Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections

Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections Heiko Pfister, Carsten Uhlig, Zsuzsanna Mayer, Eleni Polatoglou, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5678273/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The objective of this study was to investigate the features of immune protection against SARS-CoV-2 infection in a single cohort during the 6–17 months following booster immunization with an mRNA-based vaccine. The results illustrate the influence of humoral and cellular immunity on the efficacy of the vaccine. Notably, neutralizing antibody titers were found to serve as a reasonably reliable correlate of protection prior to booster immunization. However, this predictive power was largely lost after boosting. The loss appears to be due to the critical remodeling of the humoral immune response following booster immunization. Our findings support the hypothesis that immunity to both conserved and non-conserved epitopes of the viral Spike protein's receptor-binding domain (RBD) is crucial for optimal long-term protection against Omicron infection. While immunity to conserved epitopes may provide cross-variant protection, antibodies targeting non-conserved RBD epitopes play a pivotal role in achieving maximum protection. These observations highlight the critical role of repeated immunization in shaping the immune response landscape and reinforce the necessity of considering both humoral and cellular immune components, alongside intended use considerations, when assessing vaccine efficacy and developing future immunization strategies. Health sciences/Diseases/Infectious diseases/Viral infection Health sciences/Biomarkers/Predictive markers Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies Health sciences/Medical research/Outcomes research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coronavirus disease 2019 (COVID-19) has posed a major challenge to health systems worldwide. While vaccines derived from the original strain (Wuhan-Hu-1; “wild-type”) appeared to be effective against the original strain and earlier variants, the Omicron variant displays a remarkable ability to evade neutralizing antibodies (NAbs) 1 , 2 . Repeated homologous vaccinations primarily strengthen the immune response against the vaccine strain, potentially rendering the immune system less effective against variant infections 3 . The increase in breadth and potency of the humoral immune response after booster vaccination depends largely on the diversification of the memory B cell pool induced by the second vaccination 4 . Epitope masking and higher antigen availability after booster vaccination may promote off-target B cells that contribute to a broad NAb repertoire against related pathogens 5 . Despite numerous mutations, particularly those acquired by Omicron, conserved epitopes on the Spike (S) protein and within the receptor-binding domain (RBD) have been identified as promising targets for vaccination strategies effective against multiple variants 6 , 7 . While immune escape variants are able to evade the humoral immune response to varying degrees, depending on the number and position of the mutations, CD8 + T cell epitopes remain largely conserved among variants 8 , 9 . However, disease control appears to be granted by broad epitope targeting of CD4 + T cells, thereby limiting the evolution of immune evasion mutants 10 . In a study of 107 healthcare workers (HCWs), we found that the humoral immune response directed against the RBD underwent a notable rearrangement of the epitope repertoire following booster immunization. The data revealed a correlation between an elevated proportion of antibodies directed against non-conserved epitopes within the RBD and a lower incidence of infections during the 6–17 months following booster vaccination. Conversely, stimulation of T cells with specific Spike peptide pools in whole blood revealed a dominant role of conserved-epitope-specific cellular immunity in long-term protection. Results The present study was conducted with selected samples from a prospective, observational, single-center cohort study initiated in December 2020, comprising 640 healthcare workers who received two doses of the wild-type mRNA vaccine BNT162b2 between January and April 2021 and one booster dose between October 2021 and January 2022 (Suppl Fig. 1). Exclusion criteria included individuals who had received Vaxzevria (AstraZeneca, UK) due to differences in vaccination schedules. Additionally, participants had to demonstrate a lack of documented SARS-CoV-2 infection and negative results from nucleocapsid-antibody assays over the specified periods for each respective experiment. During the observation period, which commenced six months after booster vaccination in April 2022, it was reported that more than 90% of infections in Germany were attributed to the BA.1, BA.2, BA.4, and BA.5 variants through the end of 2022 11 . In early 2023, recombinant variants, most notably XBB.1, began to replace prevalent Omicron subvariants, reaching a relative prevalence of approximately 50% by the study’s conclusion in February. In the initial observation phase, the weekly incidence was reported as 1,543 cases per 100,000 citizens, dropping to a low of 75 cases per 100,000 citizens near the end of the observation phase, with intermittent spring and autumn waves peaking at 752 and 944 cases per 100,000 respectively. The median value throughout the observation period was 329 per 100,000 citizens 11 . Booster immunization does not increase long-term serum NAb activity against Omicron RBD NAb activity against multiple variants and subvariants was assessed 6 months after the second vaccination dose (TP1) and 6 months after booster immunization (TP2) by multiplex RBD-ACE2 inhibition electrochemiluminescence immunoassay (ECLIA, MSD Mesoscale). The ECLIA assays have been extensively validated and are frequently used in COVID-19 research 12 , 13 , 14 , 15 . Wild-type and all variants of concern (VOCs) relevant during the study course were included. The median inhibition ranged from 19.7% (Omicron B.1.1.529; BA.1; BA.1.15) to 31.3% (wild-type) in TP1 and from 14.0% (Omicron B.1.1.529; BA.1; BA.1.15) to 52.1% (wild-type) in TP2 (Fig. 1a, b). Thus, booster immunization induced a marked relative median increase of individual sustained NAb levels against wild-type, Alpha (B.1.1.7), Beta (B.1.351/B.1.351.1), and Delta (variants) by 53.9% (median absolute difference with 95% CI: 19.24 [12.53, 24.47]), 50.8% (12.84 [10.33, 19.61]), 46.5% (13.56 [8.631, 17.87]), and 47.8% (12.56 [10.12, 18.12]), respectively (Fig. 1c). In contrast, Omicron-specific median individual NAb levels dropped by 26.8% (B.1.1.529, BA.1, BA.1.15; median absolute difference with 95% CI: -4.571 [-8.813, -2,613]), 25.5% (BA.2 subvariants; -5.882 [-8.293, -2.771]), 0.2% (BA.2.12.1; 0.03099 [-3.202, 3.184]), 35.9% (BA.2.75; -6.985 [-10.29, -3.429]), and 6.9% (BA.4, BA.5; -1.721 [-4.666, 3.314]). TP1 levels are inversely correlated with the change in levels of all tested variants including wild-type (Fig. 1d). The correlation is strongest for the Omicron subvariants. NAb levels are not generally suitable as correlates of protection The inverse correlation between the NAb level change from TP1 to TP2 and NAb level in TP1 prompted the question of whether a reduction in NAb levels contradicts the reported initial improvement in protection against Omicron infection following booster vaccination, due to the loss of RBD-specific NAbs during affinity maturation 1 . Consequently, we correlated the data from the participants' questionnaires on confirmed cases of COVID-19 at the end of the study with the NAb levels at TP1 and TP2. 68 of 107 (63.6%) participants were infected in the median period of 3.6 months (quartile 1: 2.0 months, quartile 3: 5.3 months, max. 9.7 months) after TP2 from June 2022 until February 2023. While not all participants were informed of their variant, we estimate that approximately 90–95% of the infection symptoms were caused by BA.1, BA.2, BA.4, and BA.5 and their subvariants and less than 10% by Delta infections, since they were by far the dominating variants throughout the observation period in Germany 11 . Given that only three individuals were infected in 2023, it can be reasonably inferred that infections with recombinant XBB.1 subvariants represent single cases at most. 51 of 68 (75%) infections were confirmed by PCR, with 15 of 51 (29.4%) being typed as Omicron and two (3.9%) being typed as Delta infections. 12 (23.5%) PCRs were performed in-house, and 39 (76.5%) were performed in a certified test center. 17 (25%) infections were confirmed by antigen testing. Given that antibody titers wane over time, it was anticipated that they might not suffice to yield significant correlations with infections throughout the observation period. Accordingly, the study group was divided into two subgroups, characterized by low to medium (lower two-thirds of the scale) and high NAb levels (upper third of the scale) at TP2. In the subsequent experiment, the wild-type RBD NAb level was employed as the discriminator between subgroups. While overall NAb levels at TP2 do not appear to be associated with protection, TP1 levels do show some correlation, although not significantly in most cases (Fig. 2). Restricting the analysis to individuals with high wild-type NAb levels at TP2 (dots in the grey box in Fig. 2a and dark green circles / dark red dots in Fig. 2b and 2d representing individuals “gated through” the grey box in Fig. 2a) reveals a significant difference between those infected and uninfected at TP1 in wild-type (median infected (n = 22) / non-infected (n = 14) with 95% CI of difference: 32.66 / 44.87 [1.009, 24.27]), Alpha (29.74 / 40.19 [1.785, 20.73]), and Delta (23.75 / 36.86 [5.596, 22.30]). The substantial number of mutations in the RBD may render the Omicron test incapable of demonstrating significant distinctions between infected and uninfected individuals. Consequently, the foundation of successful booster vaccination against symptomatic variant infection may be laid by the second vaccination. However, successful booster vaccination evidently depends on an additional mechanism that cannot be resolved by solely determining serum-neutralizing capacity at TP2. Loss of RBD-specific IgG antibodies after booster vaccination is associated with infection The levels of wild-type RBD-specific IgG exhibited a marked increase in TP2, with only two infected and four uninfected individuals displaying a lower level in TP2 than in TP1. When the RBD and Spike IgG levels in the datasets were grouped alternatively by low to medium or high Spike antibody levels at TP2 (in analogy to the experiment above), uninfected individuals with high Spike TP2 levels displayed higher RBD antibody levels than infected individuals, a difference that was already apparent at TP1 (median infected (n = 46) / non-infected (n = 22) with 95% CI of difference: 0.01659 / 0.04056 [0.005196, 0.04000]) and, to a lesser extent, at TP2 (0.6903 / 0.8509 [-0.004536, 0.2051]; Fig. 3a, b). We concluded that the reduction in NAb activity within the RBD, evident in Omicron variants, must be attributed to a rearrangement of the epitope repertoire of Spike-specific antibodies from TP1 to TP2. In order to test this hypothesis, a comparative analysis was conducted between the wild-type RBD-specific and Spike-specific IgG values. A small but insignificant difference was observed between infected and non-infected individuals (Fig. 3c). In contrast, infected individuals displayed a median relative loss of RBD-specific antibodies between TP1 and TP2, overt at high Spike TP2 levels, while uninfected individuals remained stable (median infected / non-infected: 0.8720 / 1.016; 95% CI of difference [0.004328, 0.2402]; Fig. 3d). Our findings indicate that RBD-specific antibodies play a pivotal role in long-term humoral immune protection. NAbs against non-conserved RBD epitopes are associated with protection from variant infection The marked difference between wild-type and Omicron variants in NAb activity loss suggests an equilibrium shift between conserved and non-conserved epitopes. In order to gain a deeper insight into the role of RBD-specific NAbs in protecting against variant infection, we analyzed the relationship between variant- and wild-type-specific NAbs at TP2. It was anticipated that there would be an approximately linear correlation between the wild-type and the Alpha, Beta, and Delta variants, given that only a few mutations within the RBD can influence antibody binding 16 . It thus follows that the greatest degree of variation was to be expected for the Omicron subvariants, as they have by far the largest number of mutations within the RBD 17 . Based on a presumed dose-dependence of NAbs on the infection rate, samples with low to medium titers and high NAb titers were analyzed separately (analogous to the experiments described above). The Omicron scatterplots revealed a prominent bipolar distribution of infected and uninfected individuals in the group with high NAb titers (relative risk of infection with 95% CI with NAbs targeting less non-conserved epitopes of: BA.1.1.529, BA.1, BA.1.15: 2.667 [1.495, 5.535]; BA.2 subvariants and BA.4, BA.5: 2.143 [1.237, 4.201]; Fig. 4). It may therefore be concluded that maximum protection against variant infection is provided by high NAb titers with a substantial fraction of antibodies targeting non-conserved epitopes within the RBD. Cellular immunity to conserved epitopes is associated with protection from symptomatic variant infection To determine whether the humoral immune response aligns with the cellular immune response at TP2, blood cells were stimulated with peptide pools derived from wild-type and variant Spike protein in whole blood. In light of the logistical constraints and the limited availability of personnel during the ongoing COVID-19 pandemic, we proceeded to select the initial five to ten samples obtained from the daily blood draws for the planned analyses. T cell stimulation was analyzed by quantifying the mRNA expression of interferon-γ (IFN-γ) and the chemokine C-X-C motif chemokine ligand 10 (CXCL-10) by RT-qPCR. CXCL-10 has recently been introduced as a suitable proxy for quantifying cellular immunity against SARS-CoV-2, given its strong correlation with the activation of antigen-specific T cells 18 . The median level of CXCL-10 and IFN-γ mRNA expression in infected individuals was distinctly, but not statistically significantly, lower than that observed in non-infected individuals (Fig. 5a). These findings suggest that an increased number or reactivity of T cells may confer protection. Additionally, a significant association between infection and a balance shifted towards non-conserved epitopes was revealed upon relating wild-type Spike peptide pools spanning exclusively mutated sites in SARS-CoV-2 variants to whole Spike (wild-type) peptide pools (median infected (n = 8) / non-infected (n = 6) with 95% CI of difference: Alpha mutation sites (CXCL-10 // IFN-γ): 0.6035 / 0.1911 [-0.7671, -0.1917] // 0.7165 / 0.3021 [-1.163, -0.05047]; BA.1 mutation sites (CXCL-10 // IFN-γ): 1.559 / 0.4068 [-1.531, -0.5060] // 1.142 / 0.3605 [-1.463, -0.2888]; Fig. 5b). Our findings indicate that, in contrast to the humoral immune response, maintaining the balance closer to conserved-epitope-specific T cells provides superior protection from symptomatic variant infection. It is currently unclear whether our findings regarding cellular and humoral immune protection are directly correlated. Our data depict infected and uninfected individuals with varying peptide pool stimulation ratios exhibiting ratios of wild-type to variant NAb that are both above and below the median line. Of note, individuals infected prior to TP2 exhibited a pronounced tendency towards conserved epitopes in comparison to those infected subsequent to TP2 (median infected (n = 8) / previously infected (n = 11) with 95% CI of difference: CXCL-10: 1.559 / 0.9381 [-1.220, 0.01439]; IFN-γ: 1.142 / 0.5326 [-1.110, -0.08499]). This observation may be indicative of augmented protection against variant infection resulting from additional variant antigen exposure 19 . The use of peptide pools exclusively covering variant mutation sites and a corresponding wild-type reference pool demonstrated that there was no discernible difference in cross-reactivity with peptide variants between infected and uninfected individuals (Fig. 5c). In order to spatially narrow down the observed disparity between infected and uninfected individuals on the Spike protein, we conducted a separate comparison between peptide pools representing the N-terminal and C-terminal halves of all relevant Spike protein variants with the corresponding wild-type peptide pools (Fig. 5d). To map the effect of mutations on T cell stimulation as comprehensively as possible, the analysis was expanded to include Gamma variants. However, it can be reasonably assumed that the Gamma variant did not play a significant role in the incidence of infection during the course of this study 11 . Upon stimulation with the N-terminal peptide pool-1, the median cytokine ratio of variant to wild-type exceeded the value of 1 in five of six tested (sub)variants in infected individuals (median infected / non-infected with 95% CI of difference: Alpha pool-1 CXCL-10: 1.218 / 0.8041 [-1.520, -0.04511]; BA.1 pool-1 (CXCL-10 // IFN-γ): 1.443 / 0.6259 [-1.420, -0.03581] // 1.430 / 0.3510 [-1.503, -0.1223]; Fig. 5d). In contrast, only two CXCL-10 ratios of the uninfected were above 1, and none of the IFN-γ ratios. The consistent trend towards higher variant to wild-type ratios in infected individuals as compared to the non-infected localizes the source of the observed trend at least in part to the RBD-containing N-terminal half of the Spike protein. The results for peptide pool-2, which spans the C-terminal half of the Spike protein, were inconsistent. No significant difference was found for IFN-γ between infected and uninfected individuals. However, Alpha and BA.1 differed significantly in CXCL-10 values (median infected / non-infected with 95% CI of difference: Alpha pool-1: 1.323 / 0.6588 [-1.500, -0.02893]; BA.1 pool-1: 1.145 / 0.4689 [-1.277, -0.02541]), which may be due to the significant role of conserved sites on the S2 subunit and those around the S1/S2 cleavage site in protection against symptomatic infection 16 , 20 . These findings suggest that the protective effect of additional SARS-CoV-2 antigen exposure may be attributed to conserved T cell epitopes, including those in the N-terminal half of the Spike protein—specifically the RBD and N-terminal domain (NTD). A notable difference was observed in the infected groups before and after TP2, further supporting our conclusions about infection-driven T cell reactivity bias towards conserved epitopes. Cells from individuals infected with Omicron before TP2 showed cytokine ratios below 1 when stimulated with BA.1 peptides (similar to the non-infected group). In contrast, those infected after TP2 displayed ratios above 1, particularly evident with pool-1 peptides. These findings suggest that the protective effect of additional SARS-CoV-2 antigen exposure may be attributed to conserved T cell epitopes, including those in the N-terminal half of the Spike protein - specifically the RBD and N-terminal domain (NTD). This perspective is corroborated by the discovery of elevated genetic entropy levels within the T cell epitopes of the RBD and NTD, indicating viral immune evasion 20 . Discussion The majority of the immunodominant epitopes of neutralizing antibodies against SARS-CoV-2 have undergone significant mutation in the Omicron variant, which contributes to immune evasion 21 . Immune escape can be at least partially compensated for by booster immunization 22 . This gave rise to the question of whether the booster immunization merely contributes to an increase in antibody levels or whether it provides extended protection through immune adaptation processes. As demonstrated in our study, NAb levels are not an appropriate correlate of long-term protection after booster immunization against SARS-CoV-2 variant infection. Consequently, we sought to establish whether the augmented level of protection could be attributed to an expansion of adaptive processes that refine the repertoire of humoral and cellular immunity. Therefore, a six-month interval was permitted after the second and third vaccination before laboratory parameters were determined. On the supposition of a consistent reduction in antibody levels reported across a range of initial peak titers, our data substantiate the following interpretation of long-term immunity to Omicron variants induced by mRNA vaccination 23 : Following the second vaccination, RBD-specific antibodies are predominantly directed towards immunodominant epitopes. The immunologic pressure (especially on immunodominant epitopes) exerted by the vaccination program and the prevalence of wild-type infections in the population drives the evolution of the virus towards the development of immune escape variants. As a consequence, Omicron RBD NAb levels are lower compared to wild-type due to the reduced number of native immunodominant epitopes. The measured differences between the Omicron subvariants and the wild-type, as well as with all other variants tested, may be attributed to the number of mutations present within the RBD (Alpha: 1, Beta: 3, Delta: 2, Omicron: 15) rather than the mutations themselves 17 . Booster immunization does apparently not provide a sufficient trigger for the expansion of immunodominant epitope-specific B cells in all individuals, resulting in inadequate compensation for the decline in functional capacity of existing plasma cells over time (Fig. 1). This may, at least in part, be attributed to affinity maturation processes which result in antibodies with reduced affinity to mutated epitopes. As this outcome is dose-dependent, it could be caused by antibody-dependent immunoregulatory mechanisms such as antibody-mediated epitope masking. In numerous cases, particularly evident with the Omicron variant, this results in TP2 RBD NAb levels below the TP1 values. It indicates that newly formed post-booster antibodies directed at previously subdominant epitopes are also unable to compensate for the loss. It can be concluded that previously immunodominant, non-conserved epitopes make a significant contribution to the post-booster epitope spectrum in subjects with high TP1-NAb levels. The reduced median NAb levels against Omicron variant at TP2 initially seem to be at odds with the epidemiological data, which indicate an enhanced vaccine efficacy against Omicron infection following repeated immunizations 24 . However, booster vaccination may enhance cross-variant protection by elevated levels of previously subdominant epitope-specific antibodies 5 . These epitopes are less likely to be mutated in Omicron variants due to lower immunologic pressure prior to booster campaigns. Given that RBD-specific antibodies targeting non-conserved epitopes appear to be associated with protection, it seems plausible that a broad immune response, including antibodies with lowered affinity for mutated sites and antibodies directed against conserved epitopes, may contribute to maximum protection (Fig. 4). A less broad humoral immune response, which covers key epitopes that affect interactions between the virus and host cells including mutated sites such as T478K, Q493K, Q498R, and E484A, is also conceivable 25 . It is even possible that non-conserved epitopes generally exert a subordinate role in humoral immune protection after repeated vaccinations. This may be supported by the observation that the non-infected subjects in Fig. 4 exhibit elevated NAb levels at TP1 in comparison to the infected subjects (median wild-type neutralizing activity of 45% in non-infected subjects vs. 32% in infected subjects in the group with high TP2 NAb levels). Following the view that the generation of antibodies against subdominant, conserved epitopes is influenced by antibodies directed against immunodominant, non-conserved epitopes from previous immunizations, this positive correlation may lead to an underestimation of the significance of antibodies specific for conserved epitopes. Furthermore, affinity-matured antibodies are anticipated to demonstrate a diminished affinity towards mutated epitopes. This reduced affinity, when considered in conjunction with a potentially enhanced affinity of the Omicron Spike towards ACE2 relative to wild-type Spike, calls into question the assumption that booster immunization enhances humoral immune protection by exclusively engaging antibodies against non-conserved epitopes 25 . A recent mouse study has demonstrated that T cells are capable of protecting against SARS-CoV-2 independently of antibodies 26 . Accordingly, an alternative hypothesis may be posited that the primary benefit of booster immunization is conferred by the cellular immune system and not by antibodies. While antibody levels may be indicative of the protective efficacy of a less refined antibody repertoire, their utility is constrained following booster vaccination, as they do not account for alterations in the epitope repertoire, which is a pivotal element in combating variant infections. In light of the substantial influence of both titers and the repertoire post-second vaccination on the post-booster epitope repertoire, it can be inferred that NAb levels and antibody titers prior to the booster offer a more accurate indicator of infection susceptibility (Fig. 2; Fig. 3). A shift in the balance of non-RBD- and RBD-specific Spike antibodies towards a greater proportion of non-RBD Spike antibodies may occur within six months after receiving a booster. This shift in antibody profile is associated with subsequent infection, which underscores the pivotal function of RBD antibodies in humoral immune protection (Fig. 3). The data presented here support the hypothesis that conserved epitopes are a crucial element in combating symptomatic variant infection by both antibodies and T cells. Post-booster antibodies, which bind to variant epitopes spanning sites relevant for virus-host interactions, may contribute to the most effective protection from variant infection and may serve to compensate for waning antibody titers. Cellular immune protection appears to be primarily provided by conserved epitope-specific T cells. This may be because CD8 + T cells are thought to prevent a severe course of disease rather than preventing infection by blocking virus-host cell binding 27 . However, the present study is unable to contribute to this part of the hypothesis, as it does not permit the differentiation of the role played by the two branches of the immune system in immune protection. It can be concluded that vaccination strategies that exclusively target either conserved or non-conserved epitopes may not be an effective means of establishing an adequate level of protection against infection. Consequently, new vaccines should be designed according to their specific indication. Immunity providing broad cross-variant protection appears to require conserved epitopes, while superior protection may be only achieved by additionally addressing sites required for virus-host cell binding. Methods Study design The presented data were gathered as part of the Munich Observation Study of Adaptive Immune Response after COVID-19 Vaccination (MOSAIC). The MOSAIC Study was initiated as an observational cohort study in December 2020 at the German Heart Centre, Munich, Germany. The study includes 640 voluntary employees aged between 18 and 70, vaccinated either with mRNA-based Comirnaty (Pfizer-BioNTech, USA/Germany) or with vector-based COVID-19 vaccine Vaxzevria (AstraZeneca, UK). 107 participants of the MOSAIC study were selected for this sub-study according to the following criteria (Fig. S1): Vaccinated with two doses of Comirnaty followed by a single homologous booster immunization 7–13 months later. Individuals who had received the Vaxzevria vaccine were excluded from the study due to the greater diversity of vaccination schedules and the insufficient number of participants who met the other inclusion criteria. No documented SARS-CoV-2 infection until last blood draw and negative test result in two distinct SARS-CoV-2 nucleocapsid-antibody assays performed in our laboratory at TP1 and TP2. All employees were offered the option of undergoing a PCR test in our laboratory at their own discretion in the event of a suspected infection. Additionally, all employees were provided with rapid antigen tests for home use (Clungene, Clongene Biotech, Hangzhou, China) on a voluntary basis. The results of these tests were to remain negative for the entirety of the study period (uninfected individuals) or until TP2 (infected individuals). The implementation of this procedure guaranteed the monitoring of the participants with a maximum level of intensity. The selected subjects had a median age of 50.2 years (min. 23.1, max. 69.9 years), and 67.3% were female. The study was conducted following the principles of the Declaration of Helsinki, and ethical approval was obtained from the Ethics Commission/Medical Research Ethical Committee of the Technical University Munich (protocol number R20.030). All participants provided written informed consent for participation. Blood collection and infection monitoring Whole blood samples were obtained by venipuncture and collected in lithium-heparin tubes (Sarstedt, Nürmbrecht, Germany). All commercial assays were performed as single tests according to the manufacturer’s instructions. The Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics, Mannheim, Germany) was performed on a Cobas E411 Analyzer (Roche); the recom Well SARS-CoV-2 IgG (Mikrogen, Neuried, Germany) assay was processed on a fully automated Gemini ELISA plate processor (Stratec Biomedical, Birkenfeld, Germany).For virus detection, nasal and throat swabs were processed using an automated Maxwell nucleic acid extractor (Promega, Walldorf, Germany). The qPCR reaction was set up with an automated Mic liquid handling device (Bio Molecular Systems, Australia) using the ampli Cube Coronavirus SARS-CoV-2 assay (Mikrogen). The PCR was run on a Myra qPCR cycler (Bio Molecular Systems). The same equipment was used to conduct the ampli Melt SARS-CoV-2 Variants test (Mikrogen) to differentiate virus variants. Antibody assays The presence of RBD and Spike IgG antibodies and neutralizing antibodies was determined using the V-PLEX SARS-CoV-2 and V-PLEX SARS-CoV-2 (ACE2) assays (MesoScale Diagnostics, Rockville, MD), respectively, on a Meso QuickPlex SQ 120MM multiplex ECLIA plate luminometer (MesoScale) according to the manufacturer’s instructions. The following variants and subvariants were identified through the utilization of Panel 22 and Key Variant Panel 1 product variants: A (Wuhan); B.1.1.7 (Alpha); B.1.351, B.1.351.1 (Beta); AY.3 + AY.4 + AY.4.2 + AY.5 + AY.6 + AY.7 + AY.12 + AY.14 + B.1.617.2 + B.1.617.2-ΔY144 (Delta); B.1.1.529 + BA.1 + BA.1.15 (Omicron); BA.2 + BA.2.1 + BA.2.2 + BA.2.3 + BA.2.5 + BA.2.6 + BA.2.7 + BA.2.8 + BA.2.10 + BA.2.12 (BA.2 subvariants); BA.2.12.1 (Omicron); BA.2.75 (Omicron); BA.4 + BA.5 (Omicron). T cell assays Within eight hours of blood collection, T cells were stimulated in 500 µl of whole blood from randomly selected subjects at 37°C for 16 (± 0.5) hours with different peptide pools containing 150 pmol of each peptide (Table S1). The stimulation was terminated by the addition of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche) and stored at -80°C until use. RNA was extracted with a MagNA Pure 96 Cellular RNA Large Volume Kit on a MagNA Pure 96 System (Roche). The relative mRNA expression of IFN-γ, CXCL-10 with the housekeeping marker 60S acidic ribosomal protein P0 (RPLP0) as reference was analyzed with T-Track SARS-CoV-2 Quant PCR (Mikrogen) on a LightCycler 480 Instrument II (Roche) using the T-Track SARS-CoV-2 Analysis Tool (Mikrogen) according to the manufacturer's instructions. Statistical analyses Statistical analyses were performed using Prism 9.5.1 (Dotmatics, USA). Calculated p-values < 0.05 were considered statistically significant. Where indicated, data were MIN/MAX normalized. To determine if NAb values significantly decreased from TP1 to TP2 (Fig. 1c), a one-tailed Wilcoxon signed-rank test was performed against a theoretical median of 0. One-tailed p-values were derived by halving the two-tailed p-values where median differences were negative. Antibody levels and T-cell stimulation results were compared between infected and uninfected cohorts using the Mann-Whitney test (two-tailed). A median line through the origin was chosen as the classification criterion for dividing the subjects into two groups in Fig. 4, because conserved and non-conserved epitopes were assumed to contribute to the raw signal with dynamically changing ratios. The median line divides the relevant field that is shared by the subjects into two equal parts. The upper left part thus represents subjects with a higher proportion of the wild-type-specific signal than the lower right part. Statistical analyses included two-sided Fisher's exact test for contingency tables, with Koopman asymptotic score method used to calculate relative risk confidence intervals. Declarations Data availability Source data are provided with this paper. Additional research data may be shared with qualified researchers upon reasonable request to the corresponding author including a scientific proposal, subject to data protection and participant consent restrictions. Acknowledgements This research was financially supported byRoche. Mikrogen provided recom Well SARS-CoV-2 IgG test kits free of charge. The authors wish to thank the Cardiovascular Biobank - Liquids Team for the preparation of the samples and the routine lab team for antibody testing at the Institute of Laboratory Medicine at the German Heart Center, Munich. We extend our gratitude to all study participants for their voluntary involvement in this investigation. Authors and Affiliations Heiko Pfister, Carsten Uhlig, Zsuzsanna Mayer, Eleni Polatoglou, Hannah Randeu, Tabea Berchtold, Silke Burglechner-Praun, Michaela Sander, Stefan Holdenrieder Munich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center Munich, Technical University Munich, Lazarettstr. 36, D-80636 Munich, Germany. Susanne Sernetz, Felicitas Heitzer, Andrea Strötges-Achatz Occupational Medicine Service, German Heart Center Munich, Technical University Munich, Lazarettstr. 36, D-80636 Munich, Germany. Ludwig Deml Mikrogen GmbH, Anna-Sigmund-Str. 10, 82061 Neuried, Germany. Contributions S.H., M.S. and H.P. designed the study. H.P. scientifically conceptualized the study, analyzed the data and wrote the manuscript. M.S and S.H. managed the study and reviewed the manuscript. C.U. conducted quality controls and processing of the data. Z.M. typed the virus variants. E.P. performed qPCR analysis of T cell stimulation assays. H.R. collected blood samples, and anonymized and managed the test subject data. S.S. and F.H. managed and medically supervised vaccination. L.D. advised on the implementation of the T-Track system. A.S.-A. organized and carried out blood sampling and questioning. M.S., S.B.-P. and T.B. conducted T cell stimulation experiments. S.B.-P. conducted the antibody assays. Corresponding author Dr. Heiko Pfister Institute of Laboratory Medicine Deutsches Herzzentrum München Technische Universität München Lazarettstraße 36 80636 München Germany [email protected] Competing interests S.H. was part of a SARS-CoV-2 expert panel and received a honorarium from Roche. The funding agencies had no influence on study design; the collection, analysis, or interpretation of data; the writing of the report; or in the decision to submit the article for publication. All other authors declare no competing interests. References Andrews N et al (2022) Covid-19 Vaccine Effectiveness against the Omicron (B.1.1.529) Variant. N Engl J Med 386:1532–1546 Tartof SY et al (2022) Durability of BNT162b2 vaccine against hospital and emergency department admissions due to the omicron and delta variants in a large health system in the USA: a test-negative case-control study. Lancet Respir Med 10:689–699 Huang CQ, Vishwanath S, Carnell GW, Chan ACY, Heeney JL (2023) Immune imprinting and next-generation coronavirus vaccines. Nat Microbiol 8:1971–1985 Muecksch F et al (2022) Increased memory B cell potency and breadth after a SARS-CoV-2 mRNA boost. Nature 607:128–134 Yang L et al (2023) Antigen presentation dynamics shape the antibody response to variants like SARS-CoV-2 Omicron after multiple vaccinations with the original strain. Cell Rep 42:112256 Olukitibi TA, Ao Z, Warner B, Unat R, Kobasa D, Yao X (2023) Significance of Conserved Regions in Coronavirus Spike Protein for Developing a Novel Vaccine against SARS-CoV-2 Infection. Vaccines (Basel) 11 Wang Y et al (2024) Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants. Nat Commun 15:842 Meyer S et al (2023) Prevalent and immunodominant CD8 T cell epitopes are conserved in SARS-CoV-2 variants. Cell Rep 42:111995 Tarke A et al (2022) SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron. Cell 185:847–859e811 Tye EXC et al (2022) Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4(+) T cell recognition. Nat Immunol 23:1726–1734 Robert-Koch-Institut (2024) Wochenberichte zu COVID-19 (bis 8.6.2023). (ed^(eds Robert-Koch-Institut). Robert-Koch-Institut Gilbert PB et al (2022) Immune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacy clinical trial. Science 375:43–50 Skelly DT et al (2021) Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern. Nat Commun 12:5061 Bonhomme ME et al (2022) Robust validation and performance comparison of immunogenicity assays assessing IgG and neutralizing antibodies to SARS-CoV-2. PLoS ONE 17:e0262922 Shengule S et al (2024) Validation and Suitability Assessment of Multiplex Mesoscale Discovery Immunogenicity Assay for Establishing Serological Signatures Using Vaccinated, Non-Vaccinated and Breakthrough SARS-CoV-2 Infected Cases. Vaccines (Basel) 12 Chen Y, Zhao X, Zhou H, Zhu H, Jiang S, Wang P (2023) Broadly neutralizing antibodies to SARS-CoV-2 and other human coronaviruses. Nat Rev Immunol 23:189–199 Hossen ML, Baral P, Sharma T, Gerstman B, Chapagain P (2022) Significance of the RBD mutations in the SARS-CoV-2 omicron: from spike opening to antibody escape and cell attachment. Phys Chem Chem Phys 24:9123–9129 Schwarz M et al (2022) Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR. Nat Biotechnol 40:1680–1689 Bobrovitz N et al (2023) Protective effectiveness of previous SARS-CoV-2 infection and hybrid immunity against the omicron variant and severe disease: a systematic review and meta-regression. Lancet Infect Dis 23:556–567 Magazine N et al (2024) Immune Epitopes of SARS-CoV-2 Spike Protein and Considerations for Universal Vaccine Development. Immunohorizons 8:214–226 Cao Y et al (2022) Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature 602:657–663 Willett BJ et al (2022) SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway. Nat Microbiol 7:1161–1179 Jacobsen H et al (2023) Systematic review and meta-analysis of the factors affecting waning of post-vaccination neutralizing antibody responses against SARS-CoV-2. NPJ Vaccines 8:159 Song S, Madewell ZJ, Liu M, Longini IM, Yang Y (2023) Effectiveness of SARS-CoV-2 vaccines against Omicron infection and severe events: a systematic review and meta-analysis of test-negative design studies. Front Public Health 11:1195908 Shah M, Woo HG, Omicron (2021) A Heavily Mutated SARS-CoV-2 Variant Exhibits Stronger Binding to ACE2 and Potently Escapes Approved COVID-19 Therapeutic Antibodies. Front Immunol 12:830527 Fumagalli V et al (2024) Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination. Nat Immunol 25:633–643 Sette A, Sidney J, Crotty S (2023) T Cell Responses to SARS-CoV-2. Annu Rev Immunol 41:343–373 Additional Declarations Yes there is potential Competing Interest. S.H. was part of a SARS-CoV-2 expert panel and received a honorarium from Roche. All other authors declare no competing interests. Supplementary Files Sourcedata.xlsx Source data tables Supplementarydata.pdf Supplementary materials for ‘Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections’ nrreportingsummarychecked.pdf Reporting Summary nreditorialpolicychecklistchecked.pdf Editorial Policy Checklist Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5678273","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":396992690,"identity":"f1f1f267-fc56-400d-8c23-be4c7f02858d","order_by":0,"name":"Heiko Pfister","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDAC5oMNIEqO4QaQZASxDxDSwpbYAFJkTIqWBLCixAaitZizMTd//lBxL73vdncC080ddxj4jjfg12LZxtgmceBMce7MO2c3MOeeecYgeYaANQb3G9sYDrYl5G64kQvU0naYweBGAgEtxxibPxz8l5BuANdy/wFBLQ0SBxsSEhBabuDXAfHLmWMJhjOBWg4DtfBIniHgMHM29scfKmoS5Plu5G58DNQix3f8AAGHIXNAankIOAtNyygYBaNgFIwCrAAAZHhSj011HjIAAAAASUVORK5CYII=","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":true,"prefix":"","firstName":"Heiko","middleName":"","lastName":"Pfister","suffix":""},{"id":396992691,"identity":"16455de6-72c6-4cdf-98be-edf68762503a","order_by":1,"name":"Carsten Uhlig","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Carsten","middleName":"","lastName":"Uhlig","suffix":""},{"id":396992692,"identity":"a79302c9-b60e-496a-b8cd-90b2c7a06a6a","order_by":2,"name":"Zsuzsanna Mayer","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Zsuzsanna","middleName":"","lastName":"Mayer","suffix":""},{"id":396992693,"identity":"140071a3-5cf5-4e18-995d-437444aecc8d","order_by":3,"name":"Eleni Polatoglou","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Eleni","middleName":"","lastName":"Polatoglou","suffix":""},{"id":396992694,"identity":"65ea9530-fce1-4cd3-91e7-4d8c2af49c9e","order_by":4,"name":"Hannah Randeu","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Randeu","suffix":""},{"id":396992695,"identity":"efd20192-0447-484f-b954-1bb3d69fcabb","order_by":5,"name":"Silke Burglechner-Praun","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Silke","middleName":"","lastName":"Burglechner-Praun","suffix":""},{"id":396992696,"identity":"6bad0fa2-d6f1-4157-976c-4eccbeebf5c6","order_by":6,"name":"Tabea Berchtold","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Tabea","middleName":"","lastName":"Berchtold","suffix":""},{"id":396992697,"identity":"b1e0148e-daf6-4e7a-bbb9-b75ef9cb957a","order_by":7,"name":"Susanne Sernetz","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Sernetz","suffix":""},{"id":396992698,"identity":"01b80c75-0563-4b42-a122-82fefb960254","order_by":8,"name":"Felicitas Heitzer","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Felicitas","middleName":"","lastName":"Heitzer","suffix":""},{"id":396992699,"identity":"a7e506ba-6afd-4c54-b2a5-3a84daf857ee","order_by":9,"name":"Andrea Strötges-Achatz","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Strötges-Achatz","suffix":""},{"id":396992700,"identity":"dc25813d-647e-439f-9b4c-220a3909f2cd","order_by":10,"name":"Ludwig Deml","email":"","orcid":"","institution":"Mikrogen GmbH","correspondingAuthor":false,"prefix":"","firstName":"Ludwig","middleName":"","lastName":"Deml","suffix":""},{"id":396992701,"identity":"907abea4-77be-48e5-8779-ae6de975bc6d","order_by":11,"name":"Michaela Sander","email":"","orcid":"","institution":"German Heart Center Munich","correspondingAuthor":false,"prefix":"","firstName":"Michaela","middleName":"","lastName":"Sander","suffix":""},{"id":396992702,"identity":"44fe1261-70fb-420e-915b-5d867a3226c1","order_by":12,"name":"Stefan Holdenrieder","email":"","orcid":"","institution":"German Heart Centre Munich","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Holdenrieder","suffix":""}],"badges":[],"createdAt":"2024-12-19 16:05:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5678273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5678273/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73274596,"identity":"a492b678-42b5-4a1e-b50d-5552131f681f","added_by":"auto","created_at":"2025-01-08 11:36:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":272440,"visible":true,"origin":"","legend":"\u003cp\u003eNeutralizing antibody levels after second and booster vaccination.\u003c/p\u003e\n\u003cp\u003eACE2-RBD binding inhibition in serum samples (n=107) determined (a) 6 months after second vaccination or (b) 6 months after booster immunization. (c) Booster immunization induced a significant overall increase in NAb levels (p \u0026lt; 0.0001) against wild-type and all variants except Omicron. Omicron NAbs decreased significantly in the majority of subvariants as determined by Wilcoxon signed-rank test (**** p \u0026lt; 0.0001; *** p=0.0005; ** p=0.0025; ns, not significant). Whiskers denote the 1.5 × interquartile range.\u003c/p\u003e\n\u003cp\u003e(d) NAb levels at TP1 and NAb level changes from TP1 to TP2 are inversely correlated. Each sample is represented by a single spot; inverse correlation is indicated by linear regression with 95% CI shown in grey. The correlation coefficient was calculated as two-tailed Spearman’s r with p-values given in the graphs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/6f2ee12549eec1eab177b21c.png"},{"id":73273160,"identity":"9b6ea34d-206f-47f3-b5e3-9d6433e21769","added_by":"auto","created_at":"2025-01-08 11:20:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":229923,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of neutralizing antibody levels after second and booster vaccination with infection.\u003c/p\u003e\n\u003cp\u003eSome NAb levels against variants differ between infected and non-infected individuals at TP1 but not at TP2. Grey box: area with high values at TP2. Dashed lines: median of TP1 (vertical) and TP2 (horizontal). Dark green circles / light green circles: non-infected individuals from grey box in a (n=14)/ non-infected individuals from below grey box in a (n=25). Dark red dots / light red dots: infected individuals from grey box in a (n=22)/ infected individuals from below grey box in a (n=46). p-values in graphs: Mann-Whitney test between infected and non-infected individuals at TP1 (lower right in graphs) and TP2 (upper left in graphs), and at TP1 with datasets restricted to individuals within grey box in a (upper boxed) or values below (lower boxed).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/ebfb4edc2876e1445a816a51.png"},{"id":73274313,"identity":"9991f99b-7a5c-4d9d-ad90-7dc1c748f91a","added_by":"auto","created_at":"2025-01-08 11:28:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154602,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of IgG antibody levels after second and booster vaccination with infection.\u003c/p\u003e\n\u003cp\u003eNormalized antibody levels against wild-type RBD are significantly associated with the infection rate at (a) TP1 and, to a lesser extent, at (b) TP2 when Spike-specific IgG at TP2 is high. (c) While RBD/Spike IgG ratios do not differ significantly between infected and uninfected individuals, (d) the ratio of RBD IgG to Spike IgG changes from TP1 to TP2 correlates with infection at high Spike IgG TP2 levels, as determined by Mann-Whitney test (** p=0.006; * p=0.04; ns, not significant). Horizontal lines: median of each group. Green circles / red dots: non-infected (high TP2 Spike IgG: n=22; low/medium TP2 Spike IgG: n=17) / infected (high TP2 Spike IgG: n=46; low/medium TP2 Spike IgG: n=22) individuals, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/58d95a3b41bb2c0aeba1dea5.png"},{"id":73273217,"identity":"6c112bbe-9757-4a50-a778-fc3a34a6d565","added_by":"auto","created_at":"2025-01-08 11:20:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":291544,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of NAb levels against conserved and non-conserved epitopes with infection.\u003c/p\u003e\n\u003cp\u003eNormalized NAb levels (% inhibition) at TP2 against variant and wild-type RBD, divided into high NAb and medium to low NAb groups (dotted line). The median line intersecting the graph’s origins categorizes individuals into two distinct groups. Individuals above the line are indicated as having a higher proportion of NAbs directed against non-conserved epitopes than individuals below the line. Counts of individuals in each group are indicated on the left (above the line) and right (below the line) in red (infected) or green (non-infected). The proportion of NAbs directed against non-conserved epitopes is statistically significantly different between infected and uninfected individuals in several Omicron variants, as determined by Fisher’s exact test (results as indicated).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/8dedb1e75a8817594aaafb26.png"},{"id":73273189,"identity":"ddc3606f-2e56-4baa-9ea2-bbfa284e5d34","added_by":"auto","created_at":"2025-01-08 11:20:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":565539,"visible":true,"origin":"","legend":"\u003cp\u003eStimulation of T cells in whole blood of randomly selected individuals by wild-type and variant Spike protein-derived peptide pools at TP2.\u003c/p\u003e\n\u003cp\u003eExpression of CXCL-10 and IFN-γ mRNA was determined by RT-qPCR. (a) Expression levels are calculated as the ratio of the result achieved with the indicated peptide pool and an unstimulated control, both of which have been normalized to the reference marker RPLP0 (green circles / right filled red dots: non-infected (n=6)/ infected (after TP2; n=8) individuals). (b) The average ratio between wild-type Spike peptide pools spanning exclusively mutated sites in SARS-CoV-2 variants (reference pools) to whole wild-type Spike peptide pool-1 and -2 (representing the N-terminal and C-terminal half of the Spike protein, respectively) is lower in uninfected individuals. There is no apparent association of wild-type to variant NAb ratios and T cell stimulation (triangles tip up/tip down: individuals with NAb wild-type to variant ratio above/below median line in red, right-filled (infected after TP2) or green (non-infected); for NAb values, see Fig. 4). For comparative purposes, individuals infected with Omicron before TP2 are included (left-filled red dots; n=11). (c) Peptide pools exclusively spanning mutated sites elicit an equivalent relative T-cell response in infected and uninfected individuals. (d) The stimulation ratio with peptide pool-1 and -2 (representing the N-terminal and C-terminal half of the Spike protein of variant and wild-type) is lower in uninfected, especially with pool-1.\u003c/p\u003e\n\u003cp\u003eStimulation with peptide pools revealed significant differences between infected and uninfected individuals as determined by Mann-Whitney test where indicated with p-values given in the graphs (ns, not significant). Horizontal lines represent the median of each group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/6f45de03ccf2b6f4e18d1caf.png"},{"id":73274601,"identity":"f6488f15-4d59-4854-88e9-3bf2f0acc3e6","added_by":"auto","created_at":"2025-01-08 11:37:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1857987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/713f3314-b7fc-4607-a750-1af0599ce9c6.pdf"},{"id":73273164,"identity":"39ae3c9e-0909-47db-83ef-bdac8b576e85","added_by":"auto","created_at":"2025-01-08 11:20:53","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":154603,"visible":true,"origin":"","legend":"Source data tables","description":"","filename":"Sourcedata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/25e713fc157379c1344e6582.xlsx"},{"id":73273147,"identity":"02db3318-fe94-46e5-af58-6d4ce769c46c","added_by":"auto","created_at":"2025-01-08 11:20:51","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":915934,"visible":true,"origin":"","legend":"Supplementary materials for \u0026#x2018;Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections\u0026#x2019;","description":"","filename":"Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/f547b9eef9d5331869beb544.pdf"},{"id":73274312,"identity":"dbc57567-a7e3-4066-b29a-af71ef271645","added_by":"auto","created_at":"2025-01-08 11:28:54","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666635,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"nrreportingsummarychecked.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/9db0f7cb4ac80bb7b70469a5.pdf"},{"id":73273224,"identity":"bb2b5bf0-63c3-4534-9423-20ef2634c9d1","added_by":"auto","created_at":"2025-01-08 11:20:56","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1682417,"visible":true,"origin":"","legend":"Editorial Policy Checklist","description":"","filename":"nreditorialpolicychecklistchecked.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5678273/v1/2bac75e50169e4610389d738.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nS.H. was part of a SARS-CoV-2 expert panel and received a honorarium from Roche.\r\n\r\nAll other authors declare no competing interests.","formattedTitle":"Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoronavirus disease 2019 (COVID-19) has posed a major challenge to health systems worldwide. While vaccines derived from the original strain (Wuhan-Hu-1; \u0026ldquo;wild-type\u0026rdquo;) appeared to be effective against the original strain and earlier variants, the Omicron variant displays a remarkable ability to evade neutralizing antibodies (NAbs)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Repeated homologous vaccinations primarily strengthen the immune response against the vaccine strain, potentially rendering the immune system less effective against variant infections\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe increase in breadth and potency of the humoral immune response after booster vaccination depends largely on the diversification of the memory B cell pool induced by the second vaccination\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Epitope masking and higher antigen availability after booster vaccination may promote off-target B cells that contribute to a broad NAb repertoire against related pathogens\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite numerous mutations, particularly those acquired by Omicron, conserved epitopes on the Spike (S) protein and within the receptor-binding domain (RBD) have been identified as promising targets for vaccination strategies effective against multiple variants\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While immune escape variants are able to evade the humoral immune response to varying degrees, depending on the number and position of the mutations, CD8\u0026thinsp;+\u0026thinsp;T cell epitopes remain largely conserved among variants\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, disease control appears to be granted by broad epitope targeting of CD4\u0026thinsp;+\u0026thinsp;T cells, thereby limiting the evolution of immune evasion mutants\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn a study of 107 healthcare workers (HCWs), we found that the humoral immune response directed against the RBD underwent a notable rearrangement of the epitope repertoire following booster immunization. The data revealed a correlation between an elevated proportion of antibodies directed against non-conserved epitopes within the RBD and a lower incidence of infections during the 6\u0026ndash;17 months following booster vaccination. Conversely, stimulation of T cells with specific Spike peptide pools in whole blood revealed a dominant role of conserved-epitope-specific cellular immunity in long-term protection.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe present study was conducted with selected samples from a prospective, observational, single-center cohort study initiated in December 2020, comprising 640 healthcare workers who received two doses of the wild-type mRNA vaccine BNT162b2 between January and April 2021 and one booster dose between October 2021 and January 2022 (Suppl Fig.\u0026nbsp;1). Exclusion criteria included individuals who had received Vaxzevria (AstraZeneca, UK) due to differences in vaccination schedules. Additionally, participants had to demonstrate a lack of documented SARS-CoV-2 infection and negative results from nucleocapsid-antibody assays over the specified periods for each respective experiment. During the observation period, which commenced six months after booster vaccination in April 2022, it was reported that more than 90% of infections in Germany were attributed to the BA.1, BA.2, BA.4, and BA.5 variants through the end of 2022\u003csup\u003e11\u003c/sup\u003e. In early 2023, recombinant variants, most notably XBB.1, began to replace prevalent Omicron subvariants, reaching a relative prevalence of approximately 50% by the study\u0026rsquo;s conclusion in February. In the initial observation phase, the weekly incidence was reported as 1,543 cases per 100,000 citizens, dropping to a low of 75 cases per 100,000 citizens near the end of the observation phase, with intermittent spring and autumn waves peaking at 752 and 944 cases per 100,000 respectively. The median value throughout the observation period was 329 per 100,000 citizens\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBooster immunization does not increase long-term serum NAb activity against Omicron RBD\u003c/h2\u003e \u003cp\u003eNAb activity against multiple variants and subvariants was assessed 6 months after the second vaccination dose (TP1) and 6 months after booster immunization (TP2) by multiplex RBD-ACE2 inhibition electrochemiluminescence immunoassay (ECLIA, MSD Mesoscale). The ECLIA assays have been extensively validated and are frequently used in COVID-19 research\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Wild-type and all variants of concern (VOCs) relevant during the study course were included.\u003c/p\u003e \u003cp\u003eThe median inhibition ranged from 19.7% (Omicron B.1.1.529; BA.1; BA.1.15) to 31.3% (wild-type) in TP1 and from 14.0% (Omicron B.1.1.529; BA.1; BA.1.15) to 52.1% (wild-type) in TP2 (Fig.\u0026nbsp;1a, b). Thus, booster immunization induced a marked relative median increase of individual sustained NAb levels against wild-type, Alpha (B.1.1.7), Beta (B.1.351/B.1.351.1), and Delta (variants) by 53.9% (median absolute difference with 95% CI: 19.24 [12.53, 24.47]), 50.8% (12.84 [10.33, 19.61]), 46.5% (13.56 [8.631, 17.87]), and 47.8% (12.56 [10.12, 18.12]), respectively (Fig.\u0026nbsp;1c). In contrast, Omicron-specific median individual NAb levels dropped by 26.8% (B.1.1.529, BA.1, BA.1.15; median absolute difference with 95% CI: -4.571 [-8.813, -2,613]), 25.5% (BA.2 subvariants; -5.882 [-8.293, -2.771]), 0.2% (BA.2.12.1; 0.03099 [-3.202, 3.184]), 35.9% (BA.2.75; -6.985 [-10.29, -3.429]), and 6.9% (BA.4, BA.5; -1.721 [-4.666, 3.314]).\u003c/p\u003e \u003cp\u003eTP1 levels are inversely correlated with the change in levels of all tested variants including wild-type (Fig.\u0026nbsp;1d). The correlation is strongest for the Omicron subvariants.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNAb levels are not generally suitable as correlates of protection\u003c/h3\u003e\n\u003cp\u003eThe inverse correlation between the NAb level change from TP1 to TP2 and NAb level in TP1 prompted the question of whether a reduction in NAb levels contradicts the reported initial improvement in protection against Omicron infection following booster vaccination, due to the loss of RBD-specific NAbs during affinity maturation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Consequently, we correlated the data from the participants' questionnaires on confirmed cases of COVID-19 at the end of the study with the NAb levels at TP1 and TP2.\u003c/p\u003e \u003cp\u003e68 of 107 (63.6%) participants were infected in the median period of 3.6 months (quartile 1: 2.0 months, quartile 3: 5.3 months, max. 9.7 months) after TP2 from June 2022 until February 2023. While not all participants were informed of their variant, we estimate that approximately 90\u0026ndash;95% of the infection symptoms were caused by BA.1, BA.2, BA.4, and BA.5 and their subvariants and less than 10% by Delta infections, since they were by far the dominating variants throughout the observation period in Germany\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Given that only three individuals were infected in 2023, it can be reasonably inferred that infections with recombinant XBB.1 subvariants represent single cases at most. 51 of 68 (75%) infections were confirmed by PCR, with 15 of 51 (29.4%) being typed as Omicron and two (3.9%) being typed as Delta infections. 12 (23.5%) PCRs were performed in-house, and 39 (76.5%) were performed in a certified test center. 17 (25%) infections were confirmed by antigen testing.\u003c/p\u003e \u003cp\u003eGiven that antibody titers wane over time, it was anticipated that they might not suffice to yield significant correlations with infections throughout the observation period. Accordingly, the study group was divided into two subgroups, characterized by low to medium (lower two-thirds of the scale) and high NAb levels (upper third of the scale) at TP2. In the subsequent experiment, the wild-type RBD NAb level was employed as the discriminator between subgroups. While overall NAb levels at TP2 do not appear to be associated with protection, TP1 levels do show some correlation, although not significantly in most cases (Fig.\u0026nbsp;2). Restricting the analysis to individuals with high wild-type NAb levels at TP2 (dots in the grey box in Fig.\u0026nbsp;2a and dark green circles / dark red dots in Fig.\u0026nbsp;2b and 2d representing individuals \u0026ldquo;gated through\u0026rdquo; the grey box in Fig.\u0026nbsp;2a) reveals a significant difference between those infected and uninfected at TP1 in wild-type (median infected (n\u0026thinsp;=\u0026thinsp;22) / non-infected (n\u0026thinsp;=\u0026thinsp;14) with 95% CI of difference: 32.66 / 44.87 [1.009, 24.27]), Alpha (29.74 / 40.19 [1.785, 20.73]), and Delta (23.75 / 36.86 [5.596, 22.30]). The substantial number of mutations in the RBD may render the Omicron test incapable of demonstrating significant distinctions between infected and uninfected individuals. Consequently, the foundation of successful booster vaccination against symptomatic variant infection may be laid by the second vaccination. However, successful booster vaccination evidently depends on an additional mechanism that cannot be resolved by solely determining serum-neutralizing capacity at TP2.\u003c/p\u003e\n\u003ch3\u003eLoss of RBD-specific IgG antibodies after booster vaccination is associated with infection\u003c/h3\u003e\n\u003cp\u003eThe levels of wild-type RBD-specific IgG exhibited a marked increase in TP2, with only two infected and four uninfected individuals displaying a lower level in TP2 than in TP1. When the RBD and Spike IgG levels in the datasets were grouped alternatively by low to medium or high Spike antibody levels at TP2 (in analogy to the experiment above), uninfected individuals with high Spike TP2 levels displayed higher RBD antibody levels than infected individuals, a difference that was already apparent at TP1 (median infected (n\u0026thinsp;=\u0026thinsp;46) / non-infected (n\u0026thinsp;=\u0026thinsp;22) with 95% CI of difference: 0.01659 / 0.04056 [0.005196, 0.04000]) and, to a lesser extent, at TP2 (0.6903 / 0.8509 [-0.004536, 0.2051]; Fig.\u0026nbsp;3a, b). We concluded that the reduction in NAb activity within the RBD, evident in Omicron variants, must be attributed to a rearrangement of the epitope repertoire of Spike-specific antibodies from TP1 to TP2.\u003c/p\u003e \u003cp\u003eIn order to test this hypothesis, a comparative analysis was conducted between the wild-type RBD-specific and Spike-specific IgG values. A small but insignificant difference was observed between infected and non-infected individuals (Fig.\u0026nbsp;3c). In contrast, infected individuals displayed a median relative loss of RBD-specific antibodies between TP1 and TP2, overt at high Spike TP2 levels, while uninfected individuals remained stable (median infected / non-infected: 0.8720 / 1.016; 95% CI of difference [0.004328, 0.2402]; Fig.\u0026nbsp;3d). Our findings indicate that RBD-specific antibodies play a pivotal role in long-term humoral immune protection.\u003c/p\u003e\n\u003ch3\u003eNAbs against non-conserved RBD epitopes are associated with protection from variant infection\u003c/h3\u003e\n\u003cp\u003eThe marked difference between wild-type and Omicron variants in NAb activity loss suggests an equilibrium shift between conserved and non-conserved epitopes. In order to gain a deeper insight into the role of RBD-specific NAbs in protecting against variant infection, we analyzed the relationship between variant- and wild-type-specific NAbs at TP2. It was anticipated that there would be an approximately linear correlation between the wild-type and the Alpha, Beta, and Delta variants, given that only a few mutations within the RBD can influence antibody binding\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It thus follows that the greatest degree of variation was to be expected for the Omicron subvariants, as they have by far the largest number of mutations within the RBD\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on a presumed dose-dependence of NAbs on the infection rate, samples with low to medium titers and high NAb titers were analyzed separately (analogous to the experiments described above). The Omicron scatterplots revealed a prominent bipolar distribution of infected and uninfected individuals in the group with high NAb titers (relative risk of infection with 95% CI with NAbs targeting less non-conserved epitopes of: BA.1.1.529, BA.1, BA.1.15: 2.667 [1.495, 5.535]; BA.2 subvariants and BA.4, BA.5: 2.143 [1.237, 4.201]; Fig.\u0026nbsp;4). It may therefore be concluded that maximum protection against variant infection is provided by high NAb titers with a substantial fraction of antibodies targeting non-conserved epitopes within the RBD.\u003c/p\u003e\n\u003ch3\u003eCellular immunity to conserved epitopes is associated with protection from symptomatic variant infection\u003c/h3\u003e\n\u003cp\u003eTo determine whether the humoral immune response aligns with the cellular immune response at TP2, blood cells were stimulated with peptide pools derived from wild-type and variant Spike protein in whole blood. In light of the logistical constraints and the limited availability of personnel during the ongoing COVID-19 pandemic, we proceeded to select the initial five to ten samples obtained from the daily blood draws for the planned analyses. T cell stimulation was analyzed by quantifying the mRNA expression of interferon-γ (IFN-γ) and the chemokine C-X-C motif chemokine ligand 10 (CXCL-10) by RT-qPCR. CXCL-10 has recently been introduced as a suitable proxy for quantifying cellular immunity against SARS-CoV-2, given its strong correlation with the activation of antigen-specific T cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe median level of CXCL-10 and IFN-γ mRNA expression in infected individuals was distinctly, but not statistically significantly, lower than that observed in non-infected individuals (Fig.\u0026nbsp;5a). These findings suggest that an increased number or reactivity of T cells may confer protection. Additionally, a significant association between infection and a balance shifted towards non-conserved epitopes was revealed upon relating wild-type Spike peptide pools spanning exclusively mutated sites in SARS-CoV-2 variants to whole Spike (wild-type) peptide pools (median infected (n\u0026thinsp;=\u0026thinsp;8) / non-infected (n\u0026thinsp;=\u0026thinsp;6) with 95% CI of difference: Alpha mutation sites (CXCL-10 // IFN-γ): 0.6035 / 0.1911 [-0.7671, -0.1917] // 0.7165 / 0.3021 [-1.163, -0.05047]; BA.1 mutation sites (CXCL-10 // IFN-γ): 1.559 / 0.4068 [-1.531, -0.5060] // 1.142 / 0.3605 [-1.463, -0.2888]; Fig.\u0026nbsp;5b). Our findings indicate that, in contrast to the humoral immune response, maintaining the balance closer to conserved-epitope-specific T cells provides superior protection from symptomatic variant infection.\u003c/p\u003e \u003cp\u003eIt is currently unclear whether our findings regarding cellular and humoral immune protection are directly correlated. Our data depict infected and uninfected individuals with varying peptide pool stimulation ratios exhibiting ratios of wild-type to variant NAb that are both above and below the median line. Of note, individuals infected prior to TP2 exhibited a pronounced tendency towards conserved epitopes in comparison to those infected subsequent to TP2 (median infected (n\u0026thinsp;=\u0026thinsp;8) / previously infected (n\u0026thinsp;=\u0026thinsp;11) with 95% CI of difference: CXCL-10: 1.559 / 0.9381 [-1.220, 0.01439]; IFN-γ: 1.142 / 0.5326 [-1.110, -0.08499]). This observation may be indicative of augmented protection against variant infection resulting from additional variant antigen exposure\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The use of peptide pools exclusively covering variant mutation sites and a corresponding wild-type reference pool demonstrated that there was no discernible difference in cross-reactivity with peptide variants between infected and uninfected individuals (Fig.\u0026nbsp;5c).\u003c/p\u003e \u003cp\u003eIn order to spatially narrow down the observed disparity between infected and uninfected individuals on the Spike protein, we conducted a separate comparison between peptide pools representing the N-terminal and C-terminal halves of all relevant Spike protein variants with the corresponding wild-type peptide pools (Fig.\u0026nbsp;5d). To map the effect of mutations on T cell stimulation as comprehensively as possible, the analysis was expanded to include Gamma variants. However, it can be reasonably assumed that the Gamma variant did not play a significant role in the incidence of infection during the course of this study\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Upon stimulation with the N-terminal peptide pool-1, the median cytokine ratio of variant to wild-type exceeded the value of 1 in five of six tested (sub)variants in infected individuals (median infected / non-infected with 95% CI of difference: Alpha pool-1 CXCL-10: 1.218 / 0.8041 [-1.520, -0.04511]; BA.1 pool-1 (CXCL-10 // IFN-γ): 1.443 / 0.6259 [-1.420, -0.03581] // 1.430 / 0.3510 [-1.503, -0.1223]; Fig.\u0026nbsp;5d). In contrast, only two CXCL-10 ratios of the uninfected were above 1, and none of the IFN-γ ratios. The consistent trend towards higher variant to wild-type ratios in infected individuals as compared to the non-infected localizes the source of the observed trend at least in part to the RBD-containing N-terminal half of the Spike protein. The results for peptide pool-2, which spans the C-terminal half of the Spike protein, were inconsistent. No significant difference was found for IFN-γ between infected and uninfected individuals. However, Alpha and BA.1 differed significantly in CXCL-10 values (median infected / non-infected with 95% CI of difference: Alpha pool-1: 1.323 / 0.6588 [-1.500, -0.02893]; BA.1 pool-1: 1.145 / 0.4689 [-1.277, -0.02541]), which may be due to the significant role of conserved sites on the S2 subunit and those around the S1/S2 cleavage site in protection against symptomatic infection \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese findings suggest that the protective effect of additional SARS-CoV-2 antigen exposure may be attributed to conserved T cell epitopes, including those in the N-terminal half of the Spike protein\u0026mdash;specifically the RBD and N-terminal domain (NTD).\u003c/p\u003e \u003cp\u003eA notable difference was observed in the infected groups before and after TP2, further supporting our conclusions about infection-driven T cell reactivity bias towards conserved epitopes. Cells from individuals infected with Omicron before TP2 showed cytokine ratios below 1 when stimulated with BA.1 peptides (similar to the non-infected group). In contrast, those infected after TP2 displayed ratios above 1, particularly evident with pool-1 peptides. These findings suggest that the protective effect of additional SARS-CoV-2 antigen exposure may be attributed to conserved T cell epitopes, including those in the N-terminal half of the Spike protein - specifically the RBD and N-terminal domain (NTD). This perspective is corroborated by the discovery of elevated genetic entropy levels within the T cell epitopes of the RBD and NTD, indicating viral immune evasion\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe majority of the immunodominant epitopes of neutralizing antibodies against SARS-CoV-2 have undergone significant mutation in the Omicron variant, which contributes to immune evasion\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Immune escape can be at least partially compensated for by booster immunization\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This gave rise to the question of whether the booster immunization merely contributes to an increase in antibody levels or whether it provides extended protection through immune adaptation processes. As demonstrated in our study, NAb levels are not an appropriate correlate of long-term protection after booster immunization against SARS-CoV-2 variant infection. Consequently, we sought to establish whether the augmented level of protection could be attributed to an expansion of adaptive processes that refine the repertoire of humoral and cellular immunity. Therefore, a six-month interval was permitted after the second and third vaccination before laboratory parameters were determined.\u003c/p\u003e \u003cp\u003eOn the supposition of a consistent reduction in antibody levels reported across a range of initial peak titers, our data substantiate the following interpretation of long-term immunity to Omicron variants induced by mRNA vaccination\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e \u003cp\u003eFollowing the second vaccination, RBD-specific antibodies are predominantly directed towards immunodominant epitopes. The immunologic pressure (especially on immunodominant epitopes) exerted by the vaccination program and the prevalence of wild-type infections in the population drives the evolution of the virus towards the development of immune escape variants. As a consequence, Omicron RBD NAb levels are lower compared to wild-type due to the reduced number of native immunodominant epitopes. The measured differences between the Omicron subvariants and the wild-type, as well as with all other variants tested, may be attributed to the number of mutations present within the RBD (Alpha: 1, Beta: 3, Delta: 2, Omicron: 15) rather than the mutations themselves\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBooster immunization does apparently not provide a sufficient trigger for the expansion of immunodominant epitope-specific B cells in all individuals, resulting in inadequate compensation for the decline in functional capacity of existing plasma cells over time (Fig.\u0026nbsp;1). This may, at least in part, be attributed to affinity maturation processes which result in antibodies with reduced affinity to mutated epitopes. As this outcome is dose-dependent, it could be caused by antibody-dependent immunoregulatory mechanisms such as antibody-mediated epitope masking. In numerous cases, particularly evident with the Omicron variant, this results in TP2 RBD NAb levels below the TP1 values. It indicates that newly formed post-booster antibodies directed at previously subdominant epitopes are also unable to compensate for the loss. It can be concluded that previously immunodominant, non-conserved epitopes make a significant contribution to the post-booster epitope spectrum in subjects with high TP1-NAb levels.\u003c/p\u003e \u003cp\u003eThe reduced median NAb levels against Omicron variant at TP2 initially seem to be at odds with the epidemiological data, which indicate an enhanced vaccine efficacy against Omicron infection following repeated immunizations\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, booster vaccination may enhance cross-variant protection by elevated levels of previously subdominant epitope-specific antibodies\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These epitopes are less likely to be mutated in Omicron variants due to lower immunologic pressure prior to booster campaigns. Given that RBD-specific antibodies targeting non-conserved epitopes appear to be associated with protection, it seems plausible that a broad immune response, including antibodies with lowered affinity for mutated sites and antibodies directed against conserved epitopes, may contribute to maximum protection (Fig.\u0026nbsp;4). A less broad humoral immune response, which covers key epitopes that affect interactions between the virus and host cells including mutated sites such as T478K, Q493K, Q498R, and E484A, is also conceivable\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It is even possible that non-conserved epitopes generally exert a subordinate role in humoral immune protection after repeated vaccinations. This may be supported by the observation that the non-infected subjects in Fig.\u0026nbsp;4 exhibit elevated NAb levels at TP1 in comparison to the infected subjects (median wild-type neutralizing activity of 45% in non-infected subjects vs. 32% in infected subjects in the group with high TP2 NAb levels). Following the view that the generation of antibodies against subdominant, conserved epitopes is influenced by antibodies directed against immunodominant, non-conserved epitopes from previous immunizations, this positive correlation may lead to an underestimation of the significance of antibodies specific for conserved epitopes. Furthermore, affinity-matured antibodies are anticipated to demonstrate a diminished affinity towards mutated epitopes. This reduced affinity, when considered in conjunction with a potentially enhanced affinity of the Omicron Spike towards ACE2 relative to wild-type Spike, calls into question the assumption that booster immunization enhances humoral immune protection by exclusively engaging antibodies against non-conserved epitopes\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. A recent mouse study has demonstrated that T cells are capable of protecting against SARS-CoV-2 independently of antibodies\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Accordingly, an alternative hypothesis may be posited that the primary benefit of booster immunization is conferred by the cellular immune system and not by antibodies.\u003c/p\u003e \u003cp\u003eWhile antibody levels may be indicative of the protective efficacy of a less refined antibody repertoire, their utility is constrained following booster vaccination, as they do not account for alterations in the epitope repertoire, which is a pivotal element in combating variant infections. In light of the substantial influence of both titers and the repertoire post-second vaccination on the post-booster epitope repertoire, it can be inferred that NAb levels and antibody titers prior to the booster offer a more accurate indicator of infection susceptibility (Fig.\u0026nbsp;2; Fig.\u0026nbsp;3). A shift in the balance of non-RBD- and RBD-specific Spike antibodies towards a greater proportion of non-RBD Spike antibodies may occur within six months after receiving a booster. This shift in antibody profile is associated with subsequent infection, which underscores the pivotal function of RBD antibodies in humoral immune protection (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eThe data presented here support the hypothesis that conserved epitopes are a crucial element in combating symptomatic variant infection by both antibodies and T cells. Post-booster antibodies, which bind to variant epitopes spanning sites relevant for virus-host interactions, may contribute to the most effective protection from variant infection and may serve to compensate for waning antibody titers. Cellular immune protection appears to be primarily provided by conserved epitope-specific T cells. This may be because CD8\u0026thinsp;+\u0026thinsp;T cells are thought to prevent a severe course of disease rather than preventing infection by blocking virus-host cell binding\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the present study is unable to contribute to this part of the hypothesis, as it does not permit the differentiation of the role played by the two branches of the immune system in immune protection.\u003c/p\u003e \u003cp\u003eIt can be concluded that vaccination strategies that exclusively target either conserved or non-conserved epitopes may not be an effective means of establishing an adequate level of protection against infection. Consequently, new vaccines should be designed according to their specific indication. Immunity providing broad cross-variant protection appears to require conserved epitopes, while superior protection may be only achieved by additionally addressing sites required for virus-host cell binding.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe presented data were gathered as part of the \u003cem\u003eMunich Observation Study of Adaptive Immune Response after COVID-19 Vaccination\u003c/em\u003e (MOSAIC). The MOSAIC Study was initiated as an observational cohort study in December 2020 at the German Heart Centre, Munich, Germany. The study includes 640 voluntary employees aged between 18 and 70, vaccinated either with mRNA-based Comirnaty (Pfizer-BioNTech, USA/Germany) or with vector-based COVID-19 vaccine Vaxzevria (AstraZeneca, UK).\u003c/p\u003e \u003cp\u003e107 participants of the MOSAIC study were selected for this sub-study according to the following criteria (Fig. S1):\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eVaccinated with two doses of Comirnaty followed by a single homologous booster immunization 7\u0026ndash;13 months later. Individuals who had received the Vaxzevria vaccine were excluded from the study due to the greater diversity of vaccination schedules and the insufficient number of participants who met the other inclusion criteria.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNo documented SARS-CoV-2 infection until last blood draw and negative test result in two distinct SARS-CoV-2 nucleocapsid-antibody assays performed in our laboratory at TP1 and TP2.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAll employees were offered the option of undergoing a PCR test in our laboratory at their own discretion in the event of a suspected infection. Additionally, all employees were provided with rapid antigen tests for home use (Clungene, Clongene Biotech, Hangzhou, China) on a voluntary basis. The results of these tests were to remain negative for the entirety of the study period (uninfected individuals) or until TP2 (infected individuals). The implementation of this procedure guaranteed the monitoring of the participants with a maximum level of intensity.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe selected subjects had a median age of 50.2 years (min. 23.1, max. 69.9 years), and 67.3% were female.\u003c/p\u003e \u003cp\u003e The study was conducted following the principles of the Declaration of Helsinki, and ethical approval was obtained from the Ethics Commission/Medical Research Ethical Committee of the Technical University Munich (protocol number R20.030). All participants provided written informed consent for participation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBlood collection and infection monitoring\u003c/h2\u003e \u003cp\u003eWhole blood samples were obtained by venipuncture and collected in lithium-heparin tubes (Sarstedt, N\u0026uuml;rmbrecht, Germany).\u003c/p\u003e \u003cp\u003eAll commercial assays were performed as single tests according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics, Mannheim, Germany) was performed on a Cobas E411 Analyzer (Roche); the \u003cem\u003erecom\u003c/em\u003eWell SARS-CoV-2 IgG (Mikrogen, Neuried, Germany) assay was processed on a fully automated Gemini ELISA plate processor (Stratec Biomedical, Birkenfeld, Germany).For virus detection, nasal and throat swabs were processed using an automated Maxwell nucleic acid extractor (Promega, Walldorf, Germany). The qPCR reaction was set up with an automated Mic liquid handling device (Bio Molecular Systems, Australia) using the \u003cem\u003eampli\u003c/em\u003eCube Coronavirus SARS-CoV-2 assay (Mikrogen). The PCR was run on a Myra qPCR cycler (Bio Molecular Systems). The same equipment was used to conduct the \u003cem\u003eampli\u003c/em\u003eMelt SARS-CoV-2 Variants test (Mikrogen) to differentiate virus variants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibody assays\u003c/h2\u003e \u003cp\u003eThe presence of RBD and Spike IgG antibodies and neutralizing antibodies was determined using the V-PLEX SARS-CoV-2 and V-PLEX SARS-CoV-2 (ACE2) assays (MesoScale Diagnostics, Rockville, MD), respectively, on a Meso QuickPlex SQ 120MM multiplex ECLIA plate luminometer (MesoScale) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe following variants and subvariants were identified through the utilization of Panel 22 and Key Variant Panel 1 product variants: A (Wuhan); B.1.1.7 (Alpha); B.1.351, B.1.351.1 (Beta); AY.3\u0026thinsp;+\u0026thinsp;AY.4\u0026thinsp;+\u0026thinsp;AY.4.2\u0026thinsp;+\u0026thinsp;AY.5\u0026thinsp;+\u0026thinsp;AY.6\u0026thinsp;+\u0026thinsp;AY.7\u0026thinsp;+\u0026thinsp;AY.12\u0026thinsp;+\u0026thinsp;AY.14\u0026thinsp;+\u0026thinsp;B.1.617.2\u0026thinsp;+\u0026thinsp;B.1.617.2-ΔY144 (Delta); B.1.1.529\u0026thinsp;+\u0026thinsp;BA.1\u0026thinsp;+\u0026thinsp;BA.1.15 (Omicron); BA.2\u0026thinsp;+\u0026thinsp;BA.2.1\u0026thinsp;+\u0026thinsp;BA.2.2\u0026thinsp;+\u0026thinsp;BA.2.3\u0026thinsp;+\u0026thinsp;BA.2.5\u0026thinsp;+\u0026thinsp;BA.2.6\u0026thinsp;+\u0026thinsp;BA.2.7\u0026thinsp;+\u0026thinsp;BA.2.8\u0026thinsp;+\u0026thinsp;BA.2.10\u0026thinsp;+\u0026thinsp;BA.2.12 (BA.2 subvariants); BA.2.12.1 (Omicron); BA.2.75 (Omicron); BA.4\u0026thinsp;+\u0026thinsp;BA.5 (Omicron).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eT cell assays\u003c/h2\u003e \u003cp\u003eWithin eight hours of blood collection, T cells were stimulated in 500 \u0026micro;l of whole blood from randomly selected subjects at 37\u0026deg;C for 16 (\u0026plusmn;\u0026thinsp;0.5) hours with different peptide pools containing 150 pmol of each peptide (Table S1). The stimulation was terminated by the addition of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche) and stored at -80\u0026deg;C until use. RNA was extracted with a MagNA Pure 96 Cellular RNA Large Volume Kit on a MagNA Pure 96 System (Roche). The relative mRNA expression of IFN-γ, CXCL-10 with the housekeeping marker 60S acidic ribosomal protein P0 (RPLP0) as reference was analyzed with T-Track SARS-CoV-2 Quant PCR (Mikrogen) on a LightCycler 480 Instrument II (Roche) using the T-Track SARS-CoV-2 Analysis Tool (Mikrogen) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Prism 9.5.1 (Dotmatics, USA). Calculated p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Where indicated, data were MIN/MAX normalized.\u003c/p\u003e \u003cp\u003eTo determine if NAb values significantly decreased from TP1 to TP2 (Fig.\u0026nbsp;1c), a one-tailed Wilcoxon signed-rank test was performed against a theoretical median of 0. One-tailed p-values were derived by halving the two-tailed p-values where median differences were negative.\u003c/p\u003e \u003cp\u003eAntibody levels and T-cell stimulation results were compared between infected and uninfected cohorts using the Mann-Whitney test (two-tailed).\u003c/p\u003e \u003cp\u003eA median line through the origin was chosen as the classification criterion for dividing the subjects into two groups in Fig.\u0026nbsp;4, because conserved and non-conserved epitopes were assumed to contribute to the raw signal with dynamically changing ratios. The median line divides the relevant field that is shared by the subjects into two equal parts. The upper left part thus represents subjects with a higher proportion of the wild-type-specific signal than the lower right part. Statistical analyses included two-sided Fisher's exact test for contingency tables, with Koopman asymptotic score method used to calculate relative risk confidence intervals.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource data are provided with this paper. Additional research data may be shared with qualified researchers upon reasonable request to the corresponding author including a scientific proposal, subject to data protection and participant consent restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported byRoche. Mikrogen provided \u003cem\u003erecom\u003c/em\u003eWell SARS-CoV-2 IgG test kits free of charge.\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the Cardiovascular Biobank - Liquids Team for the preparation of the samples and the routine lab team for antibody testing at the Institute of Laboratory Medicine at the German Heart Center, Munich.\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to all study participants for their voluntary involvement in this investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeiko Pfister, Carsten Uhlig, Zsuzsanna Mayer, Eleni Polatoglou, Hannah Randeu, Tabea Berchtold, Silke Burglechner-Praun, Michaela Sander, Stefan Holdenrieder\u003c/p\u003e\n\u003cp\u003eMunich Biomarker Research Center, Institute of Laboratory Medicine, German Heart Center Munich, Technical University Munich, Lazarettstr. 36, D-80636 Munich, Germany.\u003c/p\u003e\n\u003cp\u003eSusanne Sernetz, Felicitas Heitzer, Andrea Str\u0026ouml;tges-Achatz\u003c/p\u003e\n\u003cp\u003eOccupational Medicine Service, German Heart Center Munich, Technical University Munich, Lazarettstr. 36, D-80636 Munich, Germany.\u003c/p\u003e\n\u003cp\u003eLudwig Deml\u003c/p\u003e\n\u003cp\u003eMikrogen GmbH, Anna-Sigmund-Str. 10, 82061 Neuried, Germany.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.H., M.S. and H.P. designed the study. H.P. scientifically conceptualized the study, analyzed the data and wrote the manuscript. M.S and S.H. managed the study and reviewed the manuscript. C.U. conducted quality controls and processing of the data. Z.M. typed the virus variants. E.P. performed qPCR analysis of T cell stimulation assays. H.R. collected blood samples, and anonymized and managed the test subject data. S.S. and F.H. managed and medically supervised vaccination. L.D. advised on the implementation of the T-Track system. A.S.-A. organized and carried out blood sampling and questioning. M.S., S.B.-P. and T.B. conducted T cell stimulation experiments. S.B.-P. conducted the antibody assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Heiko Pfister\u003c/p\u003e\n\u003cp\u003eInstitute of Laboratory Medicine\u003c/p\u003e\n\u003cp\u003eDeutsches Herzzentrum M\u0026uuml;nchen\u003c/p\u003e\n\u003cp\u003eTechnische Universit\u0026auml;t M\u0026uuml;nchen\u003c/p\u003e\n\u003cp\u003eLazarettstra\u0026szlig;e 36\u003c/p\u003e\n\u003cp\u003e80636 M\u0026uuml;nchen\u003c/p\u003e\n\u003cp\u003eGermany\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.H. was part of a SARS-CoV-2 expert panel and received a honorarium from Roche.\u003c/p\u003e\n\u003cp\u003eThe funding agencies had no influence on study design; the collection, analysis, or interpretation of data; the writing of the report; or in the decision to submit the article for publication.\u003c/p\u003e\n\u003cp\u003eAll other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndrews N et al (2022) Covid-19 Vaccine Effectiveness against the Omicron (B.1.1.529) Variant. 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NPJ Vaccines 8:159\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong S, Madewell ZJ, Liu M, Longini IM, Yang Y (2023) Effectiveness of SARS-CoV-2 vaccines against Omicron infection and severe events: a systematic review and meta-analysis of test-negative design studies. Front Public Health 11:1195908\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah M, Woo HG, Omicron (2021) A Heavily Mutated SARS-CoV-2 Variant Exhibits Stronger Binding to ACE2 and Potently Escapes Approved COVID-19 Therapeutic Antibodies. Front Immunol 12:830527\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFumagalli V et al (2024) Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination. Nat Immunol 25:633\u0026ndash;643\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSette A, Sidney J, Crotty S (2023) T Cell Responses to SARS-CoV-2. Annu Rev Immunol 41:343\u0026ndash;373\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5678273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5678273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to investigate the features of immune protection against SARS-CoV-2 infection in a single cohort during the 6\u0026ndash;17 months following booster immunization with an mRNA-based vaccine. The results illustrate the influence of humoral and cellular immunity on the efficacy of the vaccine. Notably, neutralizing antibody titers were found to serve as a reasonably reliable correlate of protection prior to booster immunization. However, this predictive power was largely lost after boosting. The loss appears to be due to the critical remodeling of the humoral immune response following booster immunization. Our findings support the hypothesis that immunity to both conserved and non-conserved epitopes of the viral Spike protein's receptor-binding domain (RBD) is crucial for optimal long-term protection against Omicron infection. While immunity to conserved epitopes may provide cross-variant protection, antibodies targeting non-conserved RBD epitopes play a pivotal role in achieving maximum protection. These observations highlight the critical role of repeated immunization in shaping the immune response landscape and reinforce the necessity of considering both humoral and cellular immune components, alongside intended use considerations, when assessing vaccine efficacy and developing future immunization strategies.\u003c/p\u003e","manuscriptTitle":"Immunity against conserved and non-conserved Spike epitopes after COVID-19 booster vaccination provides long-term protection against symptomatic Omicron infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 11:20:44","doi":"10.21203/rs.3.rs-5678273/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9cd13bc0-4fd3-4210-91cd-98561bc1edf0","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42295696,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":42295697,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":42295698,"name":"Biological sciences/Immunology/Adaptive immunity/Humoral immunity/Antibodies"},{"id":42295699,"name":"Health sciences/Medical research/Outcomes research"}],"tags":[],"updatedAt":"2025-01-08T11:20:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 11:20:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5678273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5678273","identity":"rs-5678273","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}