Distinct from vaccine-first models, infection-first exposures provide a critical context for understanding SARS-CoV-2 immune imprinting in unvaccinated populations. We analyzed neutralizing antibody responses in two independent unvaccinated Nigerian cohorts sampled in early 2023. Using a BA.1 receptor-binding domain (RBD)-based assay for Omicron exposure discrimination, we identified widespread pre-Omicron and partial Omicron exposure. Despite recent Omicron infection, plasma neutralization titers against Omicron lineages remained equal or lower compared to those against ancestral Wu-1, indicating infection-derived imprinting. Depletion of Wu-1 spike-binding antibodies abrogated neutralization of both Wu-1 and Omicron pseudoviruses, confirming dominance of ancestral cross-reactive antibodies. Following Wu-1-based vaccination, neutralizing responses increased across all variants, yet Omicron titers did not exceed Wu-1 titers even after breakthrough infection. These findings demonstrate durable infection-induced immune imprinting established before vaccination and only partially mitigated by repeated Omicron exposures, underscoring the influence of infection-first exposure sequence on antibody breadth and broader global relevance for vaccine design.

Abstract Introduction Monoclonal antibodies have emerged as a prophylactic strategy to prevent symptomatic SARS-CoV-2 infection in immunocompromised individuals. However, the evolutionary and clinical implications of breakthrough infections remain unclear. Methods An 80-year-old male with a haematological/oncological diagnosis received a 2000 mg intravenous infusion of sotrovimab in March 2023 and was diagnosed with COVID-19 by RT-qPCR from a nasopharyngeal swab in August 2023. Weekly samples (n = 24) were collected through February 2024 (171 days). All samples underwent whole-genome sequencing, with select mutations subjected to functional assessment. Results Sequencing identified the GE.1 lineage at all timepoints. An intra-host recombination event in ORF1ab (positions 8980-11203) was detected 100-107 days post-infection, followed by a 14-fold increase in viral load (7.42E+06 to 1.00E+08 RNA copies/mL) and a marked shift in the mutation landscape. E340D, a sotrovimab-resistance mutation, fluctuated over time and was selected 107 days post-infection. We assessed five spike mutations — V36M, S98F, and V213G in the N-terminal domain, Y505P in the receptor-binding domain, and P681Q near the S1/S2 cleavage site–and additionally evaluated the impact of E340D. V36M conferred the highest infectivity across all cell lines, with the most significant effect in low-TMPRSS2 cells. While all mutations showed enhanced infectivity with the addition of E340D, the effect was most pronounced in mutations with lower baseline infectivity. The addition of E340D significantly decreased relative neutralizing titres for V36M, S98F, and V213G, enabling escape from neutralizing antibodies in XBB-responsive individuals. Patient neutralizing activity was absent pre-sotrovimab, and sotrovimab-induced neutralization was further compromised by selection of E340D. Conclusion Fixation of E340D abolished the surrogate defence provided by sotrovimab, eliminating neutralizing activity and exerting a positive epistatic effect on emerging mutations. Funding Source GSK; Gates-Cambridge scholarship Topic Categories Vaccines and Immunotherapy (VAC)

Prolonged infections in immunocompromized individuals are associated with accumulation of unexpected combinations of mutations, as exemplified by the Omicron variant. Here, we show that intra-host evolution during a chronic SARS-CoV-2 infection in the pre-Omicron era was associated with mutations that modulate antibody evasion, cell entry efficiency, spike processing, and fusogenicity. Single spike N-terminal mutations S13I and W64G exerted deleterious impacts on S1/S2 cleavage, syncytia formation, and cell entry but combined through an inferred recombination event with P330S to restore function. The single mutants were never detected individually in respiratory samples, but mediated temperature-dependent evasion from neutralizing antibodies and altered reliance on TMPRSS2-mediated membrane fusion, possibly explaining their transient selection. Molecular dynamics simulations revealed that W64G induces local N-terminal domain destabilization with distal effects at the S1/S2 cleavage loop, linking structural perturbation to functional defects and immune evasion. Together, these findings demonstrate how intra-host recombination contributes to viral adaptation.

Immune suppression poses a challenge to vaccine immunogenicity. We show that serum antibody neutralization against SARS-CoV-2 Omicron descendants was largely absent post-doses 1 and 2 in individuals with vasculitis treated with rituximab. Detectable and increasing neutralizing titers were observed post-doses 3 and 4, except for XBB. Rituximab in vasculitis exacerbates neutralization deficits over standard immunosuppressive therapy, although impairment resolves over time since dosing. We observed discordance between detectable IgG binding and neutralizing activity specifically in the context of rituximab use, with high proportions of individuals showing reasonable IgG titer but no neutralization. ADCC response was more frequently detectable compared to neutralization in the context of rituximab, indicating that a notable proportion of binding antibodies are non-neutralizing. Therefore, use of rituximab is associated with severe impairment in neutralization against Omicron descendants despite repeated vaccinations, with better preservation of non-neutralizing antibody activity.

Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are a source of new variants and can provide insight into evolutionary trajectories. Here, we observe upper airway-specific evolution of SARS-CoV-2, demonstrating a fusion peptide (FP) domain mutation (S:P812S) adjacent to the S2' cleavage site that emerged during a chronic infection. Indeed, this mutation had emerged previously and been transmitted in a delta variant lineage. P812S in a spike-pseudotyped virus did not impact entry efficiency. However, cleavage at S1/S2 was reduced, and molecular dynamics simulation demonstrated altered S1/S2 loop conformations. Consistent with impaired S1/S2 cleavage, and reminiscent of Omicron BA.1, cell-cell fusogenicity was severely impaired by P812S. P812S conferred evasion of a FP-targeting monoclonal antibody, consistent with FP-region structural rearrangements. Finally, P812S-bearing viruses showed evasion of polyclonal neutralizing antibodies in sera from vaccinated individuals at 32C. These data shed light on the balance between SARS-CoV-2 upper airway adaptation/immune evasion, syncytium formation, and pathogenic potential.

Immune imprinting or original antigenic sin (OAS) originally referred to a phenomenon of suboptimal immune response to a repeat exposure to a virus that was antigenically distinct from the original virus infection. OAS has been implicated in higher mortality in young people during the 2009-10 H1N1 pandemic where the elderly (H1N1 exposure in childhood) appeared relatively well protected compared to younger individuals whose first influenza infection was not H1N1. Immune imprinting is part of a rapid recall system and is highly effective against a slowly evolving virus (drifting) but not antigenically shifting viruses such as influenza and SARS CoV-2. As predicted by OAS, suboptimal neutralization responses to the highly divergent SARS-COV-2 lineage Omicron have been observed in animal models and individuals previously vaccinated with primary course of ancestral (Wu-1) vaccine. Due to the rapid scale up of vaccine before emergence of the antigenically distinct Omicron variant, it is unknown whether immunological imprinting for occurs in the context of SARS-COV-2 infection itself. We longitudinally assessed humoral responses to primary two dose Ad26.COV2.S Wu-hu-1 based vaccination in a Nigerian population following the global emergence of Omicron. At study entry in Jan 2023, we found 93% and 58% of pre-vaccination participants previously exposed to ancestral Wu-1 and Omicron virus respectively by anti-N IgG and anti-receptor binding domain (RBD) IgG Wu-1 and Omicron -specific antibodies. In participants with no evidence of prior exposure to Omicron, neutralisation against Wu-1 was significantly higher than Omicron variants as expected. However, serum neutralisation titres in participants who were anti-RBD Omicron IgG positive were paradoxically 2-fold lower for Omicron BA.1 as compared to Wu-1. This is clear evidence for imprinted immunity from the ancestral pre-omicron lineage viruses, and remarkably these old responses to Wu-1 were able to dominate over more recent, likely multiple, Omicron lineage infections. Furthermore, in these participants with prior exposure to Omicron and evidence of imprinting, we observed that further Omicron infection and Wu-1 based vaccine was associated with boosting of responses across variants with equalisation of neutralisation titres for Wu-1 and Omicron variants. However, omicron responses did not surpass ancestral responses, suggesting persistence of imprinting and only partial mitigation. Although neutralization responses at high titres were observed post dose 1 vaccination against ancestral and Omicron variants BA.1, BA.2, BA.4 in nearly all participants, neutralisation against the highly immune evasive XBB recombinant variant remained substantially lower, with a second vaccine dose providing very modest boosting. These data highlight immune imprinting against SARS-CoV-2 prior to vaccination and its persistence thereafter. In present day unvaccinated populations where serum neutralisation responses to pre-Omicron variants dominate, use of an omicron variant based vaccine should be used in preference to Wu-1 based vaccine to override imprinting and provide broader protection for vulnerable populations such as the elderly or those with compromised immunity.

Low-oxygen conditions (hypoxia) have been associated primarily with cell-cycle arrest in dividing cells. Macrophages are typically quiescent in G0 but can proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in macrophages. Cell cycle progression is largely limited to G0-G1/S phase transition with little progression to G2/M. This cell cycle transitioning is triggered by an HIF2α-directed transcriptional program. The response is accompanied by increased expression of cell-cycle-associated proteins, including CDK1, which is known to phosphorylate SAMHD1 at T592 and thereby regulate antiviral activity. Prolyl hydroxylase (PHD) inhibitors are able to recapitulate HIF2α-dependent cell cycle entry in macrophages. Finally, tumor-associated macrophages (TAMs) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at the single-cell level. These findings have implications for inflammation and tumor progression/metastasis where low-oxygen environments are common.

An ancient conflict between hosts and pathogens has driven the innate and adaptive arms of immunity. Knowledge about this interplay can not only help us identify biological mechanisms but also reveal pathogen vulnerabilities that can be leveraged therapeutically. The humoral response to SARS-CoV-2 infection has been the focus of intense research, and the role of the innate immune system has received significantly less attention. Here, we review current knowledge of the innate immune response to SARS-CoV-2 infection and the various means SARS-CoV-2 employs to evade innate defense systems. We also consider the role of innate immunity in SARS-CoV-2 vaccines and in the phenomenon of long COVID.

Persistent SARS-CoV-2 infections are a likely source of new variants of concern. In a fatal infection in an immunocompromised patient, we find association between viral load and diversity, and abrupt diversification between contemporaneous nasopharyngeal and endotracheal aspirate samples following remdesivir and convalescent plasma treatment. Shared high proportion of G>T mutations suggests spread of viruses from the lower to the upper tract as the patient deteriorated. We identified the S:P812S spike mutation adjacent to the fusion peptide unique to the nasopharyngeal sample site. We find P812S reduced S1/S2 cleavage and decreased entry efficiency in cell lines with a range of ACE2 and TMPRSS2 expression levels. This reduction of infectivity contrasted with reduced susceptibility to neutralising antibodies in sera from vaccinated individuals conferred by P812S at both 37°C and 32°C (simulating upper tract). Thus, S:P812S is a specific adaptation during SARS-CoV-2 intrahost evolution, allowing immune evasion at lower temperatures observed in the upper respiratory tract at the expense of target cell entry efficiency. ### Competing Interest Statement The authors have declared no competing interest.

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity is recovered in the presence of heparin sulfate, which targets the 'N354 pocket' to ease restrictions of conformational transition resulting in a 'RBD-up' state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.

Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Real-world data on vaccine-elicited neutralising antibody responses for two-dose AZD1222 in African populations are limited. We assessed baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using binding antibodies analysis coupled with pseudotyped virus neutralisation assays in two cohorts from West Africa: Nigerian healthcare workers (n = 140) and a Ghanaian community cohort (n = 527) pre and post vaccination. We found 44 and 28% of pre-vaccination participants showed IgG anti-N positivity, increasing to 59 and 39% respectively with anti-receptor binding domain (RBD) IgG-specific antibodies. Previous IgG anti-N positivity significantly increased post two-dose neutralizing antibody titres in both populations. Serological evidence of breakthrough infection was observed in 8/49 (16%). Neutralising antibodies were observed to wane in both populations, especially in anti-N negative participants with an observed waning rate of 20% highlighting the need for a combination of additional markers to characterise previous infection. We conclude that AZD1222 is immunogenic in two independent West African cohorts with high background seroprevalence and incidence of breakthrough infection in 2021. Waning titres post second dose indicates the need for booster dosing after AZD1222 in the African setting despite hybrid immunity from previous infection.

Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. We have previously shown Delta spike confers rapid cell-cell fusion kinetics; here we show that increased fusogenicity can be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of successful SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.

The SARS-CoV-2 Omicron BA.1 variant emerged in 2021(1) and has multiple mutations in its spike protein(2). Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways(3) demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT‐II is involved in this process in HIV‐1 and have defined the molecular domains that are important for this. Here, using SNAP‐tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT‐II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed.