing activity and antibody levels. Consistently, sera from moderate COVID-19 patients showed higher antibodies to S, RBD, NTD and N than in severe cases.

BTI sera were collected from two Gamma variant infected patients and 14 cases of Delta variants. The median time since complete vaccination was 3.06 months. BTI sera were stratified into no/poor neutralizing and highly neutralizing sera (eight samples in each category). Five no/poor neutralizing sera showed no neutralization against strain B.1 at the highest serum concentration tested.

They noted that BTI serum samples neutralizing the B.1 strain at ≥ 1:80 dilutions either retained neutralization against corresponding infecting VOCs or showed a higher neutralization against VOCs. Conversely, most samples that failed to neutralize the B.1 strain had no neutralizing activity against the infecting variant. More than half of the BTI cases showed nAbs to Gamma or Delta VOC. Thus, serum samples had better cross-neutralizing activity against the Delta variant than B.1 strain.

All BTI sera showed a substantial decrease in neutralization against the Omicron BA.1 variant. Eight samples with high neutralizing activity against B.1. Gamma of Delta retained partial neutralization against BA.1. Anti-S, anti-RBD and anti-NTD antibody levels were higher except for one BTI sample. Neutralizing activity against B.1 strain correlated well with antibodies to RBD, NTD and S protein, but less so with anti-N antibodies.


In summary, BTI sera showed a higher neutralizing capacity against the SARS-CoV-2 B.1 strain and VOCs, including Omicron BA.1, than restorative sera. Of the eight BTI sera that neutralized B.1 and VOC, six samples partially neutralized BA.1. This showed that breakthrough infection with a pre-Omicron variant elicited antibodies that could cross neutralize the Omicron BA.1 variant.

Notably, higher neutralization was achieved with lower antibodies to S, N, RBD and NTD in BTI sera compared to restorative sera, implying greater neutralization effectiveness is achieved with lower antibody titers, perhaps because of the improved affinity of antibodies. These findings highlighted the qualitative differences in antibodies between unvaccinated recovering individuals and vaccinated recovering individuals and suggested that breakthrough infection might enhance the immune response and induce cross-neutralizing antibodies.

*Important announcement

medRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior, or be treated as established information.

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