Vaccines | Free Full-Text | Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure
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1. Introduction
Four years have passed since the World Health Organization (WHO) declared the severe acute respiratory coronavirus-2 (SARS-CoV-2) outbreak a Public Health Emergency of International Concern on 31 January 2020 and a global pandemic on 11 March 2020. Over the course of the pandemic, various vaccine platforms have been introduced, playing a crucial role in preventing infection [1,2]. However, vaccine-induced immune responses have waned rapidly, and the emergence of immune-evasive variants has contributed to the increasing number of breakthrough infections [3,4,5]. In particular, vaccines provided limited protection against the currently circulating Omicron variant. In response to this variant, a third booster dose was administered, leading to a significant increase in neutralizing capacity. However, the neutralizing antibody titers also showed a rapid decline within three months after the third dose, particularly against sublines BA.2.12.1 and BA.4/5 [6]. Consequently, each individual’s current immune profile is shaped by both vaccine-induced and infection-induced immune responses.
Previous studies have suggested that repeated exposure to the viral antigen, whether through vaccination, infection, or both, leads to the development of high-avidity cross-neutralizing activity [7,8,9]. The synergistic impact of vaccination and infection induces a particularly potent effect known as hybrid immunity. This hybrid immunity demonstrates enhanced effectiveness compared to immunity solely induced by vaccination, resulting in stronger neutralization and better adaptive immunity [5,10]. Regarding the cellular immune response, it appears to be more enduring and consistent, as opposed to the rapid rise and fall observed in the antibody response [11].
However, there remains a gap in our understanding of current immune profiles following diverse vaccination and breakthrough infection histories. In addition, there is heterogeneity amongst vaccinated individuals influenced by factors such as the total number of antigen exposures from either infection or vaccination, vaccine strategies and the specific variant responsible for the infection.
Previous longitudinal studies of SARS-CoV-2–specific immune responses have covered a period of up to one year after the completion of the primary two-dose series [5,11,12,13,14,15,16] or up to 20 months after infection [17]. However, the specific characteristics of long-term immune response metrics in infection-naive individuals remain largely unexplored. Therefore, the need for additional vaccination and the prioritization of recipients requires further study and consideration.
This study is part of a longitudinal investigation involving naïve participants who initially received two doses of vaccines and subsequently either received booster vaccinations, recovered from Omicron infections, or experienced both. We performed a comprehensive analysis of the humoral and cellular immune response to both wild-type (WT) and the Omicron subvariants (BA.1., BA.2., and BA.4/5) two years after the primary series vaccination. This study aims to analyze the immune profiles associated with SARS-CoV-2–specific adaptive immune responses in vaccinated individuals. We investigated the impact of diverse antigen exposure histories, including variations in vaccine doses, types, and Omicron exposure through breakthrough infections or bivalent vaccinations.
2. Materials and Methods
2.1. Enrolment and Sample Collection
In March 2021, we enrolled a total of 359 infection-naive healthcare workers at Seoul St. Mary’s Hospital and initiated the longitudinal monitoring of SARS-CoV-2–specific immune responses after a primary series of two-dose vaccination. All participants began their initial SARS-CoV-2 vaccination in March 2021 and completed the primary two-dose series. They received either mRNA vaccine BNT162b2 (Pfizer-BioNTech, hereafter referred to as BNT) or mRNA-1273 (Moderna) or were given the vector-based ChAdOx1 nCoV-19 (AstraZeneca, hereafter referred to as ChAd). Subsequently, in June 2023, 78 individuals from the original cohort consented to participate in this follow-up study.
In this study, we assessed the long-term SARS-CoV-2–specific immune responses following booster vaccinations and/or Omicron breakthrough infection at 25 months (T4) after completing the primary two-dose vaccination series. This study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (KC23TISI0183). All participants provided written informed consent. Additionally, they granted permission for the use of their immunogenicity test results obtained at 1 month (T1), 5 months (T2) and 11 months (T3) after completing the primary two-dose vaccination series from previous studies (KC21DIST0174). Participants were asked to complete a medical history questionnaire that included information on demographics, underlying medical conditions, SARS-CoV-2 vaccination history, and any prior experiences with breakthrough infections. Breakthrough infection was confimred based on the available reverse transcription polymerase chain reaction (RT-PCR) on the electronic medical record and anti-nucleocapsid (N) antibody positivity. Infection-naive was defined based on negative survey and negative anti-N test results.
2.2. SARS-CoV-2–Specific Humoral Immune Response Assays
Serum samples from participants were analyzed using the Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics, Basel, Switzerland) to detect binding antibodies to the receptor-binding domain (RBD) of the spike protein (anti-S/RBD) and anti-N, as per the manufacturer’s instructions. Quantification of the anti-S/RBD antibody was performed in units per mL (U/mL), and binding antibody units per mL (BAU/mL) were calculated based on the WHO International Standard for anti-SARS-CoV-2 immunoglobulin. A cut-off value of 0.8 U/mL for anti-S/RBD was utilized, as recommended by the manufacturer. The result for anti-N is presented as a cut-off index (COI), where COI > 1.0 indicates a reactive result. The reactive anti-N result was used to detect asymptomatic breakthrough infections that might have gone unreported by participants.
The SARS-CoV-2 surrogate virus neutralization test (sVNT) (GenScript cPassTM, Piscataway, NJ, USA) was conducted following previously described procedures to evaluate the binding inhibition value of neutralizing antibodies [18]. Serum samples from participants, along with positive and negative controls, were diluted 1:10 with dilution buffer. They were then mixed with horseradish peroxidase–conjugated recombinant SARS-CoV-2 RBD solution provided in the abovementioned GenScript test and incubated at 37 °C for 30 min. Following this, the mixtures were incubated for 15 min at 37 °C in a capture plate precoated with human angiotensin-converting enzyme 2 (hACE2) protein in the GenScript kit. After washing, tetramethylbenzidine (TMB) solution in the sVNT assay kit was added, and the plate was incubated in darkness at room temperature for 15 min. The reaction was halted with stop solution, and absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader. Following the manufacturer’s instructions, a cut-off of ≥30% inhibition was considered indicative of positive neutralizing activity. Each sample was tested for SARS-CoV-2 WT, Omicron BA.2 (BA.2), and Omicron BA.4 and BA.5 (BA.4/5) spike proteins.
2.3. SARS-CoV-2–Specific Cellular Immune Response Assays
The cellular immune response was assessed utilizing the enzyme-linked immunospot (ELISpot) assay. Peripheral blood mononuclear cells (PBMCs) were stimulated with PepTivator SARS-CoV-2 S1 peptide pools of WT and Omicron subvariants BA.1, BA.2, and BA.5 (Miltenyi Biotec, Bergisch Gladbach, Germany). These peptide pools consisted of 15-mer sequences with an 11 amino acid (aa) overlap, spanning aa 1-692. The S1 domain includes the RBD and N-terminal domain, well-known targets of neutralizing antibodies [9]. The 96-well plates were coated overnight at 4 °C with anti-interferon (IFN)-γ monoclonal capture antibodies sourced from Human IFN-γ ELISpot kits (BD Biosciences, San Jose, CA, USA). After adding blocking solution, 2.5 × 105 cells/mL per well were stimulated with antigens. Plates were maintained overnight at 36 °C in a CO2 incubator. After multiple washes, AEC substrate was added for 25 min and kept in dark lighting at room temperature. The IFN-γ spot forming cells were enumerated using the AID ELISpot reader system (Autoimmun Diagnostika GmbH, Strasburg, Germany). Results were quantified as IFN-γ spot forming units (SFU)/2.5 × 105 PBMCs.
2.4. Statistical Analysis
Continuous data are presented as median with interquartile ranges (IQR). Paired samples were compared by the Wilcoxon sign-rank test. Statistical comparisons between immune measures among different groups were conducted using the Mann–Whitney U test and the Kruskal–Wallis test, with Dunn’s multiple comparisons test employed post hoc as necessary, depending on the number of comparisons required. Categorical data were presented as counts and percentages and were analyzed using either the chi-square or Fisher’s exact test. The association between test results was evaluated using the Spearman rank correlation test. Data analysis and visualization were carried out using Prism version 10.0.2 for Windows (GraphPad, San Diego, CA, USA) and MedCalc statistical software version 20.114 (MedCalc Software Ltd., Ostend, Belgium). A significance threshold of p < 0.05 (two-tailed) was applied for all statistical analyses.
4. Discussion
Individual immune profiles are shaped by virus-specific vaccination and breakthrough infection. Understanding the current immune response is crucial for making future plans for controlling COVID-19. Given the dynamic nature of adaptive immune responses, the updated immune profiles reflecting various immunity-conferring events are required. This study offers a longitudinal monitoring spanning over two years after the completion of the primary two-dose vaccination. We evaluated present immune responses across groups with diverse immunogenic histories and identified individuals with diminished immune responses that may require prioritization in future vaccine plans.
Previous research has suggested that repeated antigenic stimuli contributes significantly to robust and broader humoral responses [7,8,10,13,15] in comparison to the declining humoral immune response observed in individuals who completed only two doses of BNT [14]. We also observed a robust humoral immune response following the third dose, consistent with the findings at T3 and T4. This contrasts with the rapid waning of binding antibody titers and neutralizing inhibition observed from T1 to T2 between median 26 to 161 days after the second dose administration. This finding supports previous observations that the third dose elicited the most potent humoral immune response [8,9,24,25]. Notably, an enhanced humoral response was evident in those exposed to Omicron. The stronger inhibition observed against the WT spike of SARS-CoV-2 compared to BA.2 and BA.4/5 among individuals exposed to Omicron can be attributed to the phenomenon of back-boosting associated with immune imprinting [26]. Contradictory findings exist regarding the impact of immune imprinting on SARS-CoV-2 vaccine effectiveness [27,28]. A recent study, however, provided an encouraging result suggesting that the BNT162b2 bivalent BA.4/5 vaccine elicited a broader neutralization activity, extending to currently circulating more immune-evasive Omicron sublineages such as BA.2.75.2, BA.4.6, BQ.1.1, and XBB.1 [29]. Similarly, the bivalent Omicron BA.1-containing mRNA-1273.214 vaccine (Moderna) was reported to induce broad neutralizing capacity against the alpha, beta, gamma, delta, and Omicron variants [30].
When specifically focusing on neutralization, individuals exposed to the Omicron exhibited an expanded breadth of response compared to the five infection-naïve participants in this study, whose SARS-CoV-2–specific immune response has been only induced by vaccination based on ancestral spikes. This finding might seem contradictory to a previous study [8] that reported three doses targeting the ancestral spike resulted in broader neutralizing activity to cover SARS-CoV-2 variants. Their evaluation was based on samples collected 48 days after the third dose, representing an early response. As our samples were collected a median of 744 days after second dose and 575 days after third dose vaccination, our results raise the question of whether cross-neutralization from three or more ancestral spike-targeting doses is less enduring than Omicron spike exposure. It is noteworthy that infection-naïve individuals vaccinated with bivalent BNT exhibited neutralizing activity comparable to their counterparts in the exposure number matched group.
Compared to the humoral immune response, the cellular immune response remained stable in response to vaccination and breakthrough infection in line with previous research [5,13,16,29]. However, our finding also revealed substantial increases in the magnitude of T-cell response after third exposure. This observation may indicate that the third antigenic stimuli have a substantial impact on the cellular immune response as well, suggesting that its role extends beyond influencing the duration and scope of the humoral immune response. An interesting finding was that the T cell magnitude induced by Omicron peptide pools was higher in infection-naïve individuals non-exposed to the Omicron. Initially the vaccine-induced immunity exhibits varying degrees of correlation across various immune metrics. Even infection-naïve individuals who were never exposed to the Omicron spike exhibited a degree of cross-reactive cellular immune response. Taken together, the augmented cross-reactive cellular immunity could potentially contribute to protect them against breakthrough infections. This finding is consistent with previous reports suggesting that durable and extensive cross-reactive cellular immunity may contribute to protection against infection [9,31].
In this context, it is noteworthy that an enhanced cellular response was induced in individuals who followed a heterologous vaccine schedule, comprising vector-based vaccines primed and boosted by a mRNA-based vaccine. Notably, all nine participants who remained non-infected during the observation period in this study had followed a heterologous vaccination schedule. One intriguing hypothesis is that this group may have benefited from a dual advantage: a stronger cellular response conferred by the vector-based vaccines and more robust humoral responses induced by the mRNA vaccines. However, since the heterologous vaccines did not yield statistically significantly higher rates of protection in this study, further investigation is required to confirm this hypothesis.
After breakthrough infection, a dissociation emerges between humoral immunity parameters and cellular compartment. Each component demonstrated a moderate to strong correlation within the compartment, yet a negative correlation emerges between humoral and cellular immune measures. The better correlated immune response among breakthrough infectees was previously described [5]. The inverse correlation can be explained by the hypothesis that individuals with a higher magnitude of T-cell adaptive immune response were protected from breakthrough infection, while those with lower responses experience breakthrough infections. Then, the infection itself prompted a rapid humoral response against the virus. Given the current context of widespread vaccination and breakthrough infections, depending solely on humoral immunogenicity measures as indicators of protection might not provide a comprehensive perspective for assessing vaccine effectiveness.
Our study has several limitations. As an observational study, we were unable to capture all possible combinations of the number of exposed antigens and the specific exposed variant, while the limited number of participants may affect the statistical power and reduce generalizability. Another limitation of our study is that we did not identify the causative variants in individuals with breakthrough infections through sequence analyses. However, the timing of the infection provides an indication of the likely circulating variant.
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