The SARS-CoV-2 pandemic has been marked by significant heterogeneity in immune responses, further complicated by the emergence of new variants like Omicron. While vaccination has been effective in reducing severe disease, breakthrough infections and reinfections remain a concern, particularly among vaccinated individuals. This underscores the need to understand how prior infections and vaccination influence subsequent immune responses. This study focuses on elucidating the interplay between prior immunity (from natural infection or vaccination) and the immune response to a booster vaccine dose, specifically considering the risk of Omicron breakthrough infections and the phenomenon of immune imprinting (original antigenic sin). The research investigates both humoral (antibody) and cellular (T-cell) immune responses, aiming to identify key factors contributing to varying susceptibility to reinfection and future Omicron infections. Understanding these factors is crucial for optimizing vaccination strategies and developing effective public health interventions.

Previous research has highlighted the protective effects of vaccination against severe COVID-19 but also revealed its limitations in preventing transmission. The high vaccination rates, coupled with the emergence of new variants, have led to a rise in breakthrough infections and reinfections. Studies have indicated that individual immune responses to vaccination vary widely, influenced by factors such as age, sex, comorbidities, and prior infections. The role of hybrid immunity (infection plus vaccination) has been a subject of investigation, with some studies suggesting it enhances protection, particularly IgA responses, which are often weak after vaccination alone. The concept of immune imprinting, where prior exposure to a virus variant can bias the immune response to subsequent encounters, has also been explored in the context of SARS-CoV-2, potentially impacting both B- and T-cell responses. However, the specific mechanisms and extent to which prior infection and vaccination influence susceptibility to breakthrough infection, especially with Omicron, remained largely unclear, necessitating this study.

This longitudinal study involved 1325 healthcare professionals, 955 of whom were infection-naive at the start of the study. All participants received a third dose of the BNT162b2 vaccine. Immune responses were measured before and after the booster dose at multiple time points (baseline, 21 days, 2 months, 6 months, and 12 months after the first dose). Humoral responses were assessed by measuring IgG, IgA, and neutralizing antibody levels against the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein using ELISA-based assays. Cellular responses were evaluated by measuring interferon-gamma (IFN-γ) release from T-cells stimulated with S1 peptides from the ancestral strain. Participants were categorized into groups based on their infection status: infection-naive, infected before Omicron, and infected with Omicron. Statistical analyses, including generalized linear mixed models (GLMMs) with natural cubic splines, were used to model the dynamics of immune responses over time. Subgroup analyses investigated the association between immune responses and reinfection risk, as well as the impact of immune imprinting. Multiple linear regression analyses were also performed to assess the associations between immune responses and relevant factors such as age, sex, and infection status. The study design, sample collection procedures, and assay details are thoroughly described in the supplementary materials.

The study revealed significant differences in IgG, IgA, and neutralizing antibody levels after the booster dose, influenced by previous infection status. Prior SARS-CoV-2 infection significantly increased the post-vaccination humoral immune response compared to infection-naive individuals, particularly IgA levels. Individuals with lower IgG, IgA, and neutralizing antibody levels after vaccination showed a significantly higher risk of reinfection and future Omicron infections. This association was not observed for T-cell responses (IFN-γ levels). Individuals with a prior infection before Omicron, followed by Omicron reinfection, exhibited a dampened humoral and cellular response compared to those with primary Omicron infection, confirming immune imprinting. The study also observed sex-based differences in IgG response, particularly in individuals infected before Omicron, with females exhibiting higher IgG levels than males after the boost. Age also influenced IgG levels, with older individuals showing higher IgG levels after the boost compared to younger individuals. Neutralizing antibody levels were enhanced after the booster dose, with individuals with hybrid immunity (infection plus vaccination) having the highest levels. Regarding T-cell responses, IFN-γ levels were significantly higher after the booster in infection-naive and Omicron-infected individuals, but not in those infected with earlier variants. Significant correlations were found between IgG and IFN-γ levels, indicating a potential interplay between humoral and cellular responses.

This study's findings underscore the importance of prior immunity in shaping immune responses to SARS-CoV-2 vaccination. The strong association between lower humoral responses (IgG, IgA, and neutralizing antibodies) and increased risk of breakthrough infections emphasizes the critical role of antibodies in protection against Omicron. The lack of similar association for T-cell responses suggests that while cellular immunity contributes to protection against severe disease, humoral immunity is more important for preventing infection and transmission. The significant role of IgA, particularly enhanced by previous infection, highlights the need for novel vaccine designs aiming to improve mucosal immunity. The observation of immune imprinting reinforces the need for careful consideration of previous infections when designing vaccination strategies and emphasizes the potential for waning immunity over time. The findings have implications for optimizing future vaccine strategies by considering previous infection history, including the type and timing of infection, as well as the need to better understand and enhance IgA responses.

This large-scale study demonstrates the significant impact of previous immunity on immune responses to SARS-CoV-2 booster vaccination and Omicron breakthrough infection risk. Prior infection enhances humoral and cellular responses, particularly IgA, but reinfection with Omicron can dampen these responses. Low humoral immunity is associated with increased infection risk, while cellular immunity may play a less significant role in preventing transmission. These findings highlight the need for vaccines that elicit robust humoral responses, particularly IgA, and emphasize the complex interplay between prior immunity, vaccination, and the risk of future infections. Future research could investigate variant-specific immune responses, the long-term durability of immunity, and the role of other immune factors in protection.

The study's reliance on ancestral strain antigens might have underestimated responses to Omicron. The predominantly female study population might affect generalizability. Changes in testing strategies in Denmark could have impacted the precise count of future infections. Lack of data on comorbidities and medication use could have introduced confounders. While the GLMM analysis was robust, limited data at certain time points introduced some uncertainty into predictions. Despite these limitations, the large sample size and use of GLMMs provide robust insights into the dynamics of immune responses.