The identification of antigen-specific neutralising antibodies is essential for vaccine development and therapeutic discovery, yet current methods either lack functional readouts or are impractical for polyclonal sera from large cohorts. Here, we describe an ELISA-based pulldown methodology that isolates functional antibodies from serum samples while preserving their neutralising activity for downstream applications. We optimised elution conditions using 3 M MgCl₂ in HEPES buffer, which effectively disrupts antibody-antigen interactions without dislodging immobilised antigen or impairing antibody function. The assay demonstrated 100% specificity and 77.78% sensitivity for detecting known positive samples. Compatibility with pseudotype neutralisation assays was established, with maximum tolerable MgCl₂ concentrations defined for direct use without dialysis. As proof-of-concept, we applied the method to identify domain-specific neutralising antibodies against the influenza virus haemagglutinin, distinguishing head-targeting from stem-targeting responses in human sera. This methodology provides a scalable platform for dissecting functional antibody responses with epitope-level resolution.

BackgroundSince it was first detected in 2011, antibodies to influenza D virus (IDV) have been found in several species including equids, although cattle are the major host. However, no association of IDV with naturally occurring respiratory disease in horses has yet been demonstrated.Aims/objectivesThe aim of this study was to determine whether IDV may be a causal agent of equine respiratory disease in the UK.MethodsNasopharyngeal swab extracts from horses with respiratory signs were screened by RT-qPCR for the presence of IDV RNA. Equine serum samples from apparently healthy horses were screened by haemagglutination inhibition (HI) test for the presence of IDV antibodies. Cattle and HI positive equine serum samples were screened by neutralisation test using an IDV pseudotyped virus (PVNT)ResultsNone of the 264 equine respiratory samples were positive for IDV RNA. Of the 330 serum samples from 169 Thoroughbred brood mares and 105 Thoroughbreds in training yards, only one (from a 5-year-old brood mare) was positive for HI antibodies. This single positive sample was confirmed in the PVNT. In contrast, 103 of 145 bovine serum samples (71%) were positive by PVNT, confirming widespread circulation of IDV in cattle in Great Britain and Ireland.ConclusionThis study found a low risk of horses being exposed to IDV in the UK, with no evidence that IDV contributes to equine respiratory disease.

[This corrects the article DOI: 10.1371/journal.ppat.1012763.].

Systemic administration of influenza virus-specific monoclonal antibodies achieves low concentrations in the nasal mucosa, the portal of infection. Intranasal administration may be more relevant for preventing infection, but the pharmacokinetics of intranasal influenza antibodies is unknown. We present results of preclinical studies and first-in-human phase 1 trials of the intranasally administered CR9114, an anti-hemagglutinin stem antibody that protects against influenza A and B viruses. We tested safety and tolerability of different schedules and doses; pharmacokinetics in nasal mucosal lining fluid of the nose and nasopharynx, saliva, and serum; and ex vivo functionality. We evaluated in vivo efficacy of CR9114 in mice and nonhuman primates. Intranasal CR9114 was safe and well tolerated across all doses and schedules. The half-life of CR9114 in the nose was ~3 hours. Steady-state concentrations were rapidly attained and sustained with multidosing. Trough concentrations were up to 92-fold higher with twice-daily administration compared with once-daily administration. Pharmacokinetics of intranasal CR9114 in nonhuman primates mirrored that of humans better than mice. Postdose nasal samples potently bound hemagglutinin from diverse strains of influenza A and B viruses and, particularly at the 10-milligram dose, neutralized A/H1N1, A/H5N1, and A/H3N2 more potently than baseline samples. Twice-daily administration of CR9114 protected nonhuman primates against influenza virus challenge with the same intranasal formulation and device as used in humans, providing evidence for the efficacy of intranasal multidosing. Together, these study findings characterize the pharmacokinetics of CR9114 after intranasal administration and provide proof of concept that intranasal antibodies can elicit efficacious passive immunity against influenza viruses.

Dogs have not typically been considered natural hosts for influenza viruses. However, two canine influenza subtypes, H3N8 and H3N2, have been associated with infection in this species, posing a potential zoonotic threat. In this study, a serological investigation was conducted to assess the seroprevalence of human seasonal influenza viruses (type A and B), as well as canine and avian influenza A viruses, and the H7N1 virus, in a population of adult household dogs. A total of 256 serum samples were analysed by the hemagglutination inhibition and virus neutralization assays. The seroprevalence rates were 0.78% and 77.34% for human seasonal H1N1 and H3N2 influenza viruses; 0.39% for both the B Yamagata and B Victoria lineages; and 1.17% for H7N1 virus. None of the samples tested positive for avian or canine viruses. These findings suggest that household dogs can be exposed to human seasonal influenza viruses, emphasizing the importance of continued surveillance of influenza viruses within canine populations.

BACKGROUND Understanding SARS-CoV-2 antibody dynamics is critical for pandemic preparedness, particularly where population immunity has developed through high infection rates with minimal vaccination. Whether predominantly asymptomatic infections confer protective immunity and which biomarkers best predict protection in resource-limited settings remain unclear. METHODS We conducted a household cohort study in The Gambia over 15 months (March 2021-June 2022) during Delta and Omicron waves, with weekly upper respiratory tract sampling for SARS-CoV-2 PCR testing. Serum and mucosal samples were collected at baseline, 6, and 12 months. We measured serum neutralising antibodies and mucosal IgA against five SARS-CoV-2 variants. Using Bayesian hierarchical models, we quantified antibody kinetics and evaluated correlates of protection by predictive accuracy across biomarker-variant combinations. FINDINGS Among 289 participants with 768 serological and mucosal measurements, attack rates of 53% (Delta) and 79% (Omicron) were observed, with 84% of infections asymptomatic. Delta infection produced serum neutralising antibody responses to the Delta variant (27-fold rise at peak, 95% CrI 5.0-95.2) persisting 197 days >4-fold baseline, while Delta mucosal IgA responses were shorter (9-fold rise at peak, 95% CrI 1.7-39.3, persisting 62 days). Delta infections generated substantial cross-reactive boosting to Ancestral and Alpha variants, whereas Omicron boosting was more specific to BA.1. Participants with hybrid immunity from infection and vaccination achieved higher antibody levels (24-fold rise) than those with infection alone (6-fold rise) during the Delta wave. Variant-matched neutralisation demonstrated superior predicted protection performance against infection compared to other biomarkers in both waves (Delta: AUC 0.66; Omicron BA.1: AUC 0.65). INTERPRETATION Predominantly asymptomatic SARS-CoV-2 infections in The Gambia generated robust, durable and protective antibody responses. Variant-matched serum neutralising antibody levels capture protection as effectively as complex multi-biomarker approaches, providing pragmatic guidance for serological surveillance for population immunity in resource-constrained settings. FUNDING United Kingdom Research and Innovation Grant (No. MC\_PC\_19084). ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. (MC\_PC\_19084). BK and SF are funded by the Einstein Foundation, Berlin ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Study approval was given by the joint Gambia Government and Medical Research Council Unit, The Gambia (MRCG) Ethics committee, and the London School of Hygiene and Tropical Medicine ethics committee (project ID 22556, clinicialtrials.gov [NCT05952336][1]). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced are available online at https://github.com/ccgh-idd/cop-transvir-sarscov2 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT05952336&atom=%2Fmedrxiv%2Fearly%2F2026%2F01%2F04%2F2026.01.02.26343369.atom

Abstract Influenza D virus (IDV), the most recently identified member of the Orthomyxoviridae , was first isolated from pigs but cattle have been identified as the reservoir host. To date, IDV has not been confirmed to cause human disease. Like the haemagglutinin (HA) of influenza A virus (IAV) and the haemagglutinin-esterase fusion (HEF) protein of influenza C virus (ICV), the IDV HEF is produced as a precursor protein (HEF0) that must be proteolytically cleaved by host cell proteases (into HEF1 and HEF2) to gain its fusion capacity. The proteases that activate IAV HA have been extensively studied, but those responsible for activation of IDV HEF were unknown. Identifying these proteases is key to understanding early virus–host interactions and host restriction. Therefore, we generated ICV and IDV pseudotyped viruses (PVs) in HEK 293T producer cells with or without co-transfection of plasmids expressing different type II serine proteases. Subsequent transduction of swine testicular (ST) cells indicated strong activation of both ICV and IDV PVs by the human airway trypsin-like protease (HAT) and its swine homologue (swAT). Furthermore, like influenza A/Puerto Rico/8/34 (H1N1) virus, addition of exogenous protease is not essential for IDV replication in MDCK II cells, most likely due to endogenous expression of matriptase. In conclusion, our data unveil new information on host cell proteases that activate ICV and IDV HEF proteins. Importantly, the data suggest that protease specificity is not a factor in restriction of IDV replication in the human upper respiratory tract.

Background Epidemiological surveillance of influenza D virus (IDV) has gained increased priority following recent serological findings indicating its potential zoonosis in humans. In this context, it is crucial to develop strong, reproducible, reliable and scalable immunological assays that can be quickly implemented in the surveillance of new emerging threats. Serology is a powerful tool for immune monitoring prior to infection and conducting epidemiological surveillance. However, the traditional microneutralisation (MN) assay requires wild-type viruses, considerably limiting its accessibility for some laboratories. Pseudotyped viruses (PVs) allow for expanded usage since they are safer and more flexible for adaptation to specific strains and enable application in laboratories without implementation in high biosecurity containment.Methods In this study, we conducted the qualification of a PV-based MN (pMN) assay with IDV-PVs that express the HEF glycoprotein of the D/Swine/Italy/199724-3/2015 strain. The assay functionality was examined using 14 bovine serum samples, assessing key analytical parameters including accuracy, specificity, precision, linearity and robustness.Results The findings demonstrate the IDV pMN assay to be an effective method for the detection of neutralising antibodies.Conclusions Therefore, the assay can be a valuable tool to facilitate large-scale surveillance and provide data to inform immunisation strategy development.

The increasing spread of highly pathogenic avian influenza (HPAI) A/H5 viruses poses a pandemic threat. Circulating clade 2.3.4.4b viruses have demonstrated rapid transcontinental dissemination, extensive reassortment, epizootic spread and potential sustained mammal-to-mammal transmission, signifying a heightened risk of becoming a human pathogen of high consequence. A broadly protective, future-proof vaccine against multiple clades of H5 influenza is urgently needed for pandemic preparedness. Here, we combine two novel vaccine technologies to generate a Digitally Immune Optimised and Selected H5 antigen (DIOSvax-H5inter) displayed multivalently on the mi3 nanocage using the SpyTag003/SpyCatcher003 conjugation system. Mice immunized with DIOSvax-H5inter Homotypic Nanocages at low doses demonstrate potent, cross-clade neutralizing antibody and T cell responses against diverse H5 strains. DIOSvax-H5inter Homotypic Nanocages provide a scalable vaccine candidate with the potential for pan-H5 protection against drifted or newly emergent H5 strains. This World Health Organization preferred characteristic is essential for prospective strategic stockpiling in the pre-pandemic phase.

Influenza continues to cause significant mortality globally and possesses substantial pandemic potential. Assessing pandemic risk requires a clear understanding of existing population immunity. Leveraging a unique large-scale cohort of human sera, we evaluated total and neutralising antibody-mediated immunity to multiple haemagglutinin (HA) proteins, including those from subtypes with high pandemic potential. Our analysis reveals that population immunity is heterogeneous, with distinct age-dependent differences in responses to H5, H7, and H9 avian influenza subtypes. These shifts align with historical circulation patterns of seasonal H1N1 and H3N2 human viruses. Notably, H7 viruses are primarily neutralised through head domain epitopes, while H5 viruses are targeted mainly via stem epitopes, although in both instances some neutralisation occurred via receptor binding site-adjacent epitopes. Furthermore, H7 responses were dominated by non-glycan-targeted IgG2 antibodies, whereas H5 responses were primarily IgG1-mediated. These findings highlight varying levels of susceptibility to influenza across the population, supporting vaccination approaches informed by exposure history. ### Competing Interest Statement CPT has received lecture fees from Moderna. ### Funding Statement J.S.B. was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) doctoral training programme grant [grant number BB/M011224/1]. R.S. is funded by a Medical Research Council Impact Accelerator Account grant [grant ref MR/X502674/1]. RG was funded by The Institute for Global Pandemic Planning at the University of Warwick, UK, as part of a philanthropically supported doctoral programme. K.C. was funded via the Medical Research Council doctoral training programme grant [MC\_UP\_A025_1011]. L.H. was funded by a Defence and Science Technology Laboratory grant [grant ref RQ31692]. U.O. and C.P.T. acknowledge funding from the British Council ISFP scheme [grant number 47650215]. N.C.R. is supported by a Royal Society Dorothy Hodgkin Research Fellowship [grant number DHR00620]. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was obtained for the Scottish National Blood and Transfusion Service (SNBTS) anonymous archive - IRAS project number 18005 (an NHS REC). SNBTS blood donors gave fully informed consent to virological testing, donation was made under the SNBTS Blood Establishment Authorisation and the study was approved by the SNBTS Research and Sample Governance Committee (SGS 2022-12). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors post publication and agreement by the SNBTS.

Monoclonal antibodies have two core mechanisms of protection: an antibody's antigen-binding fragment (Fab) can bind and neutralize viral pathogens and its fragment crystallizable domain (Fc) catalyzes effector functions. We investigated the relative contribution of Fab- versus Fc-mediated mechanisms of protection through passive administration of distinct forms of the pan-reactive anti-influenza antibody CR9114. We demonstrated that the contribution of Fc-independent (Fab-dependent) versus Fc-dependent mechanisms of protection is defined by the route of administration. We used CR9114 variants (wild-type, two Fc-silenced variants, or the bivalent antigen-binding fragment F(ab')2), administered either intravenously or intranasally. We found that intravenously-administered CR9114 requires the Fc domain to provide potent, pre-exposure protection against influenza A and B viral challenge. In contrast, when CR9114 was administered locally to the nasal mucosa, the main mode of protection was provided by F(ab')2, and was largely Fc-independent. Importantly, this mode of protection following intranasal administration also applied to non-neutralized influenza B strains. Moreover, intranasal administration resulted in an increase in potency against influenza A/H1N1, A/H5N1, A/H3N2, B/Yam and B/Vic compared to intravenous administration up to 50-fold. These results shed new light on the application of monoclonal antibodies such as CR9114 to combat viral infection locally, and will help inform clinical strategies of pre-exposure prophylaxis. More fundamentally, this study uncovers distinct modes of protection for systemic versus intranasally-administered prophylactic antibodies.

Effective therapeutics for severe cases of SARS-CoV-2 are still needed. As new variants of concern emerged an increase in hospitalizations was observed, especially in non-vaccinated individuals, immunocompromised individuals and the elderly, whereby treatment options became challenging. Several monoclonal antibodies (mAb) are being evaluated for approval due to their ability to neutralize the virus. Here, we report monoclonal antibodies targeting the Spike (S) with a strong neutralization profile against lentiviral and VSV pseudotypes displaying the Spikes of SARS-CoV-1, SARS-CoV-2 and variants of concern, in addition to two bat coronaviruses. We found several mAbs that were able to bind and neutralize a broad set of variants, with one mAb able to neutralize SARS-CoV-1, SARS-CoV-2, RaTG13 and WIV16. Their binding affinities were also characterized, and several mAbs were in the picomolar range against different variants. These results indicate that their use as a cocktail of monoclonal antibodies to treat patients infected with variants associated with severe disease and immune escape can be explored as an option. Furthermore, the cross reactivity observed by one mAb may reveal further insight into vulnerable portions of the Spike protein, which may become valuable future targets. ### Competing Interest Statement The authors have declared no competing interest.

H5Nx clade 2.3.4.4b high pathogenicity avian influenza viruses (HPAIVs) have decimated wild bird and poultry populations globally since the autumn of 2020. In the UK and in continental Europe, the H5N8 subtype predominated during the first epizootic wave of 2020/21, with few detections of H5N1. However, during the second (2021/22) and third (2022/23) epizootic waves, H5N1 was the dominant subtype. The rapid shift in dominance from H5N8 to H5N1 was likely driven by a combination of virological, immunological and/or host-related factors. In this study, we compared viral fitness and immunological responses in ducks, a key reservoir species, using dominant genotypes of H5N1 (genotype AB) and H5N8 (genotype A) from the second wave. While viral shedding dynamics were similar for both viruses, H5N8 was more pathogenic. Antigenic analysis of post-infection duck sera revealed that the haemagglutinin protein was antigenically similar across clade 2.3.4.4b H5 HPAIVs, but neuraminidase proteins displayed different patterns of cross-reactivity. We also modelled a scenario where ducks were pre-exposed to H5N1 (genotype C) or H5N8 (genotype A) from the first wave and subsequently challenged with either homologous or heterologous subtypes from the second wave (genotype AB or A). Despite the absence of seroconversion, pre-exposure to different subtypes resulted in varying clinical outcomes following challenge. These findings indicate that both viral and immunological factors likely played significant roles in the emergence and spread of H5Nx HPAIVs in wild bird populations.

BACKGROUND:The converging biology between enveloped viruses and extracellular vesicles (EVs) has raised interest in the application of engineered EVs as antiviral therapeutics. Following the recent COVID-19 pandemic, EVs engineered with either the ACE2-receptor or Spike-protein have been proposed as strategy to either decoy SARS-CoV-2, or to compete with its cell entry. For generic use as a platform for future pandemic preparedness, a systematic and quantitative comparison of both strategies is required to assess their limitations and benefits across different variants of concern. METHODS:Here we generated EVs decorated with either the ACE2-receptor or the Spike-protein of (Wuhan)-SARS-CoV-2 and used single vesicle imaging for in-depth quantitative characterisation. These vesicles were then systematically tested for anti-viral activity across SARS-CoV-2 variants of concern using both, pseudotype and live virus cellular infection models including primary human bronchial and nasal explants. RESULTS:Spike-protein EVs or ACE2-EVs recovered from transiently transfected HEK293T cells comprised only a small fraction of the EV secretome (5% or 20%, respectively) and were primarily derived from the plasma membrane rather than multivesicular bodies. Redirecting intracellular trafficking of the Spike protein by mutating its transmembrane or subcellular localisation domains did not increase the yields of Spike-EVs. Both types of vesicles inhibited SARS-CoV-2 (D614G) in a dose dependent manner with kinetics and immunohistochemistry consistent with an inhibition at the initial cell entry stage. ACE2-EVs were more potent than Spike-EVs and at least 500-1000 times more potent than soluble antibodies in a pseudotype model. Surprisingly, ACE2-EVs switched from an inhibitory to an enhancer activity for the Omicron BA.1 variant whereas Spike-EVs retained their activity across all variants of concern. CONCLUSIONS:While our data show that both types of engineered EVs potently inhibit SARS-CoV, the decoy versus competition strategy may result in diverging outcomes when considering viral evolution into new variants of concern. While Spike-EVs retain their competition for receptor binding even against higher affinity viral Spike mutations, the formation of complexes between ACE2-EVs and the virus may not only result in inhibition by decoy. As EVs are actively internalised by cells themselves, they may shuttle the virus into cells, resulting in a productive alternative cell entry route for variants such as Omicron, that diverge from strict plasma membrane protease cleavage to the use of endosomal proteases for release of their genome.

IntroductionAn unbalanced immune response and excessive inflammation are the major hallmarks of severe SARS-CoV-2 infection, which can result in multiorgan failure and death. The dysregulation of the complement system has been shown in various studies as a crucial factor in the immunopathology of SARS-CoV-2 infection. Complement alternative pathway has been linked to the excessive inflammation in severe SARS-CoV-2 infection in which decreased levels of factor H (FH) and elevated levels of properdin (FP) were observed. The current study investigated the potential immune protective roles of FP and FH against SARS-CoV-2 infection.MethodsThe interactions between FH and FP and the SARS-CoV-2 spike (S) and its receptor binding domain (RBD) were evaluated using direct ELISA. The cell binding and luciferase-based viral entry assays utilising S protein expressing lentiviral pseudotypes were used to evaluate the possible modulatory effects of FH, FP, and recombinant thrombospondin repeats 4 and 5 (TSR4 + 5) on SARS-CoV-2 cell entry. Using RT-qPCR, we also assessed the immunomodulatory roles of FH and FP in the cytokine response induced by SARS-CoV-2 pseudotypes.ResultsFH and FP were found to bind to both the RBD and SARS-CoV-2 S proteins. The treatment of FP or TSR4 + 5 enhanced cell binding and entry of SARS-CoV-2 pseudotypes that was administered in A549 cells expressing human ACE2 and TMPRSS2 (A549-hACE2+TMPRSS2 cells). FP increases the affinity between host ACE2 and SARS-CoV-2, according to in silico work. In A549-hACE2+TMPRSS2 cells, the effect of FP on viral cell entry and binding was counteracted by anti-FP antibody treatment. On the other hand, SARS-CoV-2 lentiviral pseudotypes’ cell entry and binding were decreased by FH treatment. The A549-hACE2+TMPRSS2 cells that were challenged with SARS-CoV-2 alphaviral pseudotypes (expressing spike, envelope, nucleocapsid, and membrane proteins) pre-treated with FP or TSR4+5 showed an upregulation of pro-inflammatory cytokine transcripts, including NF-κB and IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES. Contrary to this, the expression of these pro-inflammatory cytokines was downregulated by FH treatment. FH treatment decreased S protein-mediated NF-κB activation, but FP treatment enhanced it in A549-hACE2+TMPRSS2 cells. DiscussionThese results imply that FH may function as a SARS-CoV-2 cell entry and binding inhibitor, reducing the inflammatory response linked to infection independently of complement activation. FP could aid cell viral entry and binding and aggravate hyperinflammation that might contribute to the severity of the infection.

COVID-19 vaccine adaptation is critical to respond to continuously emerging SARS-CoV-2 variants with enhanced immune evasion. The ARVAC protein subunit vaccine, based on the receptor binding domain of the spike protein of SARS-CoV-2, has been adapted to XBB.1.5 and JN.1 variants, as monovalent and bivalent formulations. Preclinical studies in mice showed that ARVAC XBB.1.5 and JN.1 monovalent vaccines induced strong neutralizing antibodies against XBB and JN.1 lineages, though with limited efficacy against phylogenetically distant variants. By contrast, bivalent formulations combining Gamma antigen with either XBB.1.5 or JN.1 antigens demonstrated superior cross-neutralizing activity, covering variants from Ancestral to JN.1. Additionally, Gamma-containing bivalent vaccines elicited neutralizing antibodies against SARS-CoV-1, highlighting their potential for broad-spectrum immunity. Cellular immune studies confirmed robust CD4+ T cell activation across all formulations. These findings support the continued adaptation of ARVAC to current circulant variants and propose ARVAC bivalent vaccines containing the Gamma antigen as a strategy for induction of pan-sarbecovirus immunity.

SARS-CoV-2 continues to evolve antigenically under the immune pressure exerted by both natural infection and vaccination. As new variants emerge, we face the recurring challenge of updating vaccines at significant financial cost to maintain their efficacy. To address this, novel strategies are needed to enhance the breadth of protection offered by vaccines or, at a minimum, extend their effectiveness over time. One such strategy is antigen modification. In this study, we introduce a glycosylation site into a binding but non-neutralizing epitope within the SARS-CoV-2 XBB.1.5 receptor binding domain (RBD) to redirect the immune response towards more potent neutralizing epitopes. Immunization of mice with this modified antigen via the mRNA vaccine platform resulted in a dramatic increase in neutralizing antibodies compared to the wild-type XBB.1.5 RBD, showing superior protection against a range of SARS-CoV-2 Omicron variants, from BA.2 to JN.1. Our findings reinforce the power of the glycan masking approach, which in combination with the now well-established mRNA vaccine platform can contribute to broader and better vaccines.