SARS-CoV-2 infection imprints neutralising antibody responses in the absence of vaccination

SARS-CoV-2 infection imprints neutralising antibody responses in the absence of vaccination | 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 Letter SARS-CoV-2 infection imprints neutralising antibody responses in the absence of vaccination Ravindra Gupta, Adam Abdullahi, Rebecca Morse, Mark Cheng, Fehintola ige, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6347062/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 Immune imprinting refers to a phenomenon where a first viral encounter shapes subsequent immune responses against antigenically distinct variants, favouring the recall of memory B cells against cross‐reactive epitopes in lieu of de novo responses. This bias can lead to suboptimal neutralising antibody protection when subsequent infections occur with highly divergent viral lineages such as the SARS-CoV-2 Omicron lineage viruses. Given the rapid rollout of Wu-1-based vaccines in many countries before the emergence of Omicron, prior studies have demonstrated that vaccination with ancestral Wuhan-Hu-1 (Wu-1)-based vaccines results in diminished neutralisation of the Omicron variants compared to Wu-1, even after recent Omicron exposure. The effects of imprinting induced by natural infection in the absence of vaccination is unknown, however, particularly in populations where Omicron infection preceded Wu-1-based vaccination. This presents a critical gap in our knowledge given the different dominance hierarchies of neutralising antibody responses between vaccines and natural infection. We therefore assessed humoral responses in a population exposed to pre-Omicron (ancestral) and post-Omicron variants prior to receiving any vaccine. Using a binding antibody assay to distinguish variant-specific exposures, we found that 93% of participants had serological evidence of ancestral SARS-CoV-2 infection, while 58% had evidence of Omicron exposure. In individuals exposed solely to pre-Omicron variants, neutralisation titres against Wu-1 were significantly higher than those against Omicron variants, as expected. Paradoxically, participants with evidence of both pre-Omicron and Omicron infections also exhibited higher neutralisation titres for Wu-1 relative to Omicron BA.1, despite the ancestral exposure occurring ~18 months earlier and Omicron being the most recent infection. We confirmed these findings in another independent Nigerian cohort where Omicron exposure was even more prevalent (94%), and all participants had evidence of pre-Omicron infection. The findings suggest imprinted immunity from the ancestral pre-Omicron lineage viruses, and remarkably these early responses to Wu-1 were able to dominate over more recent, likely multiple, Omicron lineage infections. To directly assess imprinting, we performed serum antibody depletion experiments using Wu-1 spike protein as bait. In nearly all participants, depletion of Wu-1-specific antibodies resulted in complete abrogation of serum neutralising activity against both Wu-1 and Omicron spike pseudotyped viruses. Furthermore, during prospective follow-up, additional Omicron infection and contemporaneous administration of Wu-1-based vaccine boosted neutralising responses across variants, partially equalizing titres between Wu-1 and Omicron. However, Omicron responses did not surpass ancestral responses, suggesting only partial mitigation of imprinting. These data demonstrate the presence of immune imprinting against SARS-CoV-2 in the absence of vaccination and its persistence thereafter. The dominance of ancestral responses in unvaccinated populations in the post-Omicron era suggests the preferential use of an Omicron variant-based vaccine in lieu of Wu-1-based vaccines to override imprinting and achieve broader protection, particularly in vulnerable populations such as the elderly or those with compromised immunity. These data also inform preparation against future pandemic zoonoses with rapidly evolving viruses, cautioning against over-reliance on vaccines against ancestral viruses. Biological sciences/Immunology/Vaccines/DNA vaccines Biological sciences/Microbiology/Virology/SARS-CoV-2 Figures Figure 1 Figure 2 Figure 3 Introduction Despite high population-level SARS-CoV-2 immunity driven by previous global exposure and vaccination efforts, SARS-CoV-2 variants, evolving in immune compromised hosts 1–6 , are continuously emerging and transmitting amongst the human population. Vaccine-derived immunity remains the most robust long-term strategy for protection against SARS-CoV-2 and can be measured by binding/neutralising antibodies as surrogate measures of protective humoral immunity 7–9 . The continued circulation of SARS-CoV-2 has been driven by its ability to escape population immunity through acquiring mutations in the highly immunogenic spike protein. The significant antigenic shift represented by Omicron (B.1.1.529) resulted in a radically different virus characterized by multiple mutations in spike with extreme immune evasion of vaccines and increased transmissibility 10–17 . Immunological imprinting refers to a phenomenon where primary exposure to a pathogen shapes future immune responses to related strains 18–20 . This leads to favouring recall of existing memory B-cell responses rather than de novo responses to newer variants and thereby limits the potency and breadth of humoral responses. 21–24 Immune imprinting has been observed and well-documented for SARS-CoV-2 via a first encounter with ancestral Wu-1-based SARS-CoV-2 vaccination followed by Omicron-based vaccination or infection. The imprinting effect is marked by Wu-1-skewed neutralising antibodies despite recent Omicron exposure due to memory B cell recall and limited de novo responses to Omicron 8,25–31 , highlighting the dominant effect of prior exposure in shaping immune responses 32 . Studies have demonstrated that depletion of Wu-1 spike-binding antibodies abrogates cross-neutralising antibody responses, particularly in individuals without prior Omicron exposure, underscoring constrained neutralisation breadth due to Wu-1-based imprinting 32,33 . This effect persists even with updated vaccines targeting Omicron subvariants, such as XBB.1.5, which still elicit responses dominated by recall of pre-existing memory B cells 33 . In addition to imprinted responses against Omicron-based vaccines, immune imprinting by ancestral SARS-CoV-2 has further been shown to persist despite multiple Omicron infections 34 . Most SARS-CoV-2 imprinting studies have focused on ancestral Wu-1-based vaccination as the primary viral encounter 31,35–42 before Omicron exposure. Previous work has demonstrated that SARS-CoV-2 imprinting varies by exposure type 31,35–42 : infection skews responses toward S2 and NTD epitopes and induces stronger B cell receptor clonal expansion and higher somatic hypermutation rates 43,44 , while vaccination predominantly recalls RBD-targeting antibodies 37 . These findings underscore the importance of exposure sequence in shaping subsequent immunity, reinforcing the need to examine whether individuals with Omicron infection before vaccination exhibit distinct imprinting outcomes. Exposure sequences of Omicron infection prior to vaccination are particularly relevant for the African continent because vaccine access has been sparse across the African continent 45 with only around one-third of the continental population receiving a two-dose vaccination series as of October 2023 46 . Consequently, a significant proportion of individuals likely first encountered SARS-CoV-2 via natural infection, including with Omicron-lineage viruses, before vaccination. These circumstances provide an unprecedented opportunity to answer the question of whether and how immunological imprinting occurs in the context of prior Omicron infection without preceding vaccination. Here, we first validate the use of a serological assay to distinguish exposure to Omicron and ancestral pre-Omicron variants in an unvaccinated population receiving primary vaccination in January 2023. Thereafter, we demonstrate in two independent cohorts that despite recent Omicron exposure, Omicron neutralising antibody responses are paradoxically lower or equal to Wu-1 responses. Next, we show that depletion of antibodies in individuals with both pre-Omicron and Omicron infections leads to complete loss of neutralisation against Omicron BA.1 and BA.2, providing further evidence that imprinting occurs following infection with SARS-CoV-2. Finally, we measure neutralising antibody titres after Wu-1-based vaccination and demonstrate that imprinting by ancestral pre-Omicron variants is partially mitigated by experiencing Omicron exposures through both pre-vaccination Omicron infection and post-vaccination breakthrough infection. Results Study population characteristics and SARS-CoV-2 exposure At study entry in January 2023, our population comprised 101 confirmed HIV negative participants with no documented history of prior COVID-19 vaccination in Abuja, Nigeria (Cohort 1, Figure 1a ). The median age was 33 years (inter-quartile range: 26-41) and the cohort was 51% female ( Table 1 ). To analyse population-level exposure to SARS-CoV-2 at study entry in January 2023, when Omicron was the dominant circulating variant of concern (VOC), we tested all participants at study entry (T0, n = 101) for IgG anti-N, IgG anti-S and IgG anti-RBD against SARS-CoV-2 Wu-1 (anti-RBD-Wu-1) using a validated Luminex bead-based flow cytometric assay 47 . We found 94/101 (93%), 98/101 (97%), and 97/101 (96%) of participants positive for IgG anti-N, IgG anti-S and IgG anti-RBD-Wu-1, respectively, reflecting endemic population level exposure to SARS-CoV-2 ( Figure 1b ). To further evaluate population-level exposure to SARS-CoV-2 Omicron variants, we tested all participants for Omicron (BA.1)-specific IgG anti-RBD (anti-RBD-Omicron) and found 59/101 (58%) positive for IgG anti-RBD-Omicron (Figure 1b). Therefore, despite near-universal exposure to pre-Omicron variants, only a subset of participants was exposed to Omicron variants. The RBD mutational profile of BA.1 is similar to BA.2 (Supplementary Figure 1a). The prior exposure(s) during 2022 resulting in positive IgG anti-RBD-Omicron is/are very likely to have been BA.1 or BA.2/BA.4 based on genomic surveillance and reported cases in Nigeria (Supplementary Figure 1b) . BA.4 has two additional mutations over BA.2 at positions 452 and 486 48 . Given the global waves of SARS-CoV-2 variants, this pattern of exposure aligns with the broader epidemiological context. Since 2020, the ancestral strain accounted for ~40% of cases; followed by Alpha/Eta in early 2021 (~24%), Delta in late 2021 (18%) and various Omicron subvariants comprising ~18% of cases from 2022 onward ( Supplementary Figure 1b ). This distribution is further supported by regional genomic surveillance data, which highlights distinct lineage dynamics in Nigeria, including the dominance of the Eta lineage over Alpha and the emergence of a rare Delta AY.36 sub-lineage 49 . This timeline suggests that most study participants were likely to have encountered pre-Omicron variants before Omicron infection at study entry, consistent with near universal antibodies against Wu-1 spike observed at study entry. Imprinted immunity is evident from pre-vaccination serum neutralisation profiles We first sought to measure pre-vaccination serum neutralisation responses to evaluate imprinting in this previously unvaccinated population. We used pseudotyped viruses as previously described 47,50,51 in (n = 45) participants against ancestral Wu-1, BA.1, BA.2, BA.4 and the antigenically shifted XBB. The latter variant did not circulate until later in 2023 and therefore we expected very little neutralising antibody activity in our cohort regardless of exposure history. Characteristics and binding antibody data of this sub-population were comparable to the general cohort (Table 1, Figure 1b). In participants with pre-Omicron variant exposure but no serological evidence of prior exposure to Omicron BA.1 (IgG anti-RBD-Omicron negative, n = 16), the Geometric Mean titre (GMT) neutralisation response against Wu-1 (GMT: 1243) was relatively higher or nearly equal to Omicron variants BA.1, BA.2 and BA.4 (GMT: 658, 1099 and 1387, respectively), representing ~1.1- to 1.9-fold lower neutralisation responses relative to Wu-1. However, consistent with imprinting, serum neutralisation titres in participants with serological evidence of prior exposure to both Wu-1 and Omicron BA.1 (IgG anti-RBD-Omicron positive, n = 29) were paradoxically almost 2-fold higher for Wu-1 (GMT: 4281) compared to Omicron BA.1, BA.2 and BA.4 (GMT: 2322, 2104 and 2112, respectively) (Figure 1c) , although observations did not reach statistical significance adjusted for multiple comparisons. In the absence of imprinting, individuals with recent Omicron infection (IgG anti-RBD-Omicron positive) would be expected to elicit stronger neutralising responses against Omicron. In contrast, titres against Wu-1, reflecting exposure to a distant pre-Omicron infection as indicated by anti-RBD-Wu-1 and anti-N positivity, should have waned over time in the absence of imprinting. Titres against the antigenically shifted XBB Omicron variant were very low (mean GMT < 100) even in those with prior exposure to Omicron BA.1, BA.2 and BA.4. These data demonstrate the specificity of the screening IgG anti-RBD-Omicron antibody assay for early (pre-XBB) Omicron variants to which our study participants were exposed. In order to further demonstrate the utility of using binding for IgG anti-RBD-Omicron to assign Omicron lineage exposure, we assessed magnitude and breadth of neutralising antibody responses prior to vaccination. Magnitude-breadth curves are Kaplan-Meier curves using the neutralisation data against all tested variants, where the “event of interest” is the failure to neutralise any variant, and the “time-to-event” on the x-axis is the 50% neutralising titre (NT 50 ) for the same variant ( Figure 1d ). At baseline, we compared ancestral virus-exposed and Omicron-naïve participants to ancestral virus-exposed and Omicron-exposed participants and observed that Omicron-naïve participants exhibited 2.9-fold (p < 0.05) lower magnitude-breadth relative to Omicron-exposed participants. This clear distinction in neutralisation profiles between groups supports the robustness of our screening assay in identifying prior Omicron lineage exposure. To confirm the phenomenon of imprinted neutralising antibody responses in unvaccinated individuals, we next characterised neutralisation profiles in another independent, unvaccinated Nigerian cohort recruited in early 2023 as part of a SARS-CoV-2 fractional dosing clinical trial (PACT202206754734018) 52 (Cohort 2, Supplementary Table 1 ). In cohort 2, (n = 64) samples were available; of these 64 participants, 94% (n = 60/64) showed serological evidence of Omicron exposure characterized by positivity for IgG anti-RBD-Omicron ( Supplementary Figure 2a ). This reflects a higher degree of exposure to Omicron in this cohort relative to cohort 1. Nonetheless, the GMT neutralisation response ( Supplementary Figure 2b ) against Wu-1 (GMT: 1492) was higher than against BA.1 Omicron (GMT: 843, 1.8-fold) and was similar for BA.2 (GMT: 1316, 1.2-fold). The relatively higher BA.2 titres compared to BA.1 (~1.6-fold) likely reflect more recent exposure to BA.2 lineage viruses, which predominated circulation in 2023, and expansion of cross-reactive B cells with highest affinity for BA.2 spike ( Supplementary Figure 1b ). Notably, neutralisation titres against Wu-1 remained robust despite more than 18 months elapsing since the last exposure to an ancestral Wu-1 lineage and 94% of Cohort 2 experiencing Omicron exposure, underscoring the durability of imprinted humoral responses. Taken together, these data from disparate regions of Nigeria further support immunological imprinting via exposure to ancestral pre-Omicron lineages ( Supplementary Figure 2 ). Depletion of serum binding antibodies against ancestral Wu-1 spike confirms imprinting To test the hypothesis that observed neutralisation responses were driven by imprinted immunity, we performed targeted depletion of plasma antibodies against the Wu-1 spike protein from individuals with pre-vaccination exposure to pre-Omicron variants only (n = 8) and both pre-and post-Omicron variants (n = 21) in Cohort 1. Using a previously described method 34 , suspension Expi293 cells were transfected with Wu-1 spike-expressing plasmid and incubated for 72 hours, after which participant sera were passed over the cells ( Figure 2a ). This process allowed surface-expressed spike to selectively bind and deplete Wu-1-specific antibodies from the serum. Following depletion, we assessed residual neutralisation against Wu-1, BA.1 and BA.2 viruses. Consistent with the hypothesis of Wu-1 imprinting via infection, we observed a complete loss of neutralising activity against Wu-1, BA.1 and BA.2 viruses in 7/8 (88%) pre-Omicron exposed participants and 20/21 (95%) pre- and post-Omicron exposed participants following depletion ( Figure 2b ). We confirmed successful depletion by measuring Wu-1 spike-specific binding antibodies with Luminex in a subset (n = 9) of pre- and post-depletion samples that included two individuals who failed to show loss of neutralisation following depletion (Figure 2c ). We did not find any significant difference between ages of participants who showed neutralisation antibody abrogation (n = 27) relative to those that did not (n = 2), with a median age of 35 years (p = 0.97).abbas Impact of Omicron breakthrough infection and contemporaneous Wu-1 vaccination All participants underwent Wu-1-based vaccination. Between study entry and one month post-first dose, around half of the participants with neutralisation response data (n = 23/45) experienced a breakthrough infection (BTI, Supplementary Figure 3) . In those with prior Omicron exposure at baseline (n = 29), participants demonstrated increased neutralisation titres across ancestral and Omicron lineage viruses post-vaccination regardless of BTI status ( Supplementary Figure 4a ). In participants without prior Omicron exposure at baseline (n = 16), only those who experienced BTI between study entry and 1-month post-dose 1 demonstrated an increase in neutralisation titres across ancestral and Omicron lineage viruses, whereas those without BTI showed a tendency for increased titres, albeit nonsignificant ( Supplementary Figure 4b ). Taken together, these data suggest that Wu-1-based vaccination was associated with an increase in neutralising response activity against ancestral pre-Omicron viruses, and neutralisation against Omicron lineage viruses was boosted in participants with any prior exposure to Omicron. We first assessed individuals with no BTI post-vaccination (n = 22/45). In participants who had no prior Omicron exposure (n = 5), vaccination exacerbated the gap between neutralisation of Wu-1 (GMT:5095) and Omicron variants. Relative to Wu-1, we observed between 2.4- to 4.4-fold lower neutralisation responses for Omicron variants BA.1 (GMT:1153), BA.2 (GMT:2059) and BA.4 (GMT:2103), as well as ~58-fold lower responses for XBB (GMT:87) (Figure 3a [top, left]). In those with prior Omicron exposure at baseline (n = 17), we similarly observed a widening of the gap between neutralisation of Wu-1 (GMT:17065) versus Omicron variants following vaccination. Relative to Wu-1, we observed ~3-fold lower neutralisation responses for Omicron variants BA.1 (GMT:5073), BA.2 (GMT:5506) and BA.4 (GMT:5546), as well as ~123-fold lower responses for XBB (GMT:139) ( Figure 3a [top, right] ). We further investigated individuals with BTI post-vaccination (n = 23/45). During the pre- and post-vaccination timepoints, Omicron was the dominant circulating variant ( Supplementary Figure 1b ) and was likely responsible for breakthrough infections between vaccine administration and 1-month post-dose one. In participants with no prior Omicron exposure at baseline (n = 11), contemporaneous Wu-1-based vaccination and Omicron BTI resulted in similar neutralisation titres (~1.2-fold lower) relative to Wu-1 (GMT:8258) for Omicron variants BA.1 (GMT:6554), BA.2 (GMT:6720) and BA.4 (GMT:6960), as well as ~25-fold lower responses for XBB (GMT:331) ( Figure 3a [bottom, left]) . In individuals with both prior Omicron exposure at baseline and BTI (n = 12), neutralisation titres were again similar relative to Wu-1 (GMT:13845) for Omicron BA.1 (GMT:10022, 1.4-fold lower) and nearly equal for Omicron BA.2 (GMT:13699) and BA.4 (GMT:13810), as well as ~103-fold lower for XBB (GMT:135) ( Figure 3a [bottom, right]). The slightly lower titres of BA.1 compared to BA.2 and BA.4 are expected given that the BTI was epidemiologically most likely to be a BA.2 descendent. To quantify the magnitude and breadth of neutralising antibody responses induced by Wu-1-based vaccination, we generated magnitude-breadth curves of responses. At 1-month post-dose 1 (T1) compared to baseline (T0), we observed an approximately 3-fold increase in the magnitude-breadth AUC in the Omicron-naïve participants and a 2-fold increase in the Omicron-exposed participants, demonstrating improved neutralisation post-vaccination (Figure 3b). Next, we conducted a sub-analysis with stratification by BTI at 1-month post-dose 1 (T1). Individuals with no Omicron exposure both pre- and post-vaccination (n = 5) had persistently lower magnitude-breadth compared to those who had at least one Omicron exposure at any timepoint (5.9-fold difference, p = 0.03). Interestingly, the sequence of Omicron exposure affected magnitude-breadth outcomes: participants with pre-vaccination Omicron exposure and no BTI (n = 17) exhibited greater magnitude-breadth than participants with post-vaccination Omicron BTI exposure only (n = 11, p < 0.05). In contrast, having both pre-vaccination Omicron exposure and a BTI post-vaccination elicited the highest magnitude-breadth response ( Figure 3b) . These findings suggest that hybrid (infection and vaccine)-induced immunity with pre-Omicron variants alone isinsufficient to broaden the antibody response and target the antigenically shifted Omicron variants. Taken together, these data show partial mitigation of imprinting by repeated Omicron exposure. Discussion Understanding how sequential SARS-CoV-2 exposures shape immune responses is critical to anticipating population-level protection and informing future vaccine strategies. Devastating consequences may arise where a pathogen rapidly becomes more immune evasive and increases pathogenicity, but individuals are unable to induce effective de novo responses to neutralise the threat due to prior immune imprinting by an ancestral variant.Indeed, it is well-documented how immune imprinting by ancestral Wu-1-based SARS-CoV-2 vaccination drives responses to antigenically distinct variants against conserved epitopes and limits de novo antibody generation. However, due to the rapid scale-up of vaccination before emergence of the antigenically distinct Omicron lineage, it is not well-established whether and how immune imprinting occurs in the context of SARS-CoV-2 infection. Here we provide robust evidence for immune imprinting in the absence of vaccination. At study enrolment in early 2023 for Cohort 1, nearly all individuals demonstrated infection by pre-Omicron variants. These data are supported by previous studies showing high seroprevalence for Wu-1 binding antibodies and serum neutralising activity in Nigeria and Ghana in 2021, as well as high rates of BTI by the Delta variant post-vaccination in a 2021 cohort of Nigerian healthcare workers 16,47,53,54 . The participants with no evidence of pre-vaccination exposure to Omicron (Pre-Om inf ) demonstrated significantly lower titres of serum neutralising antibodies against Omicron variants compared to Wu-1, as expected. Paradoxically, the 58% of participants who were IgG anti-RBD-Omicron positive pre-vaccination (Pre-Om inf + Om inf ) showed similar or lower titres of serum neutralising antibodies against Omicron variants compared to Wu-1, despite pre-Omicron viruses last circulating more than a year prior. These results were further bolstered by a comparable unvaccinated Nigerian cohort (Cohort 2) in which 94% of participants had evidence of prior exposure to Omicron (Pre-Om inf + Om inf ), yet titres against Omicron BA.1 were lower compared to Wu-1, suggesting recall of Wu-1-targeting memory B cells instead of de novo response induction. We further supported these results by depleting sera of Wu-1-binding antibodies, which abrogated neutralisation against both Wu-1 and Omicron-lineage variants in 27/29 of participants. Finally, we showed that repeated Omicron exposure partially mitigated imprinting even after Wu-1 antigen delivery via vaccination. Suboptimal neutralisation responses to the highly divergent SARS-CoV-2 lineage Omicron have been observed in animal models and individuals previously vaccinated with a primary course of ancestral Wu-1-based vaccine, consistent with immunological imprinting by the vaccine 24,55,56 . Data show that individuals primed with a Wu-1-based vaccine who are then exposed to Omicron will primarily recall pre-existing memory B cells that recognise conserved multi-variant epitopes rather than generate de novo Omicron-specific responses from naïve B cells, thereby limiting breadth of responses 22–24 . For individuals primed with the attenuated live Coronavac vaccine followed by two Omicron infections, comparable neutralising antibody levels against ancestral and Omicron strains were reported, as well as increased frequencies of Omicron-specific memory B cells, suggesting mitigation of imprinting 56 . In contrast, no mitigation of imprinting was observed in individuals primed at least 3 times with Wu-1-based mRNA vaccines who were then exposed to Omicron twice via a combination of Wu-1/BA.5 bivalent vaccination and either Omicron BTI or XBB.1.5 monovalent vaccination.These studies observed the highest neutralising titres against Wu-1 and no significant Omicron-specific memory B cell response 33,55 . Interestingly, when all Wu-1 binding antibodies were absorbed and the remaining sera were tested for neutralisation, BA.5 and XBB.1.5 binding titres and neutralisation potency were ablated, which is consistent with our findings that absorption of Wu-1 antibodies abrogates neutralisation responses against BA.1 and BA.2. These results suggest that all Omicron antibodies were cross-reactive due to recall and expansion of the memory response induced by ancestral Wu-1 imprinting 33,55 . It is notable, however, that two individuals in our study who had confirmed removal of Wu-1 spike-binding antibodies were still able to neutralise Wu-1 and Omicron variants. Previous work has demonstrated de novo antibody generation after multiple Omicron exposures in individuals who received inactivated or recombinant SARS-CoV-2 vaccination 34,56 . Alternatively, the neutralisation of Wu-1 despite removal of Wu-1-binding antibodies could be due to targeting of cryptic sites of spike, which are only exposed after ACE2 binding induces conformational changes 57 and therefore would not be depleted by spike in the absence of ACE2. Future work should aim to elucidate the mechanism behind our observations, requiring cells and generation of monoclonal antibodies. With regards to mitigation of imprinting in our study participants, those exposed to Omicron both pre- and post-vaccination demonstrated neutralisation titre equalization for Wu-1 and BA.1, BA.2 and BA.4 Omicron variants. In contrast, those exposed pre-vaccination but without BTI had around 3-fold lower titres against Omicron variants. This provides evidence that infection-induced ancestral imprinting is partially mitigated by repeated exposure to Omicron variants, but that further Wu-1 antigen delivery via vaccination could exacerbate ancestral imprinting and hinder de novo responses against Omicron. Our study was subject to limitations. Our study does not include data on cellular immune responses, although we were able to demonstrate the phenomenon of imprinting using sera from two independent contemporaneous cohorts. Secondly, we were unable to distinguish by genomic sequencing the exact variants to which participants were previously exposed. Given up to half of infections are asymptomatic 58,59 , there is no practical strategy for accurately detecting and recording infections over extended periods of time. Instead, we validated the use of serology, specifically binding and neutralising antibody profiling, to determine pre- and post-Omicron variant exposure that reflected the limited population-level genomic surveillance data available in Nigeria. In summary, these data uncover immune imprinting following SARS-CoV-2 infection in the absence of vaccination. This imprinting could be mitigated by repeat infection with Omicron even with an exposure to Wu-1-based vaccination between Omicron exposures. In other present-day unvaccinated populations (and indeed vaccinated populations), use of monovalent Omicron variant-based vaccines should be used in preference to Wu-1-based and bivalent vaccines to override imprinting by ancestral pre-Omicron strains and provide broader protection for vulnerable populations such as the elderly or those with compromised immunity. As the chance of long COVID and other serious sequelae rise with each infection 58,59 and are more common in the absence of vaccination, it is crucial to understand how imprinting may be mitigated in diverse populations. More importantly, these data serve as a reminder of the sometimes-deleterious effects of imprinting and caution against over-reliance on ancestral vaccines as protective measures against rapidly evolving pathogens. Declarations Acknowledgements A.A. was supported by Cambridge-Africa award and Harvard Takemi Program in International Health. RBM was supported by the Harding Distinguished Postgraduate Scholars Programme. RKG was supported by a Wellcome Trust Senior fellowship (WT108082AIA). This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312) and Open Philanthropy. D.J.S. and S.T. are supported by the NIH NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract 75N93021C00014 as part of the SAVE program. D.J.S. and S.T. are supported by the NIH NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract 75N93021C00014 as part of the SAVE program. Competing interests The authors declare no competing interests. Author contribution: Study conceptualization and design: A.A and RKG Study management, supervision and fieldwork: A.A., E.O, M.E and A.Ab., Methodology and Investigation: A.A., R.B.M., E.O., M.T.K.C., M.E., S.T., F.I., A.K., B.S., H.W.A., E.J., O.B., A.S., H.A.-O., A.J., A.M., O.O., B.I., O.E., A.A., E.O., S.O., I.M.K., S.H.A., B.M., L.R., R.D., W.W., D.S., B.S. Research Data Curation & Analysis: A.A., R.B.M., M.T.K.C., F.I., A.K., B.S., A.Ab., and R.K.G. Funding Acquisition: A. A, A.Ab, B.S and RKG Writing : A.A. drafted the original manuscript, and R.B.M. provided critical editorial input, with contributions from all authors. All authors approved the final version of the manuscript Methods Study population and sampling: Cohort 1: HIV Negative participants across three clinical sites affiliated with Institute of Human Virology Nigeria (IHVN), Abuja, were actively recruited and asked to participate in this study. Eligible participants were men and non-pregnant women >18 years old who had no previous SARS-CoV-2 vaccination and were confirmed HIV negative using the Nigerian national HIV rapid testing algorithm 60,61 . As interim recommendations and guidelines highlighted the use of a two-dose regimen of Ad26.COV2.S vaccine given 2 months apart due to improved vaccine efficacy 62 , participants were administered a second dose two months following the first dose. Following signed informed consent, participants were recruited in this prospective observational cohort study. The study was comprised of adult participants eligible to receive their first-dose vaccination between 23 rd January 2023 to 20 th April 2023. Participants were recruited by i) local community outreach and ii) through phone calls to previously registered patients across three health facilities in Abuja, Nigeria. Participants provided plasma samples at baseline (prior to first-dose, T0) and 1-month post-dose 1 (T1). Cohort 2: Pre-vaccination samples from individuals in the SIFCoVAN trial across Nigeria recruited in 2023 were tested for binding antibodies and pseudotyped virus neutralisation across VOC. The protocol was registered with the Pan African Clinical Trials Registry (PACTR) PACTR 202206754734018. Laboratory methods and sample testing: Binding antibody measurement Binding IgG antibodies (Abs) against SARS-CoV-2 trimeric spike protein (S), nucleocapsid protein (N), Wu-1 D614G and Omicron (BA.1)-specific receptor-binding domain (RBD) were measured using the Luminex-based SARS-CoV-2-IgG assay by flow cytometry as previously detailed 16,47,53,54 . The assay was validated using pre-pandemic serum samples and robustness of the defined cut-off is shown in Supplementary Figure 5 . We defined positive total anti-S antibody (anti-S) as anti-S IgG above cut-off of 226.48 mean fluorescence intensity (MFI), positive total RBD as anti-RBD-Wu-1 above cut-off of 411.9, and positive total anti-RBD-Omicron above cut-off of 729. Cut-offs were defined based on analysis of ‘true’ positive (convalescent) and negative pre-pandemic samples. We defined previous SARS-CoV-2 infection as positive anti-N IgG above a cut-off of 1472.8. Neutralisation assays For plasma neutralising antibody measurement, SARS-CoV-2 lentiviral pseudotyped viruses were prepared by transfecting HEK293T cells with Wu-1 D614G, BA.1, BA.2, BA.4 and XBB plasmids as previously described in detail 50,63 . There is evidence to show a high correlation between pseudotyped virus and live virus neutralisation 64,65 . All neutralisation assays were repeated in two independent experiments containing two technical replicates for each condition. Antibody depletion assay Cell culture Expi293F suspension cells were maintained at 37°C and 8% CO2 with constant rocking in 125-ml vented Erlenmeyer flasks with Expi293 Expression Medium. One day prior to serum absorption-neutralisation experiments, 96-well F-bottom cell culture microplates were plated with 2 x 10 4 HeLa-ACE2 cells per well and maintained in DMEM supplemented with 10% FBS. Expi293F cell transfection Expi293F cells were sub-cultured at least three times before transfection. Four days prior to experiments, cells were passaged to a density of 2.5-3 x 10 6 viable Expi293F cells/ml. Three days prior to experiments, Expi293F cells were transfected with a mixture of Opti-MEM Reduced Serum Medium, Fugene HD transfection reagent, and either plasmids expressing Wuhan-Hu-1 spike or no plasmid (mock transfection control). Serum absorption On the day of the experiment, 72 hours post-transfection, Expi293F cells were centrifuged and washed twice with warm Expi293 Expression Medium, then resuspended at 40 x 10 6 cells/ml. Heat-inactivated serum samples were initially diluted 1:10 in warm Expi293 Expression Medium and split into two fractions for absorption by Wuhan-Hu-1 spike-transfected Expi293F cells or mock-transfected Expi293F cells. For each individual, Expi293 Expression Medium containing 8 x 10 6 Expi293F cells were mixed 1:1 (v/v) with 1:10 diluted serum in 1.5-ml microcentrifuge tubes. The mixtures were incubated for 1 hour at 37°C and shaken at 900 rpm in an Eppendorf Thermomixer. After incubation, Expi293F cells were centrifuged, and the supernatant was transferred to a new microcentrifuge tube for re-centrifugation. Expi293 Expression Medium was added to the clarified supernatant such that all absorbed serum samples were now diluted 1:20. Absorbed serum with a dilution of 1:20 were then added to 96-well cell culture F-bottom plates in duplicate and serially diluted 1:3 in DMEM supplemented with 10% FBS. Pseudotyped Wuhan-Hu-1, BA.1, and BA.2 viruses were added as previously described to the serial dilutions of absorbed sera, mixed and incubated at 37°C and 5% CO2 for 1 hour. After the incubation of absorbed sera and pseudotyped viruses, these mixtures were added to the plates of HeLa-ACE2 cells prepared the day prior to experiments. The final dilution of serum was 1:58.33. Neutralisation plates were incubated and read as previously described. RBD Alignment: The Spike sequences for Wuhan-Hu-1 (NC_045512.2), BA.1 (OL672836.1), BA.2 (OM371884.1), BA.4 (ON373214.1), BA.2.86 (OR775659.1) and XBB (XBB 1.5 OP790748.1 + S: P486S) were aligned using MAFFT 66 . SARS-CoV-2 spike RBD residues 319-541 were aligned. Asterisks indicate completely conserved columns in the alignment. The receptor binding motif (RBM) at residue 438-506 is indicated by + highlighted in yellow. Epigenomic map Genomic epidemiological data from GISAID was representatively sampled and visualised using Nextstrain. 67 To further break down the cases in Nigeria in both the pre-Omicron and post-Omicron study periods, the Global initiative on Sharing All Influenza Data (GISAID) database 68 was queried using Outbreak.info API 69 for all SARS-CoV-2 sequences collected from Nigeria. Subclades of WHO-assigned Variants of Concern (VOCs) were assigned their WHO name and Nextstrain-assigned clade. To account for the changing number of SARS-CoV-2 cases, we weighed the number of sequences attributed to each VOC by weekly new cases reported to the WHO (https://data.who.int/dashboards/covid19/data): Magnitude breadth analysis To capture the relationship between the magnitude (NT50) and breadth (percentage of VOCs neutralised) of serum neutralisation, we plotted magnitude-breadth curves, a framework previously used in analysing HIV 70–72 and SARS-CoV-2 73 humoral responses against multiple strains. Magnitude-breadth curves are Kaplan-Meier curves using the neutralisation data against all tested variants, where the “event of interest” is the failure of neutralisation of any variant, and the “time-to-event” on the x-axis is the NT 50 for the same variant. Curves are modelled using the R package Survival v3.8-3 74,75 , and plotted using the R package survminer 0.5.0. 76 In addition, the areas under the curve (AUC) were calculated for both individual curves and aggregated curves were calculated using the “km” method in package AdjustedCurves v0.11.2 to facilitate comparison 77 . Definition of terms and statistical analysis: We defined vaccine breakthrough infection as a ≥2-fold increase in IgG anti-RBD-Omicron levels relative to study entry timepoint ( Supplementary Figure 3 ). Geometric Mean Titre (GMT) with standard deviation (SD) of neutralisation antibody was calculated across time points. Baseline characteristics of participants were expressed as proportions and percentages for categorical variables and median inter-quartile range (IQR) for continuous variables. Two-tailed Wilcoxon signed-rank tests or Friedman’s Test with Holm-corrected post-Hoc Wilcoxon test was used to compare neutralisation antibody titres. 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USA","correspondingAuthor":false,"prefix":"","firstName":"Leah","middleName":"","lastName":"Rosenzweig","suffix":""},{"id":438511619,"identity":"5e158a4a-57e8-4ad4-a1da-3e91048c2cf2","order_by":26,"name":"Derek Smith","email":"","orcid":"https://orcid.org/0000-0002-2393-1890","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Derek","middleName":"","lastName":"Smith","suffix":""},{"id":438511620,"identity":"2fa35379-b8c8-41a6-b8be-795b3c9ac454","order_by":27,"name":"Rainer Doffinger","email":"","orcid":"","institution":"CUH NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Rainer","middleName":"","lastName":"Doffinger","suffix":""},{"id":438511621,"identity":"cfffbae7-0877-4577-8826-cdcf0183a9f6","order_by":28,"name":"Witold Więcek","email":"","orcid":"","institution":"University of Chicago, Chicago, IL, USA","correspondingAuthor":false,"prefix":"","firstName":"Witold","middleName":"","lastName":"Więcek","suffix":""},{"id":438511622,"identity":"9f5fc0f6-1d8a-4e66-b5b1-7d55541693f0","order_by":29,"name":"Babatunde Salako","email":"","orcid":"","institution":"NIMR","correspondingAuthor":false,"prefix":"","firstName":"Babatunde","middleName":"","lastName":"Salako","suffix":""},{"id":438511623,"identity":"399b3fe0-0242-4256-b350-e71bd7a1efc7","order_by":30,"name":"Alash'le Abimuku","email":"","orcid":"","institution":"IHVN","correspondingAuthor":false,"prefix":"","firstName":"Alash'le","middleName":"","lastName":"Abimuku","suffix":""}],"badges":[],"createdAt":"2025-03-31 17:50:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6347062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6347062/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80047914,"identity":"cf158c40-48a5-4dff-8ef7-44cee92812d0","added_by":"auto","created_at":"2025-04-07 09:59:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":219619,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-6347062/v1/e15e1363daba8f9182544d6c.png"},{"id":80047917,"identity":"f88c0f41-3c49-4096-a934-6c9cb331cf1d","added_by":"auto","created_at":"2025-04-07 09:59:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256473,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for 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10:07:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14264,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6347062/v1/4dd888043f26a2ab4dc06dad.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"SARS-CoV-2 infection imprints neutralising antibody responses in the absence of vaccination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite high population-level SARS-CoV-2 immunity driven by previous global exposure and vaccination efforts, SARS-CoV-2 variants, evolving in immune compromised hosts\u003csup\u003e1–6\u003c/sup\u003e, are continuously emerging and transmitting amongst the human population. Vaccine-derived immunity remains the most robust long-term strategy for protection against SARS-CoV-2 and can be measured by binding/neutralising antibodies as surrogate measures of protective humoral immunity\u003csup\u003e7–9\u003c/sup\u003e.\u0026nbsp;The continued circulation of SARS-CoV-2 has been driven by its ability to escape population immunity through acquiring mutations in the highly immunogenic spike protein. \u0026nbsp; The significant antigenic shift represented by Omicron (B.1.1.529) resulted in a radically different virus\u0026nbsp;characterized by multiple mutations in spike with extreme immune evasion of vaccines and increased transmissibility\u003csup\u003e10–17\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunological imprinting refers to a phenomenon where primary exposure to a pathogen shapes future immune responses to related strains\u003csup\u003e18–20\u003c/sup\u003e. This leads to favouring recall of existing memory B-cell responses rather than \u003cem\u003ede novo\u003c/em\u003e responses to newer variants and thereby limits the potency and breadth of humoral responses.\u003csup\u003e21–24\u003c/sup\u003e Immune imprinting has been observed and well-documented for SARS-CoV-2 via a first encounter with ancestral Wu-1-based SARS-CoV-2 vaccination followed by Omicron-based vaccination or infection. The imprinting effect is marked by Wu-1-skewed neutralising antibodies despite recent Omicron exposure due to memory B cell recall and limited \u003cem\u003ede novo\u003c/em\u003e responses to Omicron\u003csup\u003e8,25–31\u003c/sup\u003e, highlighting the dominant effect of prior exposure in shaping immune responses\u003csup\u003e32\u003c/sup\u003e. Studies have demonstrated that depletion of Wu-1 spike-binding antibodies abrogates cross-neutralising antibody responses, particularly in individuals without prior Omicron exposure, underscoring constrained neutralisation breadth due to Wu-1-based imprinting\u003csup\u003e32,33\u003c/sup\u003e. This effect persists even with updated vaccines targeting Omicron subvariants, such as XBB.1.5, which still elicit responses dominated by recall of pre-existing memory B cells\u003csup\u003e33\u003c/sup\u003e.\u0026nbsp;In addition to imprinted responses against Omicron-based vaccines, immune imprinting by ancestral SARS-CoV-2 has further been shown to persist despite multiple Omicron infections\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMost SARS-CoV-2 imprinting studies have focused on ancestral Wu-1-based vaccination as the primary viral encounter\u003csup\u003e31,35–42\u003c/sup\u003e before Omicron exposure. Previous work has demonstrated that SARS-CoV-2 imprinting varies by exposure type\u003csup\u003e31,35–42\u003c/sup\u003e: infection skews responses toward S2 and NTD epitopes and induces stronger B cell receptor clonal expansion and higher somatic hypermutation rates\u003csup\u003e43,44\u003c/sup\u003e, while vaccination predominantly recalls RBD-targeting antibodies\u003csup\u003e37\u003c/sup\u003e. These findings underscore the importance of exposure sequence in shaping subsequent immunity, reinforcing the need to examine whether individuals with Omicron infection before vaccination exhibit distinct imprinting outcomes. Exposure sequences of Omicron infection prior to vaccination are particularly relevant for the African continent because vaccine access has been sparse across the African continent\u003csup\u003e45\u003c/sup\u003e with only around one-third of the continental population receiving a two-dose vaccination series as of October 2023\u003csup\u003e46\u003c/sup\u003e. \u0026nbsp;Consequently, a significant proportion of individuals likely first encountered SARS-CoV-2 via natural infection, including with Omicron-lineage viruses, before vaccination. These circumstances provide an unprecedented opportunity to answer the question of whether and how immunological imprinting occurs in the context of prior Omicron infection without preceding vaccination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we first validate the use of a serological assay to distinguish exposure to Omicron and ancestral pre-Omicron variants in an unvaccinated population receiving primary vaccination in January 2023. Thereafter, we demonstrate in two independent cohorts that despite recent Omicron exposure, Omicron neutralising antibody responses are paradoxically lower or equal to Wu-1 responses. Next, we show that depletion of antibodies in individuals with both pre-Omicron and Omicron infections leads to complete loss of neutralisation against Omicron BA.1 and BA.2, providing further evidence that imprinting occurs following infection with SARS-CoV-2. Finally, we measure neutralising antibody titres after Wu-1-based vaccination and demonstrate that imprinting by ancestral pre-Omicron variants is partially mitigated by experiencing Omicron exposures through both pre-vaccination Omicron infection and post-vaccination breakthrough infection.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStudy population characteristics and SARS-CoV-2 exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt study entry in January 2023, our population comprised 101 confirmed HIV negative participants with no documented history of prior COVID-19 vaccination in Abuja, Nigeria (Cohort 1, \u003cstrong\u003eFigure 1a\u003c/strong\u003e). The median age was 33 years (inter-quartile range: 26-41) and the cohort was 51% female (\u003cstrong\u003eTable 1\u003c/strong\u003e). To analyse population-level exposure to SARS-CoV-2 at study entry in January 2023, when Omicron was the dominant circulating variant of concern (VOC), we tested all participants at study entry (T0, n = 101) for IgG anti-N, IgG anti-S and IgG anti-RBD against SARS-CoV-2 Wu-1 (anti-RBD-Wu-1) using a validated Luminex bead-based flow cytometric assay\u003csup\u003e47\u003c/sup\u003e. We found 94/101 (93%), 98/101 (97%), and 97/101 (96%) of participants positive for IgG anti-N, IgG anti-S and IgG anti-RBD-Wu-1, respectively, reflecting endemic population level exposure to SARS-CoV-2 (\u003cstrong\u003eFigure 1b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further evaluate population-level exposure to SARS-CoV-2 Omicron variants, we tested all participants for Omicron (BA.1)-specific IgG anti-RBD (anti-RBD-Omicron) and found 59/101 (58%) positive for IgG anti-RBD-Omicron\u003cstrong\u003e\u0026nbsp;(Figure 1b). \u0026nbsp;\u003c/strong\u003eTherefore, despite near-universal exposure to pre-Omicron variants, only a subset of participants was exposed to Omicron variants. The RBD mutational profile of BA.1 is similar to BA.2 \u003cstrong\u003e(Supplementary Figure 1a).\u003c/strong\u003e The prior exposure(s) during 2022 resulting in positive IgG anti-RBD-Omicron is/are very likely to have been BA.1 or BA.2/BA.4 based on genomic surveillance and reported cases in Nigeria \u003cstrong\u003e(Supplementary Figure 1b)\u003c/strong\u003e. BA.4 has two additional mutations over BA.2 at positions 452 and 486\u003csup\u003e48\u003c/sup\u003e. Given the global waves of SARS-CoV-2 variants, this pattern of exposure aligns with the broader epidemiological context. Since 2020, the ancestral strain accounted for ~40% of cases; followed by Alpha/Eta in early 2021 (~24%), Delta in late 2021 (18%) and various Omicron subvariants comprising ~18% of cases from 2022 onward (\u003cstrong\u003eSupplementary Figure 1b\u003c/strong\u003e). This distribution is further supported by regional genomic surveillance data, which highlights distinct lineage dynamics in Nigeria, including the dominance of the Eta lineage over Alpha and the emergence of a rare Delta AY.36 sub-lineage\u003csup\u003e49\u003c/sup\u003e. This timeline suggests that most study participants were likely to have encountered pre-Omicron variants before Omicron infection at study entry, consistent with near universal antibodies against Wu-1 spike observed at study entry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImprinted immunity is evident from pre-vaccination serum neutralisation profiles\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first sought to measure pre-vaccination serum neutralisation responses to evaluate imprinting in this previously unvaccinated population. We used pseudotyped viruses as previously described\u003csup\u003e47,50,51\u003c/sup\u003e in (n = 45) participants against ancestral Wu-1, BA.1, BA.2, BA.4 and the antigenically shifted XBB. The latter variant did not circulate until later in 2023 and therefore we expected very little neutralising antibody activity in our cohort regardless of exposure history. Characteristics and binding antibody data of this sub-population were comparable to the general cohort \u003cstrong\u003e(Table 1, Figure 1b).\u0026nbsp;\u003c/strong\u003eIn participants with pre-Omicron variant exposure but no serological evidence of prior exposure to Omicron BA.1 (IgG anti-RBD-Omicron negative, n = 16), the Geometric Mean titre (GMT) neutralisation response against Wu-1 (GMT: 1243) was relatively higher or nearly equal to Omicron variants BA.1, BA.2 and BA.4 (GMT: 658, 1099 and 1387, respectively), representing ~1.1- to 1.9-fold lower neutralisation responses relative to Wu-1. However, consistent with imprinting, serum neutralisation titres in participants with serological evidence of prior exposure to both Wu-1 and Omicron BA.1 (IgG anti-RBD-Omicron positive, n = 29) were paradoxically almost 2-fold higher for Wu-1 (GMT: 4281) compared to Omicron BA.1, BA.2 and BA.4 (GMT: 2322, 2104 and 2112, respectively) \u003cstrong\u003e(Figure 1c)\u003c/strong\u003e, although observations did not reach statistical significance adjusted for multiple comparisons. In the absence of imprinting, individuals with recent Omicron infection (IgG anti-RBD-Omicron positive) would be expected to elicit stronger neutralising responses against Omicron. In contrast, titres against Wu-1, reflecting exposure to a distant pre-Omicron infection as indicated by anti-RBD-Wu-1 and anti-N positivity, should have waned over time in the absence of imprinting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTitres against the antigenically shifted XBB Omicron variant were very low (mean GMT \u0026lt; 100) even in those with prior exposure to Omicron BA.1, BA.2 and BA.4. These data demonstrate the specificity of the screening IgG anti-RBD-Omicron antibody assay for early (pre-XBB) Omicron variants to which our study participants were exposed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to further demonstrate the utility of using binding for IgG anti-RBD-Omicron to assign Omicron lineage exposure, we assessed magnitude and breadth of neutralising antibody responses prior to vaccination.\u0026nbsp;Magnitude-breadth curves are Kaplan-Meier curves using the neutralisation data against all tested variants, where the “event of interest” is the failure to neutralise any variant, and the “time-to-event” on the x-axis is the 50% neutralising titre (NT\u003csub\u003e50\u003c/sub\u003e) for the same variant (\u003cstrong\u003eFigure 1d\u003c/strong\u003e). At baseline, we compared ancestral virus-exposed and Omicron-naïve participants to ancestral virus-exposed and Omicron-exposed participants and observed that Omicron-naïve participants exhibited 2.9-fold (p \u0026lt; 0.05) lower magnitude-breadth relative to Omicron-exposed participants. This clear distinction in neutralisation profiles between groups supports the robustness of our screening assay in identifying prior Omicron lineage exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm the phenomenon of imprinted neutralising antibody responses in unvaccinated individuals, we next characterised neutralisation profiles in another independent, unvaccinated Nigerian cohort recruited in early 2023 as part of a SARS-CoV-2 fractional dosing clinical trial (PACT202206754734018)\u003csup\u003e52\u003c/sup\u003e (Cohort 2, \u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e). In cohort 2, (n = 64) samples were available; of these 64 participants, 94% (n = 60/64) showed serological evidence of Omicron exposure characterized by positivity for IgG anti-RBD-Omicron (\u003cstrong\u003eSupplementary Figure 2a\u003c/strong\u003e). This reflects a higher degree of exposure to Omicron in this cohort relative to cohort 1. Nonetheless, the GMT neutralisation response (\u003cstrong\u003eSupplementary Figure 2b\u003c/strong\u003e) against Wu-1 (GMT: 1492) was higher than against BA.1 Omicron (GMT: 843, 1.8-fold) and was similar for BA.2 (GMT: 1316, 1.2-fold). The relatively higher BA.2 titres compared to BA.1 (~1.6-fold) likely reflect more recent exposure to BA.2 lineage viruses, which predominated circulation in 2023, and expansion of cross-reactive B cells with highest affinity for BA.2 spike (\u003cstrong\u003eSupplementary Figure 1b\u003c/strong\u003e). \u0026nbsp;Notably, neutralisation titres against Wu-1 remained robust despite more than 18 months elapsing since the last exposure to an ancestral Wu-1 lineage and 94% of Cohort 2 experiencing Omicron exposure, underscoring the durability of imprinted humoral responses. Taken together, these data from disparate regions of Nigeria further support immunological imprinting via exposure to ancestral pre-Omicron lineages (\u003cstrong\u003eSupplementary Figure 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepletion of serum binding antibodies against ancestral Wu-1 spike confirms imprinting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that observed neutralisation responses were driven by imprinted immunity, we performed targeted depletion of plasma antibodies against the Wu-1 spike protein from individuals with pre-vaccination exposure to pre-Omicron variants only (n = 8) and both pre-and post-Omicron variants (n = 21) in Cohort 1. Using a previously described method\u003csup\u003e34\u003c/sup\u003e, suspension Expi293 cells were transfected with Wu-1 spike-expressing plasmid and incubated for 72 hours, after which participant sera were passed over the cells (\u003cstrong\u003eFigure 2a\u003c/strong\u003e). This process allowed surface-expressed spike to selectively bind and deplete Wu-1-specific antibodies from the serum. Following depletion, we assessed residual neutralisation against Wu-1, BA.1 and BA.2 viruses.\u003c/p\u003e\n\u003cp\u003eConsistent with the hypothesis of Wu-1 imprinting via infection, we observed a complete loss of neutralising activity against Wu-1, BA.1 and BA.2 viruses in 7/8 (88%) pre-Omicron exposed participants and 20/21 (95%) pre- and post-Omicron exposed participants following depletion (\u003cstrong\u003eFigure 2b\u003c/strong\u003e). We confirmed successful depletion by measuring Wu-1 spike-specific binding antibodies with Luminex in a subset (n = 9) of pre- and post-depletion samples that included two individuals who failed to show loss of neutralisation following depletion \u003cstrong\u003e(Figure 2c\u003c/strong\u003e). We did not find any significant difference between ages of participants who showed neutralisation antibody abrogation (n = 27) relative to those that did not (n = 2), with a median age of 35 years (p = 0.97).abbas\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of Omicron breakthrough infection and contemporaneous Wu-1 vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants underwent Wu-1-based vaccination. Between study entry and one month post-first dose, around half of the participants with neutralisation response data (n = 23/45) experienced a breakthrough infection (BTI, \u003cstrong\u003eSupplementary Figure 3)\u003c/strong\u003e. In those with prior Omicron exposure at baseline (n = 29), participants demonstrated increased neutralisation titres across ancestral and Omicron lineage viruses post-vaccination regardless of BTI status (\u003cstrong\u003eSupplementary Figure 4a\u003c/strong\u003e). In participants without prior Omicron exposure at baseline (n = 16), only those who experienced BTI between study entry and 1-month post-dose 1 demonstrated an increase in neutralisation titres across ancestral and Omicron lineage viruses, whereas those without BTI showed a tendency for increased titres, albeit nonsignificant (\u003cstrong\u003eSupplementary Figure 4b\u003c/strong\u003e). Taken together, these data suggest that Wu-1-based vaccination was associated with an increase in neutralising response activity against ancestral pre-Omicron viruses, and neutralisation against Omicron lineage viruses was boosted in participants with any prior exposure to Omicron.\u003c/p\u003e\n\u003cp\u003eWe first assessed individuals with no BTI post-vaccination (n = 22/45). In participants who had no prior Omicron exposure (n = 5), vaccination exacerbated the gap between neutralisation of Wu-1 (GMT:5095) and Omicron variants. Relative to Wu-1, we observed between 2.4- to 4.4-fold lower neutralisation responses for Omicron variants BA.1 (GMT:1153), BA.2 (GMT:2059) and BA.4 (GMT:2103), as well as ~58-fold lower responses for XBB (GMT:87) \u003cstrong\u003e(Figure 3a [top, left]).\u0026nbsp;\u003c/strong\u003eIn those with prior Omicron exposure at baseline (n = 17), we similarly observed a widening of the gap between neutralisation of Wu-1 (GMT:17065) versus Omicron variants following vaccination. Relative to Wu-1, we observed ~3-fold lower neutralisation responses for Omicron variants BA.1 (GMT:5073), BA.2 (GMT:5506) and BA.4 (GMT:5546), as well as ~123-fold lower responses for XBB (GMT:139) (\u003cstrong\u003eFigure 3a [top, right]\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eWe further investigated individuals with BTI post-vaccination (n = 23/45). During the pre- and post-vaccination timepoints, Omicron was the dominant circulating variant (\u003cstrong\u003eSupplementary Figure 1b\u003c/strong\u003e) and was likely responsible for breakthrough infections between vaccine administration and 1-month post-dose one. \u0026nbsp;In participants with no prior Omicron exposure at baseline (n = 11), contemporaneous Wu-1-based vaccination and Omicron BTI resulted in similar neutralisation titres (~1.2-fold lower) relative to Wu-1 (GMT:8258) for Omicron variants BA.1 (GMT:6554), BA.2 (GMT:6720) and \u0026nbsp;BA.4 (GMT:6960), as well as ~25-fold lower responses for XBB (GMT:331) (\u003cstrong\u003eFigure 3a [bottom, left])\u003c/strong\u003e. In individuals with both prior Omicron exposure at baseline and BTI (n = 12), neutralisation titres were again similar relative to Wu-1 (GMT:13845) for Omicron BA.1 (GMT:10022, 1.4-fold lower) and nearly equal for Omicron BA.2 (GMT:13699) and BA.4 (GMT:13810), as well as ~103-fold lower for XBB (GMT:135) (\u003cstrong\u003eFigure 3a [bottom, right]).\u0026nbsp;\u003c/strong\u003eThe slightly lower titres of BA.1 compared to BA.2 and BA.4 are expected given that the BTI was epidemiologically most likely to be a BA.2 descendent.\u003c/p\u003e\n\u003cp\u003eTo quantify the magnitude and breadth of neutralising antibody responses induced by Wu-1-based vaccination, we generated\u0026nbsp;magnitude-breadth curves of responses. At 1-month post-dose 1 (T1) compared to baseline (T0), we observed an approximately 3-fold increase in the magnitude-breadth AUC in the Omicron-naïve participants and a 2-fold increase in the Omicron-exposed participants, demonstrating improved neutralisation post-vaccination \u003cstrong\u003e(Figure 3b).\u003c/strong\u003e Next, we conducted a sub-analysis with stratification by BTI at 1-month post-dose 1 (T1). Individuals with no Omicron exposure both pre- and post-vaccination (n = 5) had persistently lower magnitude-breadth compared to those who had at least one Omicron exposure at any timepoint (5.9-fold difference, \u003cem\u003ep\u003c/em\u003e = 0.03). Interestingly, the sequence of Omicron exposure affected magnitude-breadth outcomes: participants with pre-vaccination Omicron exposure and no BTI (n = 17) exhibited greater magnitude-breadth than participants with post-vaccination Omicron BTI exposure only (n = 11, p \u0026lt; 0.05). In contrast, having both pre-vaccination Omicron exposure and a BTI post-vaccination elicited the highest magnitude-breadth response (\u003cstrong\u003eFigure 3b)\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e These findings suggest that hybrid (infection and vaccine)-induced immunity with pre-Omicron variants alone isinsufficient to broaden the antibody response and target the antigenically shifted Omicron variants. Taken together, these data show partial mitigation of imprinting by repeated Omicron exposure.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding how sequential SARS-CoV-2 exposures shape immune responses is critical to anticipating population-level protection and informing future vaccine strategies. Devastating consequences may arise where a pathogen rapidly becomes more immune evasive and increases pathogenicity, but individuals are unable to induce effective \u003cem\u003ede novo\u003c/em\u003e responses to neutralise the threat due to prior immune imprinting by an ancestral variant.Indeed, it is well-documented how immune imprinting by ancestral Wu-1-based SARS-CoV-2 vaccination drives responses to antigenically distinct variants against conserved epitopes and limits \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eantibody generation. However, due to the rapid scale-up of vaccination before emergence of the antigenically distinct Omicron lineage, it is not well-established whether and how immune imprinting occurs in the context of SARS-CoV-2 infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we provide robust evidence for immune imprinting in the absence of vaccination. At study enrolment in early 2023 for Cohort 1, nearly all individuals demonstrated infection by pre-Omicron variants. These data are supported by previous studies showing high seroprevalence for Wu-1 binding antibodies and serum neutralising activity in Nigeria and Ghana in 2021, as well as high rates of BTI by the Delta variant post-vaccination in a 2021 cohort of Nigerian healthcare workers\u003csup\u003e16,47,53,54\u003c/sup\u003e. The participants with no evidence of pre-vaccination exposure to Omicron (Pre-Om\u003csub\u003einf\u003c/sub\u003e) demonstrated significantly lower titres of serum neutralising antibodies against Omicron variants compared to Wu-1, as expected. Paradoxically, the 58% of participants who were IgG anti-RBD-Omicron positive pre-vaccination (Pre-Om\u003csub\u003einf\u003c/sub\u003e + Om\u003csub\u003einf\u003c/sub\u003e) showed similar or lower titres of serum neutralising antibodies against Omicron variants compared to Wu-1, despite pre-Omicron viruses last circulating more than a year prior. These results were further bolstered by a comparable unvaccinated Nigerian cohort (Cohort 2) in which 94% of participants had evidence of prior exposure to Omicron (Pre-Om\u003csub\u003einf\u003c/sub\u003e + Om\u003csub\u003einf\u003c/sub\u003e), yet titres against Omicron BA.1 were lower compared to Wu-1, suggesting recall of Wu-1-targeting memory B cells instead of \u003cem\u003ede novo\u003c/em\u003e response induction. We further supported these results by depleting sera of Wu-1-binding antibodies, which abrogated neutralisation against both Wu-1 and Omicron-lineage variants in 27/29 of participants. Finally, we showed that repeated Omicron exposure partially mitigated imprinting even after Wu-1 antigen delivery via vaccination.\u003c/p\u003e\n\u003cp\u003eSuboptimal neutralisation responses to the highly divergent SARS-CoV-2 lineage Omicron have been observed in animal models and individuals previously vaccinated with a primary course of ancestral Wu-1-based vaccine, consistent with immunological imprinting by the vaccine\u003csup\u003e24,55,56\u003c/sup\u003e. Data show that individuals primed with a Wu-1-based vaccine who are then exposed to Omicron will primarily recall pre-existing memory B cells that recognise conserved multi-variant epitopes rather than generate \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003eOmicron-specific responses from naïve B cells, thereby limiting breadth of responses\u003csup\u003e22–24\u003c/sup\u003e. For individuals primed with the attenuated live Coronavac vaccine followed by two Omicron infections, comparable neutralising antibody levels against ancestral and Omicron strains were reported, as well as increased frequencies of Omicron-specific memory B cells, suggesting mitigation of imprinting\u003csup\u003e56\u003c/sup\u003e. In contrast, no mitigation of imprinting was observed in individuals primed at least 3 times with Wu-1-based mRNA vaccines who were then exposed to Omicron twice via a combination of Wu-1/BA.5 bivalent vaccination and either Omicron BTI or XBB.1.5 monovalent vaccination.These studies observed the highest neutralising titres against Wu-1 and no significant Omicron-specific memory B cell response\u003csup\u003e33,55\u003c/sup\u003e. Interestingly, when all Wu-1 binding antibodies were absorbed and the remaining sera were tested for neutralisation, BA.5 and XBB.1.5 binding titres and neutralisation potency were ablated, which is consistent with our findings that absorption of Wu-1 antibodies abrogates neutralisation responses against BA.1 and BA.2. These results suggest that all Omicron antibodies were cross-reactive due to recall and expansion of the memory response induced by ancestral Wu-1 imprinting\u003csup\u003e33,55\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is notable, however, that two individuals in our study who had confirmed removal of Wu-1 spike-binding antibodies were still able to neutralise Wu-1 and Omicron variants. Previous work has demonstrated \u003cem\u003ede novo\u003c/em\u003e antibody generation after multiple Omicron exposures in individuals who received inactivated or recombinant SARS-CoV-2 vaccination\u003csup\u003e34,56\u003c/sup\u003e. Alternatively, the neutralisation of Wu-1 despite removal of Wu-1-binding antibodies could be due to targeting of cryptic sites of spike, which are only exposed after ACE2 binding induces conformational changes\u003csup\u003e57\u003c/sup\u003e and therefore would not be depleted by spike in the absence of ACE2. Future work should aim to elucidate the mechanism behind our observations, requiring cells and generation of monoclonal antibodies.\u003c/p\u003e\n\u003cp\u003eWith regards to mitigation of imprinting in our study participants, those exposed to Omicron both pre- and post-vaccination demonstrated neutralisation titre equalization for Wu-1 and BA.1, BA.2 and BA.4 Omicron variants. In contrast, those exposed pre-vaccination but without BTI had around 3-fold lower titres against Omicron variants. This provides evidence that infection-induced ancestral imprinting is partially mitigated by repeated exposure to Omicron variants, but that further Wu-1 antigen delivery via vaccination could exacerbate ancestral imprinting and hinder \u003cem\u003ede novo\u003c/em\u003e responses against Omicron.\u003c/p\u003e\n\u003cp\u003eOur study was subject to limitations. Our study does not include data on cellular immune responses, although we were able to demonstrate the phenomenon of imprinting using sera from two independent contemporaneous cohorts. Secondly, we were unable to distinguish by genomic sequencing the exact variants to which participants were previously exposed. Given up to half of infections are asymptomatic\u003csup\u003e58,59\u003c/sup\u003e, there is no practical strategy for accurately detecting and recording infections over extended periods of time. Instead, we validated the use of serology, specifically binding and neutralising antibody profiling, to determine pre- and post-Omicron variant exposure that reflected the limited population-level genomic surveillance data available in Nigeria.\u003c/p\u003e\n\u003cp\u003eIn summary, these data uncover immune imprinting following SARS-CoV-2 infection in the absence of vaccination. This imprinting could be mitigated by repeat infection with Omicron even with an exposure to Wu-1-based vaccination between Omicron exposures. In other present-day unvaccinated populations (and indeed vaccinated populations), use of monovalent Omicron variant-based vaccines should be used in preference to Wu-1-based and bivalent vaccines to override imprinting by ancestral pre-Omicron strains and provide broader protection for vulnerable populations such as the elderly or those with compromised immunity. As the chance of long COVID and other serious sequelae rise with each infection\u003csup\u003e58,59\u003c/sup\u003e and are more common in the absence of vaccination, it is crucial to understand how imprinting may be mitigated in diverse populations. More importantly, these data serve as a reminder of the sometimes-deleterious effects of imprinting and caution against over-reliance on ancestral vaccines as protective measures against rapidly evolving pathogens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.A. was supported by Cambridge-Africa award and Harvard Takemi Program in International Health.\u0026nbsp;RBM was supported by the Harding Distinguished Postgraduate Scholars Programme. RKG was supported by a Wellcome Trust Senior fellowship (WT108082AIA). This research was supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312) and Open Philanthropy. D.J.S. and S.T. are supported by the NIH NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract 75N93021C00014 as part of the SAVE program. D.J.S. and S.T. are supported by the NIH NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract 75N93021C00014 as part of the SAVE program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy conceptualization and design:\u0026nbsp;\u003c/strong\u003eA.A and RKG\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy management, supervision and fieldwork:\u0026nbsp;\u003c/strong\u003eA.A., E.O, M.E and A.Ab.,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology and Investigation:\u0026nbsp;\u003c/strong\u003eA.A., R.B.M., E.O., M.T.K.C., M.E., S.T., F.I., A.K., B.S., H.W.A., E.J., O.B., A.S., H.A.-O., A.J., A.M., O.O., B.I., O.E., A.A., E.O., S.O., I.M.K., S.H.A., B.M., L.R., R.D., W.W., D.S., B.S.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Data Curation \u0026amp; Analysis:\u0026nbsp;\u003c/strong\u003eA.A., R.B.M., M.T.K.C., F.I., A.K., B.S., A.Ab., and R.K.G.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Acquisition:\u0026nbsp;\u003c/strong\u003eA. A, A.Ab, B.S and RKG\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting\u003c/strong\u003e: A.A. drafted the original manuscript, and R.B.M. provided critical editorial input, with contributions from all authors. All authors approved the final version of the manuscript\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy population and sampling:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCohort 1:\u0026nbsp;\u003c/strong\u003eHIV Negative participants across three clinical sites affiliated with Institute of Human Virology Nigeria (IHVN), Abuja, were actively recruited and asked to participate in this study. Eligible participants were men and non-pregnant women \u0026gt;18 years old who had no previous SARS-CoV-2 vaccination and were confirmed HIV negative using the Nigerian national HIV rapid testing algorithm\u003csup\u003e60,61\u003c/sup\u003e. As interim recommendations and guidelines highlighted the use of a\u0026nbsp;two-dose regimen of Ad26.COV2.S vaccine given 2 months apart due to improved vaccine efficacy\u003csup\u003e62\u003c/sup\u003e, participants were administered a second dose two months following the first dose. Following signed informed consent, participants were recruited in this prospective observational cohort study. The study was comprised of adult participants eligible to receive their first-dose vaccination between 23\u003csup\u003erd\u003c/sup\u003e January 2023 to 20\u003csup\u003eth\u003c/sup\u003e April 2023. Participants were recruited by i) local community outreach and ii) through phone calls to previously registered patients across three health facilities in Abuja, Nigeria. Participants provided plasma samples at baseline (prior to first-dose, T0) and 1-month post-dose 1 (T1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCohort 2:\u0026nbsp;\u003c/strong\u003ePre-vaccination samples from individuals in the\u0026nbsp;SIFCoVAN trial across Nigeria recruited in 2023 were tested for binding antibodies and pseudotyped virus neutralisation across VOC. The protocol was registered with the Pan African Clinical Trials Registry (PACTR) PACTR 202206754734018.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eLaboratory methods and sample testing:\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eBinding antibody measurement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBinding IgG antibodies (Abs) against SARS-CoV-2 trimeric spike protein (S),\u0026nbsp;nucleocapsid protein (N), Wu-1 D614G and Omicron (BA.1)-specific receptor-binding domain (RBD) were measured using the Luminex-based SARS-CoV-2-IgG assay by flow cytometry as previously detailed\u003csup\u003e16,47,53,54\u003c/sup\u003e. \u0026nbsp; The assay was validated using pre-pandemic serum samples and robustness of the defined cut-off is shown in \u003cstrong\u003eSupplementary Figure 5\u003c/strong\u003e. We defined positive total anti-S antibody (anti-S) as anti-S IgG above cut-off of 226.48 mean fluorescence intensity (MFI), positive total RBD as anti-RBD-Wu-1 above cut-off of 411.9, and positive total anti-RBD-Omicron above cut-off of 729. Cut-offs were defined based on analysis of \u0026lsquo;true\u0026rsquo; positive (convalescent) and negative pre-pandemic samples. We defined previous SARS-CoV-2 infection as positive anti-N IgG above a cut-off of 1472.8.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNeutralisation assays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor plasma neutralising antibody measurement, SARS-CoV-2 lentiviral pseudotyped viruses were prepared by transfecting HEK293T cells with Wu-1 D614G, BA.1, BA.2, BA.4 and XBB plasmids as previously described in detail\u003csup\u003e50,63\u003c/sup\u003e. There is evidence to show a high correlation between pseudotyped virus and live virus neutralisation\u003csup\u003e64,65\u003c/sup\u003e. \u0026nbsp;All neutralisation assays were repeated in two independent experiments containing two technical replicates for each condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody depletion assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExpi293F suspension cells were maintained at 37\u0026deg;C and 8% CO2 with constant rocking in 125-ml vented Erlenmeyer flasks with Expi293 Expression Medium. One day prior to serum absorption-neutralisation experiments, 96-well F-bottom cell culture microplates were plated with 2 x 10\u003csup\u003e4\u003c/sup\u003e HeLa-ACE2 cells per well and maintained in DMEM supplemented with 10% FBS.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExpi293F cell transfection\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExpi293F cells were sub-cultured at least three times before transfection. Four days prior to experiments, cells were passaged to a density of 2.5-3 x 10\u003csup\u003e6\u003c/sup\u003e viable Expi293F cells/ml. Three days prior to experiments, Expi293F cells were transfected with a mixture of Opti-MEM Reduced Serum Medium, Fugene HD transfection reagent, and either plasmids expressing Wuhan-Hu-1 spike or no plasmid (mock transfection control).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSerum absorption\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOn the day of the experiment, 72 hours post-transfection, Expi293F cells were centrifuged and washed twice with warm Expi293 Expression Medium, then resuspended at 40 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml. Heat-inactivated serum samples were initially diluted 1:10 in warm Expi293 Expression Medium and split into two fractions for absorption by Wuhan-Hu-1 spike-transfected Expi293F cells or mock-transfected Expi293F cells.\u003c/p\u003e\n\u003cp\u003eFor each individual, Expi293 Expression Medium containing 8 x 10\u003csup\u003e6\u003c/sup\u003e Expi293F cells were mixed 1:1 (v/v) with 1:10 diluted serum in 1.5-ml microcentrifuge tubes. The mixtures were incubated for 1 hour at 37\u0026deg;C and shaken at 900 rpm in an Eppendorf Thermomixer. After incubation, Expi293F cells were centrifuged, and the supernatant was transferred to a new microcentrifuge tube for re-centrifugation. Expi293 Expression Medium was added to the clarified supernatant such that all absorbed serum samples were now diluted 1:20.\u003c/p\u003e\n\u003cp\u003eAbsorbed serum with a dilution of 1:20 were then added to 96-well cell culture F-bottom plates in duplicate and serially diluted 1:3 in DMEM supplemented with 10% FBS. Pseudotyped Wuhan-Hu-1, BA.1, and BA.2 viruses were added as previously described to the serial dilutions of absorbed sera, mixed and incubated at 37\u0026deg;C and 5% CO2 for 1 hour. After the incubation of absorbed sera and pseudotyped viruses, these mixtures were added to the plates of HeLa-ACE2 cells prepared the day prior to experiments. The final dilution of serum was 1:58.33. Neutralisation plates were incubated and read as previously described.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRBD Alignment:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Spike sequences for Wuhan-Hu-1 (NC_045512.2), BA.1 (OL672836.1), BA.2 (OM371884.1), BA.4 (ON373214.1), BA.2.86 (OR775659.1) and XBB (XBB 1.5 OP790748.1 + S: P486S) were aligned using MAFFT\u003csup\u003e66\u003c/sup\u003e. SARS-CoV-2 spike RBD residues 319-541 were aligned. Asterisks indicate completely conserved columns in the alignment. The receptor binding motif (RBM) at residue 438-506 is indicated by + highlighted in yellow.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpigenomic map\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic epidemiological data from GISAID was representatively sampled and visualised using Nextstrain.\u003csup\u003e67\u003c/sup\u003e\u0026nbsp; To further break down the cases in Nigeria in both the pre-Omicron and post-Omicron study periods, the Global initiative on Sharing All Influenza Data (GISAID) database\u003csup\u003e68\u003c/sup\u003e was queried using Outbreak.info API\u003csup\u003e69\u003c/sup\u003e for all SARS-CoV-2 sequences collected from Nigeria. Subclades of WHO-assigned Variants of Concern (VOCs) were assigned their WHO name and Nextstrain-assigned clade. To account for the changing number of SARS-CoV-2 cases, we weighed the number of sequences attributed to each VOC by weekly new cases reported to the WHO (https://data.who.int/dashboards/covid19/data):\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMagnitude breadth analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo capture the relationship between the magnitude (NT50) and breadth (percentage of VOCs neutralised) of serum neutralisation, we plotted magnitude-breadth curves, a framework previously used in analysing HIV\u003csup\u003e70\u0026ndash;72\u003c/sup\u003e and SARS-CoV-2\u003csup\u003e73\u003c/sup\u003e humoral responses against multiple strains. Magnitude-breadth curves are Kaplan-Meier curves using the neutralisation data against all tested variants, where the \u0026ldquo;event of interest\u0026rdquo; is the failure of neutralisation of any variant, and the \u0026ldquo;time-to-event\u0026rdquo; on the x-axis is the NT\u003csub\u003e50\u0026nbsp;\u003c/sub\u003efor the same variant. Curves are modelled using the R package Survival v3.8-3\u003csup\u003e74,75\u003c/sup\u003e, and plotted using the R package survminer 0.5.0.\u003csup\u003e76\u003c/sup\u003e In addition, the areas under the curve (AUC) were calculated for both individual curves and aggregated curves were calculated using the \u0026ldquo;km\u0026rdquo; method in package AdjustedCurves v0.11.2 to facilitate comparison\u003csup\u003e77\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDefinition of terms and statistical analysis:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe defined vaccine breakthrough infection as a \u0026ge;2-fold increase in IgG anti-RBD-Omicron levels relative to study entry timepoint (\u003cstrong\u003eSupplementary Figure 3\u003c/strong\u003e). Geometric Mean Titre (GMT) with standard deviation (SD) of neutralisation antibody was calculated across time points. Baseline characteristics of participants were expressed as proportions and percentages for categorical variables and median inter-quartile range (IQR) for continuous variables. Two-tailed Wilcoxon signed-rank tests or Friedman\u0026rsquo;s Test with Holm-corrected post-Hoc Wilcoxon test was used to compare neutralisation antibody titres. Statistical analysis was performed using GraphPad Prism version 9.3.1 and Rstatix v0.7.2\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthics:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the FCT Health Research Ethics Committee (FCT HREC) with approval FHREC/2022/01/193/18-10-22 and London-Surrey Research Ethics Committee [23/PR/0586] with IRAS ID: \u0026nbsp; 309527. Approvals were also obtained from the secondary health centres \u0026ndash; Asokoro District Hospital [Approval ID: FCTA/HHSS/HMB/ADH/111/22] and Maitama District Hospital [Approval ID: FCTA/HHSS/HMB/GEN/038/T].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTable 1: Baseline characteristics of study participants in Cohort 1\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"559\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eTotal number, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e101 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eAge, median years (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e33 (26, 42)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eFemale sex, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e51 (51)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eTime between vaccine doses, days (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e62 (59, 65)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 559px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Participants with neutralisation response data\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eTotal number, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e45 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eAge, median years (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e36 (28, 42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eFemale sex, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e25 (56)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 401px;\"\u003e\n \u003cp\u003eTime between vaccine doses, days (IQR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 158px;\"\u003e\n \u003cp\u003e62 (59, 63)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKemp, S. 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A comparison of different methods to adjust survival curves for confounders. \u003cem\u003eStat Med\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1461\u0026ndash;1479 (2023).\u003c/li\u003e\n\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-6347062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6347062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmune imprinting refers to a phenomenon where a first viral encounter shapes subsequent immune responses against antigenically distinct variants, favouring the recall of memory B cells against cross‐reactive epitopes in lieu of \u003cem\u003ede novo\u003c/em\u003eresponses. This bias can lead to suboptimal neutralising antibody protection when subsequent infections occur with highly divergent viral lineages such as the SARS-CoV-2 Omicron lineage viruses. Given the rapid rollout of Wu-1-based vaccines in many countries before the emergence of Omicron, prior studies have demonstrated that vaccination with ancestral Wuhan-Hu-1 (Wu-1)-based vaccines results in diminished neutralisation of the Omicron variants compared to Wu-1, even after recent Omicron exposure. The effects of imprinting induced by natural infection in the absence of vaccination is unknown, however, particularly in populations where Omicron infection preceded Wu-1-based vaccination. This presents a critical gap in our knowledge given the different dominance hierarchies of neutralising antibody responses between vaccines and natural infection. We therefore assessed humoral responses in a population exposed to pre-Omicron (ancestral) and post-Omicron variants prior to receiving any vaccine. Using a binding antibody assay to distinguish variant-specific exposures, we found that 93% of participants had serological evidence of ancestral SARS-CoV-2 infection, while 58% had evidence of Omicron exposure. In individuals exposed solely to pre-Omicron variants, neutralisation titres against Wu-1 were significantly higher than those against Omicron variants, as expected. Paradoxically, participants with evidence of both pre-Omicron and Omicron infections also exhibited higher neutralisation titres for Wu-1 relative to Omicron BA.1, despite the ancestral exposure occurring ~18 months earlier and Omicron being the most recent infection. We confirmed these findings in another independent Nigerian cohort where Omicron exposure was even more prevalent (94%), and all participants had evidence of pre-Omicron infection. The findings suggest imprinted immunity from the ancestral pre-Omicron lineage viruses, and remarkably these early responses to Wu-1 were able to dominate over more recent, likely multiple, Omicron lineage infections. To directly assess imprinting, we performed serum antibody depletion experiments using Wu-1 spike protein as bait. In nearly all participants, depletion of Wu-1-specific antibodies resulted in complete abrogation of serum neutralising activity against both Wu-1 and Omicron spike pseudotyped viruses. Furthermore, during prospective follow-up, additional Omicron infection and contemporaneous administration of Wu-1-based vaccine boosted neutralising responses across variants, partially equalizing titres between Wu-1 and Omicron. However, Omicron responses did not surpass ancestral responses, suggesting only partial mitigation of imprinting. These data demonstrate the presence of immune imprinting against SARS-CoV-2 in the absence of vaccination and its persistence thereafter. The dominance of ancestral responses in unvaccinated populations in the post-Omicron era suggests the preferential use of an Omicron variant-based vaccine in lieu of Wu-1-based vaccines to override imprinting and achieve broader protection, particularly in vulnerable populations such as the elderly or those with compromised immunity. These data also inform preparation against future pandemic zoonoses with rapidly evolving viruses, cautioning against over-reliance on vaccines against ancestral viruses.\u003c/p\u003e","manuscriptTitle":"SARS-CoV-2 infection imprints neutralising antibody responses in the absence of vaccination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 09:59:20","doi":"10.21203/rs.3.rs-6347062/v1","editorialEvents":[],"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":"5e83ef2c-84d9-42f9-b5af-b5d3b2a13f96","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46697148,"name":"Biological sciences/Immunology/Vaccines/DNA vaccines"},{"id":46697149,"name":"Biological sciences/Microbiology/Virology/SARS-CoV-2"}],"tags":[],"updatedAt":"2025-06-17T22:35:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 09:59:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6347062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6347062","identity":"rs-6347062","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}