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 coronavirus disease 2019 (COVID-19) patients.

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

The causal pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has evolved throughout the pandemic, emerging as mutant variants with enhanced characteristics. SARS-CoV-2 variants that are highly transmissible or immune-evasive easily outcompete previously dominant strains. Vaccines such as Pfizer’s BNT162b2, Moderna’s mRNA-1273, Janssen’s Ad.26COV2.S, and AstraZeneca’s ChAdOx1 are effective in protecting against severe disease.

Yet, protection against infection is poor, particularly for SARS-CoV-2 Delta and Omicron variants. The protection conferred by prior infection with pre-Omicron variants of concern (VOCs) against the Omicron variant is debatable. Some reports indicated that breakthrough infection with Omicron BA.1 variant induces neutralizing antibodies (nAbs) against BA.1 and previous VOCs; still, these nAbs exhibit poor cross-neutralizing activity against Omicron sub-lineages.

Omicron infection in non-vaccinated people elicits nAbs with insufficient neutralization against all VOCs. The extensive dissemination of SARS-CoV-2 Omicron warrants a better understanding of the sensitivity to neutralization by vaccination or infection-induced antibodies. 

About the study

In the present study, researchers compared the neutralization of Omicron BA.1 by sera from non-vaccinated convalescents and those who had vaccine breakthrough infections. Serum samples were obtained from 70 convalescent individuals (henceforth convalescent sera) infected with ancestral B.1 strain and from 16 vaccinated subjects (BTI sera) who experienced breakthrough infection with Gamma or Delta VOC. COVID-19 severity was categorized into mild/asymptomatic, moderate, and severe disease.

The research team isolated the D614G strain and Gamma, Delta, or Omicron VOCs from the left-over nasopharyngeal swabs of patients. They added residual swab 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 left-over swabs were sequenced using next-generation sequencing (NGS), and the spike (S) gene was re-sequenced post passage 2 to verify sequence conservation. Lastly, they performed a live-virus neutralization test.  


Most samples of convalescent sera were collected from patients with moderate or severe disease. The half-maximal inhibitory concentration (IC50) values of convalescent sera exhibited a broad range of neutralizing activity against B.1, Gamma, and Delta variants with comparable geometric means.

All convalescent samples except one did not neutralize Omicron at the highest tested serum concentration.

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

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 against S, RBD, NTD, and N than in severe cases.

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

They noted that BTI serum samples that neutralized B.1 strain at ≥ 1:80 dilutions retained neutralization against corresponding infecting VOC or exhibited higher neutralization against VOCs. Conversely, most samples, which failed to neutralize B.1 strain, had no neutralizing activity against the infecting variant. Over half the BTI cases demonstrated nAbs against 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. However, eight samples with high neutralizing activity against B.1. Gamma or 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 against RBD, NTD, and S protein, but less with anti-N antibodies.


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

Notably, higher neutralization was attained with lower antibodies against S, N, RBD, and NTD in BTI sera relative to convalescent sera, implying that greater neutralization efficacy is achieved with lower antibody titers, perhaps, due to improved affinity of antibodies. These findings highlighted the qualitative differences in antibodies between non-vaccinated convalescent individuals and vaccinated convalescents and suggested that breakthrough infection could boost immune response and induce cross-neutralizing antibodies.

*Important notice

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

Journal reference:

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.


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