Antibody Response and Variant Cross-Neutralization After SARS-CoV-2 Breakthrough Infection

Breakthrough infections after vaccination against SARS-CoV-2 are increasingly reported, possibly due to waning of vaccine-induced antibody levels.¹ Moreover, emerging variants of concern with diminished susceptibility to vaccine-induced antibodies are responsible for most new cases.^2,3 Studies have focused on determining the rate of vaccine breakthrough based on antibody levels after standard vaccination practices.^4,5 We assessed antibody levels and variant cross-neutralization after breakthrough infection.

Fully vaccinated health care workers subsequently diagnosed with SARS-CoV-2 breakthrough infection based on a positive polymerase chain reaction (PCR) test result were sequentially recruited at the Oregon Health & Science University between January 31, 2021, and August 18, 2021. Only those with no history of previous infection whose test results were negative for nucleocapsid antibodies were included. Controls were fully vaccinated individuals without a breakthrough infection matched on sex, age, time between vaccine doses, and time between sample collection and most recent antigen exposure (PCR confirmation for those with breakthrough infection and final vaccine dose for controls). Full-length viral genomic sequencing was used to determine SARS-CoV-2 variant identity. Enzyme-linked immunosorbent assays were used to determine serum dilution titers with a 50% effective concentration (EC₅₀) of IgG, IgA, and IgM antibodies specific to the SARS-CoV-2 spike receptor–binding domain. Live SARS-CoV-2 neutralizing serum dilution titers were determined by 50% focus reduction neutralization tests (FRNT₅₀) against isolates of the original SARS-CoV-2 strain (WA1) and variants of concern (Alpha, Beta, Gamma, and Delta). Median breakthrough and control serum values were calculated in GraphPad Prism and compared with the Wilcoxon matched-pairs signed rank test with the Holm-Šídák correction. Delta-neutralizing potency was determined by comparing Delta- and WA1-neutralizing titers for sequence-confirmed Delta variant breakthrough cases, non-Delta breakthrough cases, and controls using the Kruskal-Wallis test with Dunn correction. Statistical significance was defined as a 2-tailed P < .05. Additional laboratory methods are provided in the Supplement. The Oregon Health & Science University institutional review board approved this study. Written informed consent was obtained.

Twenty-six participants with breakthrough infections (mean age, 38 years; 20 [77%] women; 24 [92%] were vaccinated with BNT162b2, sampled a median 28 days after PCR date and 213.5 days after final vaccination; 21 [81%] with mild symptoms) were matched to 26 controls (mean age, 39 years; 21 [81%] women; 26 [100%] were vaccinated with BNT162b2, sampled a median 28 days after final vaccination). Total receptor-binding domain–specific immunoglobulin increased in participants with breakthrough infection with a median EC₅₀ of 2152 (95% CI, 961-3596) compared with 668 (95% CI, 473-892) in controls (322% increase; P < .001) (Figure 1A). Median serum dilutions increased for both IgG and IgA. For example, the median IgA EC₅₀ after breakthrough infection was 120 (95% CI, 44-246), compared with 24 (95% CI, 24-24) for controls (502% increase; P < .001). IgM levels were not significantly different between groups (Figure 1B).

Among sequence-confirmed breakthrough cases, 10 were Delta and 9 were non-Delta infections. Among breakthrough cases, the median FRNT₅₀ against WA1 was 4646 (95% CI, 2283-7053) vs 489 (95% CI, 272-822) for controls (950% increase; P < .001). FRNT₅₀ results for Alpha, Beta, and Gamma variants are shown in Figure 2A. In breakthrough cases, median FRNT₅₀ against the Delta variant was 2482 (95% CI, 1072-4923), compared with 243 (95% CI, 118-336) for controls (1021% increase; P < .001) (Figure 2A). Sera from Delta breakthrough cases showed improved potency against the Delta variant at 99% (95% CI, 73-151) of WA1 neutralization for each participant, compared with 36% (95% CI, 33-52) for non-Delta cases and 41% (95% CI, 24-56) for controls (Figure 2B).

Results of this study showed substantial boosting of humoral immunity after breakthrough infection, despite predominantly mild disease. Boosting was most notable for IgA, possibly due to the differences in route of exposure between vaccination and natural infection. In addition, breakthrough sera demonstrated improved variant cross-neutralization, and Delta breakthrough infections in particular exhibited improved potency against Delta vs WA1, suggesting that the protective immune response may be broadened through development of variant boosters with antigenic inserts matching the emerging SARS-CoV-2 variants. Limitations of this study include the small number of samples and the difference in time from initial vaccination to serum collection between the breakthrough and control groups, which emerging evidence suggests may contribute to the development of variant cross-neutralizing antibody responses.⁶

Corresponding Author: Fikadu G. Tafesse, PhD, Department of Molecular Microbiology & Immunology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239 (tafesse@ohsu.edu).

Accepted for Publication: December 3, 2021.

Published Online: December 16, 2021. doi:10.1001/jama.2021.22898

Author Contributions: Dr Tafesse had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Bates, McBride, Winders, Curlin, Tafesse.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Bates.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Bates, McBride, Schoen.

Obtained funding: Curlin, Tafesse.

Administrative, technical, or material support: Bates, McBride, Schoen, Curlin, Tafesse.

Supervision: Curlin, Tafesse.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was funded in part by an unrestricted grant from the M. J. Murdock Charitable Trust, an unrestricted grant from the Oregon Health & Science University (OHSU) Foundation, National Institutes of Health training grant T32HL083808 on Multidisciplinary Research Training in Pulmonary Medicine, and OHSU Innovative IDEA grant 1018784.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We acknowledge the efforts of the OHSU COVID-19 serology research team for their assistance with sample acquisition and data collection. We also want to thank the Oregon SARS-CoV-2 Genome Sequencing Center at OHSU for their help in sequencing our samples, including Xuan Qin, PhD, Brian O’Roak, PhD, Andrew Adey, PhD, and Benjamin Bimber, PhD. None of the above individuals were compensated for their contributions. We are grateful for the Oregon Health & Science University faculty, staff, and patients who contributed to this study.