Cross-clade vaccination to overcome sarbecovirus or merbecovirus neutralization gaps

Wan Ni Chia; Feng Zhu; Samuel Mo Sheng Cheng; Wee Chee Yap; Abeer N Alshukairi; Yun Yan Mah; Mayfong Mayxay; Vilada Chansamouth; Andrew Letizia; Beng Lee Lim; Yidan Wang; Madeline Sheng Si Kwek; Tierra Smiley Evans; Ohnmar Aung; Hlaing Myat Thu; Christine K Johnson; Yanqun Wang; Jincun Zhao; Jessica E Manning; Christina Yek; Chanthap Lon; Seng Heng; Sovann Ly; Rithea Leang; Rekol Huy; Aghnianditya Kresno Dewantari; Frilasita Aisyah Yudhaputri; Khin Saw Aye Myint; Matthew P Rubach; John A Crump; Venance P Maro; Nicole J Moreland; Aimee Paterson; Richard Charlewood; Jingjing Song; Shiwei Chen; Wan Yi Tan; Chris Ka Pun Mok; Catherine Hong Huan Hor; David Lye; David SC Hui; Barnaby E Young; Malik Peiris; Lin-Fa Wang; Chee Wah Tan

doi:10.1016/j.celrep.2026.117444

. Author manuscript; available in PMC: 2026 Sep 26.

Published in final edited form as: Cell Rep. 2026 May 28;45(6):117444. doi: 10.1016/j.celrep.2026.117444

Cross-clade vaccination to overcome sarbecovirus or merbecovirus neutralization gaps

Wan Ni Chia ^1,^2,³⁶, Feng Zhu ^1,³⁶, Samuel Mo Sheng Cheng ^3,³⁶, Wee Chee Yap ^1,⁴, Abeer N Alshukairi ^5,⁶, Yun Yan Mah ^1,⁴, Mayfong Mayxay ^7,^8,^9,¹⁰, Vilada Chansamouth ⁷, Andrew Letizia ¹¹, Beng Lee Lim ^1,⁴, Yidan Wang ^1,¹², Madeline Sheng Si Kwek ¹, Tierra Smiley Evans ^13,¹⁴, Ohnmar Aung ¹³, Hlaing Myat Thu ¹⁵, Christine K Johnson ¹³, Yanqun Wang ¹⁶, Jincun Zhao ¹⁶, Jessica E Manning ^17,¹⁸, Christina Yek ^17,¹⁸, Chanthap Lon ¹⁸, Seng Heng ¹⁹, Sovann Ly ¹⁹, Rithea Leang ²⁰, Rekol Huy ²⁰, Aghnianditya Kresno Dewantari ²¹, Frilasita Aisyah Yudhaputri ²¹, Khin Saw Aye Myint ²¹, Matthew P Rubach ^22,²³, John A Crump ^22,^23,^24,²⁵, Venance P Maro ²², Nicole J Moreland ²⁶, Aimee Paterson ²⁶, Richard Charlewood ^26,²⁷, Jingjing Song ⁴, Shiwei Chen ¹, Wan Yi Tan ⁴, Chris Ka Pun Mok ²⁸, Catherine Hong Huan Hor ²⁹, David Lye ^30,^31,^32,^33,³⁴, David SC Hui ²⁸, Barnaby E Young ^31,^33,³⁴, Malik Peiris ³, Lin-Fa Wang ^1,^*, Chee Wah Tan ^4,^35,^37,^*

¹Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore

²Leyden Labs Asia, Singapore, Singapore

³Public Health Laboratory Sciences, School of Public Health, LKS Faculty of Medicine, Hong Kong University, Pokfulam, Hong Kong

⁴Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore

⁵King Faisal Specialist Hospital and Research Center, Jeddah, Kingdom of Saudi Arabia

⁶College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi Arabia

⁷Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Ministry of Health, Vientiane, Laos

⁸Unit for Health Evidence and Policy (UHEP), Institute of Research and Education Development, University of Health Sciences, Ministry of Health, Vientiane, Lao People’s Democratic Republic

⁹Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK

¹⁰Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore

¹¹Naval Medical Research Unit INDO PACIFIC, Singapore, Singapore

¹²Biomedical Synthetic Biology Research Center, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

¹³Epicenter for Disease Dynamics, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA

¹⁴Infectious Diseases and Vaccinology Division, School of Public Health & Department of Integrative Biology, University of California, Berkeley, Berkeley, CA, USA

¹⁵Department of Medical Research, Dagon Township, Yangon, Myanmar

¹⁶Guangzhou National Laboratory, Guangzhou, China

¹⁷Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD, USA

¹⁸International Center of Excellence in Research Cambodia, National Institute of Allergy and Infectious Diseases, Phnom Penh, Cambodia

¹⁹Communicable Disease Control Department, Ministry of Health, Phnom Penh, Cambodia

²⁰National Center for Parasitology, Entomology and Malaria Control, Ministry of Health, Phnom Penh, Cambodia

²¹Exeins Health Initiative, South Jakarta, Indonesia

²²Kilimajaro Christian Medical Centre, Moshi, Tanzania

²³Duke Global Health Institute, Duke University, Durham, NC, USA

²⁴Centre for International Health, University of Otago, Dunedin, New Zealand

²⁵Division of Infectious Diseases and International Health, Department of Medicine, Duke University Medical Centre, Durham, NC, USA

²⁶Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand

²⁷New Zealand Blood Service, Auckland, New Zealand

²⁸S. H. Ho Research Centre for Infectious Diseases, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong

²⁹Department of Chemistry, Faculty of Science, Hong Kong Baptist University, Kowloon, Hong Kong

³⁰Communicable Disease Agency, National Centre for Infectious Diseases, Singapore, Singapore

³¹National Centre for Infectious Diseases, Singapore, Singapore

³²Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

³³Lee Kong Chian School of Medicine, Nanyang Technology University, Singapore, Singapore

³⁴Tan Tock Seng Hospital, Singapore, Singapore

³⁵Infectious Diseases Research & Training Office, National Centre for Infectious Diseases, Singapore, Singapore

³⁶These authors contributed equally

³⁷Lead contact

AUTHOR CONTRIBUTIONS

Conceptualizing, C.W.T. and L.-F.W.; investigating – performing experiment and data analysis, W.N.C., F.Z., S.M.S.C., W.C.Y., Y.Y.M., B.L.L., Y.W., M.S.S.K., J.S., W.Y.T., Y.W., and C.H.H.H.; providing samples, A.N.A., M.M., V.C., A.L., T.S.E., O.A., H.M.T., C.K.J., J.Z., J.E.M., C.Y., C.L., S.H., S.L., R.L., R.H., A.K.D., M.P.R., J.A.C., V.P.M., N.J.M., C.K.P.M., D.S.C.H., and M.P.; writing – original draft, C.W.T.; writing – editing, C.W.T., L.-F.W., W.N.C., and F.Z.; reviewing and approving the draft, all authors.

Correspondence: linfa.wang@duke-nus.edu.sg (L.-F.W.), gmstanc@nus.edu.sg (C.W.T.)

PMCID: PMC13614252 NIHMSID: NIHMS2187404 PMID: 42213772

SUMMARY

The coronavirus disease 2019 (COVID-19) pandemic highlights the importance of identifying high risk pathogens, defining the immunity gaps and developing preemptive vaccination strategies. Here, we establish a high-resolution surrogate virus neutralization test detecting neutralizing antibodies against multiple virus families simultaneously, demonstrating good concordance with traditional assays. Extensive serosurveillance of pre-pandemic sera from different continents reveals low prevalence human exposures to different beta-coronaviruses, and identifies individuals with prior exposure to ACE2-binding MERS-like viruses. Furthermore, COVID-19 vaccination induces significant cross-neutralizing antibodies against clade 1b, 1c, and 3 but not clade 1a sarbecoviruses. Similarly, MERS and Nipah convalescent sera neutralize cognate viruses but have limited cross-neutralization against other related merbecoviruses and henipaviruses that utilize DPP4 and ephrin B2 receptors. Finally, a cross-clade prime-and-boost vaccination strategy using antigenically distinct antigens could induce broadly neutralizing antibodies against related viruses beyond vaccine antigens, supporting broad-spectrum beta coronavirus vaccine development.

Graphical Abstract

graphic file with name nihms-2187404-f0001.webp

In brief

Chia et al. develop a high-resolution multiplex surrogate neutralization assay to map human immunity gaps across high-risk beta-coronavirus and henipavirus families. Using multi-country pre-pandemic serosurveillance and vaccination/convalescent samples, they show limited pre-existing cross-immunity and demonstrate that cross-clade prime-and-boost strategies can elicit broader subgenus beta-coronavirus neutralizing antibodies.

INTRODUCTION

Over the last few decades, we have witnessed multiple zoonotic virus outbreaks,^1–8 some of which led to epidemics or pandemics, causing devastating loss of life and major disruptions to global health and socio-economic stability. The impact of the COVID-19 pandemic was particularly catastrophic—not only did it claim millions of lives, it also significantly weakened the global economy.⁹ These events underscore the inadequacy of our current pandemic preparedness strategies in countering the threat of a future “disease X” pandemic.

Most emerging viral diseases in humans originate from other animal species.¹⁰ Some mammals, such as bats, harbor more viruses than others.¹¹ Bats are known reservoirs for a number of viruses with significant health risks, such as the Nipah virus (NiV),^12,13 Ebola virus,¹⁴ severe acute respiratory syndrome (SARS)-related coronaviruses,^15,16 and Middle East respiratory syndrome (MERS)-related coronaviruses.^17,18 Increasing evidence suggests that land use for agricultural production and livestock activities, coupled with climate change, can be associated with an increased risk of zoonotic virus spillover events.^19–21 A recent study by Sanchez et al.²² estimated that over 60,000 people are exposed annually to animal SARS-related coronaviruses in the Southeast Asia region, highlighting that spillover events happen more often than we previously thought.

Active surveillance of zoonotic viruses at human-animal interfaces is critical for the early detection of spillover events and for mitigating disease transmission risk in the population.²³ Since the onset of the COVID-19 pandemic, the scientific and pharmaceutical communities have accelerated efforts to develop vaccines against high-priority pathogens like NiV and MERS-CoV. However, the question remains: are pathogen-specific vaccines enough to protect against future related threats, such as a hypothetical MERS-CoV-2? The emergence of SARS-CoV-2—distinct from SARS-CoV-1—demonstrated that pandemic threats can arise from antigenically novel viruses, reinforcing the urgent need to invest in broad-spectrum, pan-subgenus vaccine candidates.

Here, we invented a multi-family multiplex surrogate virus neutralization test (sVNT) that detects sarbecovirus, merbecovirus, and henipavirus neutralizing antibodies that block spike glycoprotein-host receptor interactions in the same reaction. Using this platform, we identified low population immunity to these viruses, revealed the limitations of existing pathogen-specific vaccination strategies, and designed cross-clade prime-boost strategies for broad-spectrum vaccine candidates.

RESULTS

Multi-family multiplex surrogate virus neutralization test

During the COVID-19 pandemic, we developed a SARS-CoV-2 sVNT that detects neutralizing antibodies, which block the interaction between the receptor-binding domain (RBD) and angiotensin-converting enzyme receptor 2 (ACE2).²⁴ Since then, we have transformed this test into a multiplex sVNT that covers sarbecoviruses from clades 1a and b.^25,26 In this study, we have established a multiplex sVNT capable of detecting neutralizing antibodies against a broad panel of sarbecoviruses, merbecoviruses, and henipaviruses (Figures 1A and S1A–S1C). This platform includes ten ACE2-using sarbecoviruses (SARS-CoV-2, bat CoV BANAL-52, pangolin CoV GD-1, pangolin CoV GX-P5L, bat CoV RaTG13, bat CoV RsSHC014, bat CoV WIV-1, SARS-CoV-1, bat CoV RsYN04, and bat CoV Khosta-2) (Figure 1B), five merbecoviruses using either DPP4 or ACE2 receptor (MERS-CoV, camel MERSr-CoV, bat CoV 422, pangolin HKU4r-CoV, and NeoCoV) (Figure 1C), and four ephrin-B2-binding henipaviruses, including NiV, hendra virus (HeV), cedar virus (CedV), and Ghanaian henipavirus (GhV) (Figure 1D), As shown in Figure 1E, the representative spike/glycoproteins bind to their corresponding human receptors in a dose-dependent manner. Clade 2 sarbecoviruses, which do not bind to human ACE2,^27,28 were excluded from this study. Some merbecoviruses showed weak binding to either DPP4 or ACE2 but were incompatible with the multi-family multiplex surrogate virus neutralization test (MM-sVNT) due to very low binding affinity for reliable analysis. We demonstrated that the MM-sVNT correlates well with plaque reduction neutralization test (PRNT) or pseudotyped virus neutralization test (pVNT), with R² values of 0.9687 and 0.9366 for MERS-CoV and NiV, respectively (Figures S1D–S1F). Naïve healthy serum samples were used to establish the assay cut-off, and samples with neutralization titer 50% (NT50) values >1:100 were considered positive (Figure 1F). We further validated the platform using virus-specific monoclonal antibodies (Figure 1G). The E7 monoclonal antibody, isolated from a SARS survivor vaccinated against COVID-19,²⁹ neutralized all sarbecoviruses in the panel, with inhibition concentration 50% (IC50) value ranging from 6.6 to 100.2 ng/mL (Figure 1G). M102.4³⁰ and HENV-26³¹ neutralized both HeV and NiV (IC50: 21.4 to 43.9 ng/mL) but not CedV or GhV (Figure 1G). Four MERS-specific monoclonal antibodies were tested, all of which neutralized human MERS-CoV and camel MERSr-CoV NV1405. Notably, JC57 also cross-neutralized bat CoV-422 with an IC50 value of 40.9 ng/mL (Figure 1G), as described previously.³²

Figure 1. — (A) Schematic illustration of the MM-sVNT platform, capable of detecting neutralizing antibodies against sarbecoviruses, merbecoviruses, and henipaviruses in a single reaction.

(B–D) Phylogenetic trees of (B) sarbecovirus RBDs, (C) merbecovirus RBDs, and (D) henipavirus glycoproteins, generated using the general-time reversible (GTR) model with 1,000 bootstrap replicates. Bootstrap values > 70% are shown above or below the branches. Scale bars represent the number of substitutions per site. Viruses included in the MM-sVNT panel are indicated in blue.

(E) Mean fluorescence intensity (MFI) of sarbecovirus, merbecovirus, and henipavirus RBD/glycoprotein binding to human ACE2, DPP4, and ephrin-B2 receptors, respectively.

(F) Specificity of the MM-sVNT platform, tested using naïve healthy human serum samples. A cut-off dilution of 1:100 was used; samples with NT50 values below this threshold were considered negative.

(G) Heatmap showing sVNT IC50 values for 12 published monoclonal antibodies against sarbecoviruses, merbecoviruses, and henipaviruses.

Limited cross-neutralizing antibodies against pre-emergent related viruses

To assess the breadth of neutralizing antibodies induced by SARS-CoV-2 vaccination, we determined the NT50 of serum samples collected from COVID-19 vaccine recipients, SARS survivors, and SARS survivors who subsequently received a COVID-19 mRNA vaccine. COVID-19 vaccines induced significant cross-neutralizing antibodies against clade 1b (with geometric mean NT50 values ranging from 2,871 to 176), clade 1c (geometric mean NT50 of 357 against RsYN04), and clade 3 (geometric mean NT50 of 128 against Khosta-2) but showed limited neutralization of clade 1a sarbecoviruses (geometric mean NT50 values ranging from 31 to 100) (Figure 2A). Serum samples from SARS survivors neutralized only SARS-CoV-1 and WIV-1, with geometric mean NT50 values of 92 and 137, respectively (Figure 2B). In contrast, SARS survivors who had received a COVID-19 mRNA vaccine produced broadly neutralizing antibodies capable of targeting sarbecoviruses from clades 1a, 1b, 1c, and 3, with geometric mean NT50 values ranging from 140 to 860 (Figure 2C). Despite RsSHC014 being more genetically similar to SARS-CoV-2 than to sarbecoviruses from other clades (1a, 1c, and 3) (Figure 2D), it remained the least susceptible to cross-neutralizing antibodies induced by SARS-CoV-2 vaccination (Figures 2A–2C). We next investigated whether the XBB booster vaccine could enhance the breadth of neutralizing antibodies against animal sarbecoviruses. As shown in Figure 2E, the XBB booster vaccine induced a 6-fold increase in neutralizing antibodies against SARS-CoV-2 and a 2.1- to 3.5-fold increase in cross-neutralizing antibodies against clade 1a sarbecoviruses. Similarly, we observed enhanced cross-neutralization activity against RsYN04 and Khosta-2 (Figure 2E), with 4.1- and 5-fold increases in geometric mean NT50 value, respectively, in individuals who received the XBB booster vaccine. To determine whether improved cross-neutralization was due to reduced binding affinity to human ACE2, we performed surface plasmon resonance analysis of the RsYN04 and Khosta-2 RBDs. We found that both RBDs bound to human ACE2 with dissociation constants (Kd) of 9.96 × 10⁻¹⁰ M and 2.69 × 10⁻⁷ M, respectively. Notably, RsYN04 exhibited the strongest binding affinity to ACE2 among all RBDs tested (Figures S2A–S2F), highlighting that binding affinity does not correlate with neutralization activity. Lastly, we demonstrated that booster vaccination and late Omicron breakthrough infections shaped cross-neutralization responses toward Omicron variants but had minimal impact on SARS-CoV-1 (Figures S3A and S3B), further underscoring the need for pan-sarbecovirus vaccine candidates.

We then characterized the cross-neutralizing antibodies induced by MERS-CoV. As shown in Figure 2F, human MERS survivors produced neutralizing antibodies against MERS-CoV (geometric mean NT50 of 149) and camel MERSr-CoV (geometric mean NT50 of 144), with limited cross-neutralization against bat CoV-422 (geometric mean NT50 of 26) and mjHKU4r-CoV (geometric mean NT50 of 16). Bat CoV-422 and mjHKU4r-CoV share 63.1% and 58.9% amino acid identity, respectively, with the MERS-CoV spike (Figure 2G). MERS sera failed to neutralize NeoCoV, which shares only 33.9% amino acid identity in RBD with MERS-CoV (Figures 2G and S4).

Similarly, NiV survivors produced NiV-specific neutralizing antibodies, with a geometric mean NT50 of 152 against NiV, and also cross-neutralized HeV, though with a 5.8-fold reduction in activity compared to that against NiV (Figure 2H). No cross-neutralizing activity was observed against CedV or GhV (Figure 2H). Ferrets or horses infected by CedV or HeV produced CedV-specific or HeV-specific neutralizing antibodies with no cross-neutralization of other henipaviruses (Figures S5A and S5B). Despite all tested henipaviruses using ephrin B2 as an entry receptor, CedV and GhV glycoproteins share only 30.9% and 24.7% amino acid identity, respectively, with the NiV glycoprotein (Figure 2I).

Serosurveillance revealed low to no pre-existing immunity against the three virus groups

We screened pre-COVID-19 pandemic serum samples collected from various regions, including New Zealand, Southeast Asia, and Tanzania, and found a low prevalence of antibodies against these viruses (Figures 3A–3C). Notably, seropositivity rates for sarbecoviruses were higher in Southeast Asian countries, suggesting possible spillover of related coronaviruses in the region (Figure 3A). Interestingly, more merbecovirus-positive samples were detected in Tanzania than in Southeast Asia or New Zealand (Figure 3B). The median percentage inhibition for MERS-CoV in Tanzania was 26.7%, compared to 12.4% and 4.7% in Southeast Asia and New Zealand, respectively (Figure 3B).

Figure 3. — MM-sVNT analysis was performed using serum samples collected before the COVID-19 pandemic from Southeast Asia (SEA), New Zealand (NZ), and Tanzania (TZ), diluted 1:20, to assess neutralization against (A) sarbecoviruses, (B) merbecoviruses, (C) henipaviruses, and (D) NeoCoV. Data are presented as violin plots, with the percentage of samples showing >40% inhibition indicated above each plot. Statistical analysis in (A), (B), and (C) was performed using two-tailed unpaired t test in GraphPad Prism 10. Two NeoCoV-positive samples were further analyzed by (E) a merbecovirus-specific MM-sVNT and (F) western blot. Western blot analysis was performed using serum diluted 1:400.

To investigate further, we analyzed additional human samples collected in Nigeria and Morocco, which had previously tested negative for MERS-CoV using pVNTs. These samples consistently showed high baseline inhibition against MERS-CoV and related viruses, with median inhibition ranging from 25.0% to 30.0% (Figure S6A). Notably, a higher number of MERS-CoV-positive samples were identified in human cohorts from Africa, further indicating potential spillover of related viruses into the human population (Figures S6A and S6B). Similarly, camel serum samples that tested negative for MERS-CoV via pVNT revealed that a few animals in Kenya, Pakistan, Morocco, and Uganda had neutralizing antibodies against MERS-CoV and MERS-related coronavirus (MERSr-CoV) NV1405, suggesting possible prior exposure to MERS-CoV related viruses (Figure S6C).

Through retrospective surveillance, we identified two individuals from a Southeast Asian country who had prior exposure to NeoCoV (or a related virus)—a MERS-like virus originally identified in Neoromicia capensis bats from South Africa³³—with NT50 values of 447.4 and 514, respectively (Figures 3D and 3E). The presence of MERS-like RBD-specific antibodies was confirmed by western blot analysis (Figure 3F). This finding highlights that zoonotic spillover events may occur more frequently than we could reliably assess previously, emphasizing the need for active molecular and serological surveillance in this region using improved tools. Notably, sera samples that tested positive for NeoCoV failed to neutralize bat HKU5-CoV-2, indicating that the two viruses are antigenically distinct (Figure 3E).

Cross-clade prime-and-boost with antigenically distinct vaccine candidates induces broadly neutralizing antibodies in mice

In this study, we demonstrated that a virus-specific vaccination strategy is unable to induce broadly neutralizing antibodies against antigenically distinct but closely related viruses within the same genus or sub-genus (Figures 2A and 2B). Given the limited and uneven cross-neutralizing antibody responses revealed by our serosurveillance, we next asked whether a sequential, cross-clade prime-and-boost vaccination strategy could overcome these gaps and elicit broadly protective antibodies. Inspired by the observations in COVID-19-vaccinated SARS survivors (Figure 2C), we designed pan-sarbecovirus vaccines based on a cross-clade prime-and-boost strategy (Figures 4A and 4B), postulating that priming and boosting the immune system with antigenically distinct antigens would elicit responses targeting conserved but otherwise less immunogenic epitopes (Figure 4B). Given that NiV-specific infections did not induce cross-neutralizing antibodies (Figures 2H and S5) and that there is significant sequence divergence between NiV and bat-borne henipaviruses (Figure 2I), we focused our cross-clade prime-and-boost vaccination study on sarbecoviruses and merbecoviruses. To this end, we designed and produced consensus spike proteins from known ACE2-binding and non-binding sarbecoviruses (C1.25, see also Figure S7A). Additional Omicron mutations were incorporated into consensus spike C1.26 to improve breadth against Omicron variants (Figure S7A). The C1.28 sequence employed a more mosaic antigen design, merging spike sequences from clades 1c, 2, and 3. Together, C1.25, C1.26, and C1.28 constituted a rationally engineered vaccine series that anticipates viral evolution by targeting conserved epitopes across sarbecovirus clades 1–3, providing the foundation for true pan-sarbecovirus protection.

Figure 4. — (A) Coronavirus family tree and schematic of the pan-sarbecovirus and pan-merbecovirus cross-clade prime-and-boost vaccination strategy.

(B) Illustration of the proposed mechanism underlying the pan-sarbecovirus vaccine.

(C) Graphical overview of the animal experiment involving a control group receiving either three or four doses of BNT162b2 and a test group receiving pan-sarbecovirus booster vaccines, C1.26 and C1.28.

(D) MM-sVNT analysis of serum samples collected 14 days after the third and fourth booster doses. Error bars represent standard error; data are shown as mean NT50 values from five mice.

(E) Graphical overview of the animal experiment involving the pan-merbecovirus vaccination strategy. Mice were primed with 10 μg of subunit homologous vaccine at day 0, 21 and 49, and boosted with 20 μg heterologous subunit vaccine.

(F) Multiplex analysis of serum samples collected 14 days after the third and fourth booster doses. Error bars represent standard error; data are shown as mean NT50 values from five mice.

(G) sVNT analysis of mice receiving pan-merbecovirus vaccines against BtHKU5-CoV-2. Red arrow indicates the time point when the mice received BtHKU5-CoV-2 RBD booster.

(H) The cross-neutralization analysis was performed on sera collected from mice before and after receiving the BtHKU5-CoV-2 RBD booster, targeting MERS-CoV, camel MERSr-CoV NV1405, mjHKU4r-CoV, and bat CoV 422.

Error bars represent standard error, and the geometric mean titer is indicated above each bar.

Statistical analysis in (G) and (H) was performed using two-tailed unpaired t test in GraphPad Prism 10.

Mice receiving three or four doses of the BNT162b2 mRNA vaccine developed robust neutralizing antibodies against SARS-CoV-2, its variants, and clade 1b sarbecoviruses but showed limited cross-neutralization against clade 1a and clade 3 sarbecoviruses (Figures 4C, 4D, and S7B). In contrast, mice that received two doses of BNT162b2 followed by C1.26 and C1.28 boosters (Figure 4D) developed broadly neutralizing antibodies capable of neutralizing all sarbecoviruses tested in our panel, with geometric mean NT50 titers ranging from 681 to 25,286. Notably, mice boosted with C1.26 and C1.28 exhibited higher neutralization activity against all SARS-CoV-2 variants, including immune-evasive Omicron subvariants, probably due to the presence of Omicron-specific mutations in the C1.26 construct (Figures S7A and S7B).

To further test whether a similar strategy could be applied to different viruses in the subgenus Merbecovirus, we immunized mice with three doses of a homologous subunit vaccine followed by a heterologous subunit booster (Figure 4E). As shown in Figure 4F, virus-specific vaccines induced neutralizing antibodies with limited to no cross-neutralization of antigenically distinct related viruses. However, a booster antigen from a different clade was sufficient to elicit broad-spectrum neutralizing antibodies capable of neutralizing all merbecoviruses tested (Figure 4F). While preparing this manuscript, an ACE2-binding MERS-like virus, BtHKU5-CoV-2, was reported.¹⁷ Using serum samples from mice primed and boosted with antigenically distinct MERS-like viruses, we observed cross-neutralization activity against BtHKU5-CoV-2 (Figure 4G). Remarkably, a single dose of a BtHKU5-CoV-2 RBD booster was sufficient to induce high levels of neutralizing antibodies against BtHKU5-CoV-2 (Figure 4G) and other merbecoviruses (Figure 4H).

DISCUSSION

In this study, we described a high-throughput and high-resolution serological tool that enables the rapid detection of neutralizing antibodies that block receptor-RBD protein-protein interactions^24–26 of multiple viruses from different viral families simultaneously in a single tube. This MM-sVNT demonstrated a strong correlation with live or pseudotyped neutralization tests, highlighting its potential application for high-throughput serosurveillance of zoonotic spillover events in human populations. Such surveillance can enhance our understanding of regional infectious disease threats and population immunity levels, enabling better preparedness for future outbreaks or pandemics. Using this platform, we analyzed serum samples collected from individuals infected with or vaccinated against SARS-CoV-2, NiV, or MERS-CoV. Our data revealed little or no cross-neutralizing antibodies against antigenically distinct but phylogenetically related viruses.

Serosurveillance of archived samples collected before the COVID-19 pandemic from Southeast Asian countries, New Zealand, and Tanzania revealed limited to no pre-existing immunity to sarbecoviruses, merbecoviruses, and henipaviruses. This highlights the susceptibility of these populations to these viruses, should they spill over into humans. Samples collected from Tanzania exhibited a high baseline immune response to MERS-CoV or related viruses, suggesting prior exposure to related pathogens. Study by Mok et al.,³⁴ using samples from individuals exposed to dromedary camels in Nigeria, demonstrated that while these individuals had no detectable neutralizing antibodies, they displayed positive MERS-CoV T cell responses, implying prior exposure to MERS-related viruses in these populations. Moreover, neutralizing antibody levels were correlated with disease severity, with individuals experiencing mild disease failing to generate detectable neutralizing antibodies.³⁵ This suggests that individuals with asymptomatic MERS-related infections may not develop detectable levels of neutralizing antibodies.

Samples collected in Southeast Asian countries exhibit higher seropositive rates for SARS-like viruses, suggesting more frequent spillover of related viruses in this region, likely due to the abundance of Rhinolophus sp. bats.^22,27,36,37 Notably, samples collected from the New Zealand population consistently show low seropositivity for the tested viruses. New Zealand is home to only two bat species—the lesser short-tailed bat (Mystacina tuberculata) and the long-tailed bat (Chalinolobus tuberculatus)³⁸—neither of which are known to carry the viruses tested in our panel.^{15,18,39–41} A recent study by Waller et al.³⁸ revealed that bat guano collected in New Zealand tested positive only for alphacoronaviruses. Our findings suggest that spillover of zoonotic viruses may occur more frequently, and it is associated with the distribution of reservoir species.

With this platform, we identified individuals who possibly had prior exposure to MERS-like viruses capable of using human ACE2 as an entry receptor,³³ suggesting that spillover of such viruses may be occurring in Southeast Asia. Notably, NeoCoV-positive sera failed to neutralize another recently identified ACE2-binding MERS-like virus, BtHKU5-CoV-2,¹⁷ underscoring that even ACE2-using merbecoviruses can be antigenically distinct from each other. Comparative structural analysis of the RBD-ACE2 binding footprint revealed that BtHKU5-CoV-2 shares an overlapping ACE2-binding footprint on the RBD with SARS-CoV-1, SARS-CoV-2, and NL63, but not with NeoCoV,¹⁷ which might explain the lack of cross-neutralization between these ACE2-binding MERS-like viruses. Evidence of spillover of ACE2-binding MERS-like viruses in human population is concerning because among the seven known human coronaviruses, three use human ACE2 as an entry receptor, indicating that ACE2 binding may pose a higher risk of disease transmission in the population.^42,43 Considering that DPP4 expression is primarily localized in the alveolar regions and is rarely detected on the surface epithelium from the nasal cavity to the conducting airway,⁴⁴ DPP4-binding MERS-CoV or MERS-like viruses are less likely to sustain effective human-to-human transmission, making a pandemic on the scale of SARS-CoV-2 less probable. However, we speculate that ACE2-binding MERS-like viruses may pose a particularly high pandemic risk. This speculation is supported by multiple observations: the lack of population immunity to such viruses, the recent discovery of BtHKU5-CoV-2 (another ACE2-using MERS-like coronavirus),¹⁷ and evidence that a bat HKU5-related virus can transmit across species (to mink) via ACE2 interaction.⁴⁵ Together, these findings highlight the urgent need for effective therapeutics and broadly protective pan-merbecovirus vaccine.

The lack of cross-neutralization activity in individuals vaccinated against or infected with COVID-19 suggests that virus-specific vaccines may not be effective against pre-emergent viruses, highlighting the need for next-generation pan-subgenus vaccine candidates. It is also worth noting that despite being more closely related to SARS-CoV-2 (clade 1b) than bat coronaviruses from clade 1c, SARS-like viruses (clade 1a), such as SARS-CoV-1, WIV-1, and RsSHC014, are the most immune-evasive to SARS-CoV-2-specific neutralizing antibodies. This underscores the need for an effective vaccine against this clade of sarbecoviruses. Additionally, the mechanism by which SARS-CoV-2-specific neutralizing antibodies neutralize Khosta-2 and RsYN04 warrants further investigation. Inspired by the potent and broad “super immunity” observed in SARS survivors who were vaccinated against SARS-CoV-2,²⁵ we have designed and tested pan-sarbecovirus and pan-merbecovirus vaccine candidates preclinically by using a cross-clade prime-and-boost strategy. Mice primed and boosted with antigenically distinct vaccine candidates developed broadly neutralizing antibodies against all tested sarbecoviruses or merbecoviruses, highlighting the feasibility of a sequential immunization strategy as pan-subgenus vaccine design. Although previous studies demonstrated that mosaic RBD nanoparticles can induce pan-sarbecovirus immunity,^46,47 sequential immunization remains the more cost-effective approach.

In conclusion, using the customizable multiplex sVNT platform we developed, we identified a lack of population immunity against emerging zoonotic viruses, highlighting the vulnerability of the human population to future spillover events. Our findings also suggest that virus-specific vaccines may not offer sufficient protection against future coronavirus or henipavirus spillovers involving related but antigenically distinct viruses.^25,26,48,49 This underscores the urgent need for next-generation vaccines that are broadly protective and agnostic to future variants.

Limitations of the study

This study has several limitations. Our serosurveillance analysis was based on retrospective cohorts with uneven sample sizes and heterogeneous exposure backgrounds, which may limit cross-population comparisons. In addition, several human cohorts were small. Another key limitation of our study is that we did not perform in vivo viral challenge experiments or evaluate vaccine-induced cellular immunity to confirm that the broadly neutralizing antibodies elicited by cross-clade prime-and-boost regimens translate into actual protection against infection or disease. Consequently, broader validation of these findings is necessary to fully establish the protective efficacy of this strategy.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chee Wah Tan (gmstanc@nus.edu.sg).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

This paper analyzes existing, publicly available data. Accession numbers are provided in the key resources table.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
This paper does not report original code.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
E7 monoclonal antibody	This paper	PDB ID: 7Y71
S309 monoclonal antibody	This paper	PDB ID: 6WPS
CR3022 monoclonal antibody	This paper	PDB ID: 6W7Y
S2H97 monoclonal antibody	This paper	PDB ID: 7M7W
S2X324 monoclonal antibody	This paper	PDB ID: 8ERR
SA55 monoclonal antibody	This paper	PDB ID: 7Y0W
M102.4 monoclonal antibody	This paper	PDB ID: 6CMG
HENV-26 monoclonal antibody	This paper	PDB ID: 6VY5
MERS-27 monoclonal antibody	This paper	PDB ID: 4ZS6
M336 monoclonal antibody	This paper	PDB ID: 4XAK
LCA60 monoclonal antibody	This paper	PDB ID: 6NB5
JC57-11 monoclonal antibody	This paper	PDB ID: 8SAK
Anti-VSV-G [8G5F11] Antibody	Kerafast	UniProt: P03522; Cat# EB0010; RRID: AB_2811223
Bacterial and virus strains
MERS-CoV EMC/2012	Dr. R Fouchier (Erasmus University Medical Center, Rotterdam, Netherlands)	GenBank: JX869059
VSV–Nipah G/F pseudovirus	This paper	GenBank: AY988601
Chemicals, peptides, and recombinant proteins
Pierce^™ Protein A/G Agarose	Thermo Scientific	Cat# 20422
Pierce^™ Avidin	Thermo Scientific	Cat# 21121
SARS-CoV-2 Wuhan Hu-1 receptor-binding domain	This paper	GenBank: NC_045512
Bat CoV BANAL-20-52 receptor-binding domain	This paper	GenBank: MZ937000
Pangolin CoV GD/1/2019 receptor-binding domain	This paper	GISAID: EPI_ISL_410721
Pangolin CoV GX-P5L receptor-binding domain	This paper	GISAID: EPI_ISL_410540
Bat CoV RaTG13 receptor-binding domain	This paper	GenBank: MN996532
Bat CoV RsSHC014 receptor-binding domain receptor-binding domain	This paper	GenBank: KC881005
Bat CoV WIV1 receptor-binding domain	This paper	GenBank: KF367457
SARS-CoV BJ01 receptor-binding domain	This paper	GenBank: AY278488
Bat CoV RsYN04 receptor-binding domain	This paper	GenBank: MZ081380
Bat CoV Khosta-2 receptor-binding domain	This paper	GenBank: MZ190138
MERS-CoV EMC/2012 receptor-binding domain	This paper	GenBank: NC_019843
Camel MERSr-CoV Nigeria/NV1405/2016 (MERSr-CoV NV1405) receptor-binding domain	This paper	GenBank: MG923474
Bat CoV NL140422 (BatCoV 422) receptor-binding domain	This paper	GenBank: MG021452
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) receptor-binding domain	This paper	GenBank: OM009282
Neoromicia/PML-PHE1/RSA/2011 (NeoCoV) receptor-binding domain	This paper	GenBank: KC869678
Nipah virus isolate UMMC1 (NiV) soluble G glycoprotein	This paper	GenBank: AY029767
Hendra virus (HeV) soluble G glycoprotein	This paper	GenBank: NC_001906
Cedar virus isolate CG1a (CedV) soluble G glycoprotein	This paper	GenBank: NC_025351
Ni Sepharose^™ High Performance histidine-tagged protein purification resin	Cytiva	Cat# 17526802
Recombinant human ACE2	GenScript	Cat# Z03516
Recombinant human DPP4	GenScript	Cat# Z03324
Recombinant human ephrin B2	GenScript	Cat# Z05259
MagPlex-C Microspheres, Region 12	Luminex	Cat# MC10012-01
MagPlex-C Microspheres, Region 13	Luminex	Cat# MC10013-01
MagPlex-C Microspheres, Region 15	Luminex	Cat# MC10015-01
MagPlex-C Microspheres, Region 19	Luminex	Cat# MC10019-01
MagPlex-C Microspheres, Region 20	Luminex	Cat# MC10020-01
MagPlex-C Microspheres, Region 26	Luminex	Cat# MC10026-01
MagPlex-C Microspheres, Region 27	Luminex	Cat# MC10027-01
MagPlex-C Microspheres, Region 30	Luminex	Cat# MC10030-01
MagPlex-C Microspheres, Region 34	Luminex	Cat# MC10034-01
MagPlex-C Microspheres, Region 36	Luminex	Cat# MC10036-01
MagPlex-C Microspheres, Region 46	Luminex	Cat# MC10046-01
MagPlex-C Microspheres, Region 56	Luminex	Cat# MC10056-01
MagPlex-C Microspheres, Region 64	Luminex	Cat# MC10064-01
MagPlex-C Microspheres, Region 72	Luminex	Cat# MC10072-01
MagPlex-C Microspheres, Region 76	Luminex	Cat# MC10076-01
MagPlex-C Microspheres, Region 37	Luminex	Cat# MC10037-01
MagPlex-C Microspheres, Region 66	Luminex	Cat# MC10066-01
MagPlex-C Microspheres, Region 44	Luminex	Cat# MC10044-01
MagPlex-C Microspheres, Region 28	Luminex	Cat# MC10028-01
FuGene 6	Promega	Cat# E5911
Series S Sensor Chip protein A	Cytiva Biacore	Cat# 29127555
C1.25 trimeric spike proteins	Genscript	This study
C1.26 trimeric spike proteins	Genscript	This study
C1.28 trimeric spike proteins	Genscript	This study
MERS-CoV EMC/2012 spike	This paper	GenBank: NC_019843
Bat CoV NL140422 (BatCoV 422) spike	This paper	GenBank: MG021452
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) spike	This paper	GenBank: OM009282
Sigma Adjuvant System	Sigma aldrich	Cat# S6322
BNT162b2 mRNA vaccine	Pfizer-BioNTech	BNT162b2 mRNA vaccine (Pfizer-BioNTech)
Recombinant human ACE-2, hFc	Genscript	Cat# Z03484
Critical commercial assays
Enzymatic Protein Biotinylation Kit	Sigma-Aldrich	Cat# CS0008
PE / R-Phycoerythrin Conjugation Kit - Lightning-Link^®	abcam	Cat# ab102918
ONE-Glo^™ Luciferase Assay System	Promega	Cat# E6110
Deposited data
SARS-CoV BJ01	NCBI	GenBank: AY278488
Bat CoV Rs4231	NCBI	GenBank: KY417146
Bat CoV RsSHC014	NCBI	GenBank: KC881005
Bat CoV Rs3367	NCBI	GenBank: KC881006
Bat CoV WIV1	NCBI	GenBank: KF367457
Bat CoV LYRa11	NCBI	GenBank: KF569996
SARS-CoV-2 Wuhan Hu-1	NCBI	GenBank: NC_045512
Bat CoV RaTG13	NCBI	GenBank: MN996532
Pangolin CoV GD/1/2019	GISAID	EPI_ISL_410721
Pangolin CoV GX-P5L	GISAID	EPI_ISL_410540
Bat CoV BANAL-20-52	NCBI	GenBank: MZ937000
Bat CoV BANAL-20-236	NCBI	GenBank: MZ937003
Bat CoV RsYN04	NCBI	GenBank: MZ081380
Bat CoV RaTG15	NGDC	CNGB: GWHBAUP01000001
Bat CoV RacCS203	NCBI	GenBank: MW251308
Bat CoV RmYN02	GISAID	EPI_ISL_412977
Bat CoV Anlong-103	NCBI	GenBank: KY770858
Bat CoV Rp3	NCBI	GenBank: DQ071615
Bat CoV JTMC15	NCBI	GenBank: KU182964
Bat CoV Rf1	NCBI	GenBank: DQ412042
Bat CoV SL-ZXC21	NCBI	GenBank: MG772934
Bat CoV SL-ZC45	NCBI	GenBank: MG772933
Bat CoV BM48-31	NCBI	GenBank: GU190215
Bat CoV BtKY72	NCBI	GenBank: KY352407
Bat CoV Khosta-1	NCBI	GenBank: MZ190137
Bat CoV Khosta-2	NCBI	GenBank: MZ190138
MERS-CoV EMC/2012	NCBI	GenBank: NC_019843
MERS-CoV England 1	NCBI	GenBank: KC164505
MERS-CoV England-Qatar/2012	NCBI	GenBank: KC667074
MERS-CoV THA/CU/17/06/2015	NCBI	GenBank: KT225476
Camel MERSr-CoV Burkina Faso/CIRAD-HKU697/2015	NCBI	GenBank: MG923473
Camel MERSr-CoV Nigeria/NV1405/2016 (MERSr-CoV NV1405)	NCBI	GenBank: MG923474
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV)	NCBI	GenBank: OM009282
Pangolin GX/HKU4-GX/2020	NCBI	GenBank: OQ297693
Pangolin HKU4/GX-2020	NCBI	GenBank: OP288969
Bat CoV NL140422 (BatCoV 422)	NCBI	GenBank: MG021452
Erinaceus hedgehog CoV HKU31 strain Rs13	NCBI	GenBank: MK907287
Neoromicia/PML-PHE1/RSA/2011 (NeoCoV)	NCBI	GenBank: KC869678
Bat CoV PREDICT/PDF-2180	NCBI	GenBank: KX574227
BtVs-BetaCoV/SC2013	NCBI	GenBank: KJ473821
BtPa-BetaCoV/GD2013	NCBI	GenBank: KJ473820
BtTp betaCoV/Gx2012	NCBI	GenBank: KJ473822
Pipistrellus bat CoV HKU5	NCBI	GenBank: EF065509
Pipistrellus bat CoV HKU5 BY140562	NCBI	GenBank: MH002341
Pipistrellus bat CoV HKU5 BY140535	NCBI	GenBank: MH002340
Pipistrellus bat CoV HKU5 YD13403	NCBI	GenBank: MH002342
Tylonycteris bat CoV HKU4 CZ07	NCBI	GenBank: MH002338
Tylonycteris bat CoV HKU4 CZ01	NCBI	GenBank: MH002337
Bat CoV JPDB144	NCBI	GenBank: KU182965
Tylonycteris bat CoV HKU4 GZ131656	NCBI	GenBank: MN611519
Tylonycteris bat CoV HKU4 SZ140324	NCBI	GenBank: MH002339
Bat coronavirus HKU4-4	NCBI	GenBank: EF065508
Tylonycteris bat CoV HKU4 SM3A	NCBI	GenBank: MW218395
Bat coronavirus HKU4-3	NCBI	GenBank: EF065507
Bat coronavirus HKU4-2	NCBI	GenBank: EF065506
Tylonycteris bat CoV HKU4	NCBI	GenBank: EF065505
HKU5-CoV-2 441	NGDC	CNGB: C_AA085194.1
HKU5-CoV-2 023	NGDC	CNGB: C_AA085189.1
HKU5-CoV-2 381	NGDC	CNGB: C_AA085193.1
Measles virus (out goup)	NCBI	GenBank: NC_001498
Melian virus strain GN/Meliandou/Cg/1/2018	NCBI	GenBank: OK623353
Denwin virus strain BE/Ninove/Cr/1/2019	NCBI	GenBank: OK623354
Henipavirus sp. strain Cs17-65	NCBI	GenBank: MZ574409
Jingmen Crocidura shantungensis henipavirus 2	NCBI	GenBank: OM030315
Langya virus	NCBI	GenBank: OM101125
Mojiang virus isolate Tongguan1	NCBI	GenBank: NC_025352
Wenzhou Apodemus agrarius henipavirus 1	NCBI	GenBank: MZ328275
Henipavirus sp. strain Cl17-46	NCBI	GenBank: MZ574408
Henipavirus sp. strain Cl17-32	NCBI	GenBank: MZ574407
Jingmen Crocidura shantungensis henipavirus 1	NCBI	GenBank: OM030314
Wufeng Crocidura attenuata henipavirus 1	NCBI	GenBank: OM030317
Wufeng Chodsigoa smithii henipavirus 1	NCBI	GenBank: OM030316
Hendra henipavirus HeV-G2	NCBI	GenBank: MZ229746
Hendra virus (HeV)	NCBI	GenBank: NC_001906
Nipah virus isolate UMMC1 (NiV)	NCBI	GenBank: AY029767
Cedar virus isolate CG1a (CedV)	NCBI	GenBank: NC_025351
Bat Paramyxovirus Eid_hel/GH-M74a/GHA/2009 (GhV)	NCBI	GenBank: NC_025256
Experimental models: Cell lines
Homo sapiens: HEK293T	ATCC	RRID: CVCL_0063; Cat# CRL-3216
Mesocricetus auratus: BHK-21	ATCC	RRID: CVCL_1915;Cat# CCL-10
Homo sapiens: Expi293T	Thermo Fisher	RRID: CVCL_D615
Cercopithecus aethiops: Vero	ATCC	RRID: CVCL_0059
Chlorocebus sabaeus: Vero E6	ATCC	RRID: CVCL_0574
Experimental models: Organisms/strains
Mus musculus strain Balb/c	InVivos	BALB/cJInv
Recombinant DNA
pTRIOZ-hIgG1 E7	This paper	PDB ID: 7Y71
pTRIOZ-hIgG1S309	This paper	PDB ID: 6WPS
pTRIOZ-hIgG1CR3022	This paper	PDB ID: 6W7Y
pTRIOZ-hIgG1S2H97	This paper	PDB ID: 7M7W
pTRIOZ-hIgG1 S2X324	This paper	PDB ID: 8ERR
pTRIOZ-hIgG1SA55	This paper	PDB ID: 7Y0W
pTRIOZ-hIgG1M102.4	This paper	PDB ID: 6CMG
pTRIOZ-hIgG1HENV-26	This paper	PDB ID: 6VY5
pTRIOZ-hIgG1MERS-27	This paper	PDB ID: 4ZS6
pTRIOZ-hIgG1M336	This paper	PDB ID: 4XAK
pTRIOZ-hIgG1LCA60	This paper	PDB ID: 6NB5
pTRIOZ-hIgG1 JC57-11	This paper	PDB ID: 8SAK
pCAGGS NiV-G	This paper	GenBank: AY988601
pCAGGS-NiV-F	This paper	GenBank: AY988601
pCAGGS MERS-CoV EMC/2012 spike	This paper	GenBank: NC_019843
pCAGGS Bat CoV NL140422 (BatCoV 422) spike	This paper	GenBank: MG021452
pCAGGS Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) spike	This paper	GenBank: OM009282
Software and algorithms
GraphPad Prism	Dotmatics	RRID: SCR_002798
R	https://www.r-project.org/	RRID: SCR_001905
Geneious Prime	Dotmatics	RRID: SCR_010519
MAFFT	https://mafft.cbrc.jp/alignment/server/	RRID: SCR_011811
Racmacs	GitHub/CRAN	https://acorg.github.io/Racmacs/

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STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Human and animal serum panels

Naïve sera (n = 20) were used as controls. The vaccinated panels consisted of sera from individuals who received a monovalent COVID-19 mRNA vaccine (n = 20), were boosted with a bivalent mRNA vaccine (n = 11), or were boosted with an XBB-specific vaccine (n = 21); and sera from SARS survivors who received two doses of BNT162b2 mRNA vaccine (n = 5). The convalescent panel included sera from individuals infected with SARS in 2003 (n = 13), MERS (n = 43), Nipah virus (n = 10), and SARS followed by COVID-19 BNT162b2 vaccination (n = 6). Naïve sera collected prior to the COVID-19 pandemic from Southeast Asian countries (Singapore, Indonesia, Cambodia, Laos, and Myanmar, n = 845), New Zealand (n = 181), and African countries (Tanzania, n = 172; Nigeria, n = 50; Morocco, n = 35) were used to assess baseline population immunity. One limitation of the current study is the lack of detail on sex, gender, and age information of participants as all samples were fully deidentified with no personally identifiable information accessible to the investigators. Samples were randomly allocated for inclusion in the study analysis. Camel sera collected in Ethiopia (n = 18), Kenya (n = 46), Uganda (n = 20), Morocco (n = 41), and Pakistan (n = 20) were used for merbecovirus sVNT analysis. All sera were heat-inactivated at 56°C for 30 min prior to the sVNT. Ethics statements, Institutional Review Board approvals, and references to previous studies are provided in Table S1. Camel samples were collected from freshly slaughtered camels in abattoirs. No Institutional Animal Care and Use Committee (IACUC) approvals required.

Cell line

Human embryonic kidney (HEK293T, ATCC CRL-3216) and baby hamster kidney (BHK-21, ATCC CCL-10) were grown and maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cell lines were tested for mycoplasma contamination.

Ethical approval

Ethical approval was given by the Ethics Committee at the First Affiliated Hospital of Guangzhou Medical University (ES-2024-137-01). Written informed consent was obtained from all patients. All experiments with authentic MERS-CoV were performed at the Guangzhou Customs District Technology Center BSL-3 Laboratory.

METHOD DETAILS

Monoclonal antibodies

All monoclonal antibodies used in this study were produced in-house using Expi293T mammalian expression system. In brief, the heavy and light chain sequences of the E7,²⁹ S309,⁵⁰ CR3022,⁵¹ S2H97,⁵² S2X324,⁵³ SA55,⁵⁴ M102.4,³⁰ HENV-26,³¹ MERS-27,²⁹ M336,⁵⁵ LCA60⁵⁶ and JC57–11^32,57 were cloned into pTRIOZ-hIgG1 (InvivoGen) expression vector. This plasmid was transfected into Expi293T cells according to manufacturer’s instructions, and recombinant RBD/glycoproteins were harvested 7 days post-transfection. The recombinant proteins were purified using Pierce Protein A/G agarose (Thermo Scientific) and desalted using Amicon Ultra-4, 30 KDa cutoff (Merck).

Protein expression, purification and enzymatic biotinylation

All RBDs and glycoproteins were codon optimized and gene synthesized from either Twist Bioscience or GenScript. All RBDs and glycoproteins used in this study were produced using the Expi293T expression system (Thermo Scientific) according to the manufacturer’s instructions. Briefly, the RBD/glycoprotein gene was cloned downstream of the chicken β-actin promoter and signal peptide, and upstream of AviTag and HisTag. This plasmid was transfected into Expi293T cells, and recombinant RBD/glycoproteins were harvested 7 days post-transfection. The recombinant proteins were purified using Ni-sepharose (Cytiva) and desalted using Amicon Ultra-4, 10 KDa cutoff (Merck). Enzymatic biotinylation was performed using the BirA biotinylation kit (Sigma) according to the manufacturer’s protocol. Excessive biotin was removed using Amicron Ultra-4, 10 KDa cutoff (Merck). Protein concentration was determined by Nanodrop (Thermo Scientific).

Multiplex sVNT

To set up the multiplex sVNT platform, avidin (Pierce) was coupled to MagPlex microspheres using the antibody coupling kit (Luminex). Enzymatically biotinylated RBDs/glycoproteins were coated onto the corresponding MagPlex microsphere regions at a concentration of 5 μg per 1 million microspheres. Recombinant ACE2, DPP4, and ephrin B2 were custom-made by GenScript, and R-PE labeling was performed using the Lightning-Link R-PE conjugation kit (Abcam) according to the manufacturer’s instructions.

MM-sVNT was performed by pre-incubating 600 beads/antigen with serially diluted serum in assay buffer (phosphate buffer saline (PBS) supplemented with 1% bovine serum albumin (BSA)) for 1 hour at 37°C with agitation at 800 rpm. An equal volume of receptors (2000 ng/ml of R-PE-labelled human ACE2, DPP4, and ephrin B2) was added and incubated for another hour at 37°C with agitation at 800 rpm. The beads were washed twice with assay buffer before signal acquisition using the MAGPIX instrument.

MERS-CoV plaque reduction neutralization test

Three-fold serially diluted plasma and sera in Dulbecco’s modified Eagle medium (DMEM) were mixed with 100 plaque forming unit of MERS-CoV (1:1). After 1 hour of incubation at 37°C, the mixture was added to Vero 81 cells for an additional one h to permit absorption. Cells were then overlaid with 1.2% agarose (containing 2% fetal bovine serum and DMEM). After further incubation for 3 days, culture cells were fixed in 37% formaldehyde for at least 20 min, agarose plugs were removed and the remaining plaques were visualized by 0.1% crystal violet staining.

Pseudotyped neutralization test

NiV full-length glycoprotein-Fusion protein-pseudotyped viruses were produced and packaged as previously described. Briefly, 5 million HEK293T cells were transfected with 20 μg of pCAGGS NiV-G and pCAGGS-NiV-F plasmid using FuGene6 (Promega). At 24 h post transfection, cells were incubated with VSVΔG luc seed virus (at a multiplicity of infection of 5) for 2 h. Following two PBS washes, infected cells were replenished with complete growth media supplemented with 1:5,000 diluted anti-VSV-G mAb (Clone 8GF11, Kerafast). At 24 h post infection, pseudoviruses were collected by centrifugation at 2,000 × g for 5 min. For pVNT assay, 3 × 10⁶ relative light units of pseudoviruses were pre-incubated with 4-fold serial-diluted test serum in a final volume of 50 μl for 1 h at 37°C, followed by infection of BHK-21 cells. At 20–24 h post infection, an equal volume of ONE-Glo luciferase substrate (Promega) was added and the luminescence signal was measured using the Cytation 5 microplate reader (BioTek) with Gen5 software version 3.10.

Surface plasmon resonance

Surface plasmon resonance experiments were conducted using a Biacore 1S+ system (Cytiva, Marlborough, MA, USA). The following materials were used: Series S Sensor Chip protein A (cat. no. 29127555), 1 × HBS-P buffer (10 × HBS-P, cat. no. BR100827) supplemented with 3 mM ethylenediaminetetraacetic acid (EDTA) (0.5 M EDTA, pH 8.0, Invitrogen^™, cat. no. 15575020), and regeneration buffer (10 mM glycine-HCl, pH 1.5). All RBD proteins and Fc-tagged ACE2 proteins were produced in-house using mammalian expression systems.

For kinetic analysis of RBD/Fc-tagged ACE2 interactions, the protein A capture method was used. Fc-tagged ACE2 proteins were captured onto the protein A sensor chip surface by injection at 2.6 μg/mL for 120 s at a flow rate of 10 μL/min. To reduce non-specific binding, 0.1 mg/mL BSA in 1 × HEPES-buffered saline with EDTA and surfactant p20 buffer was injected as a blocking step prior to RBD injection (60 s at a flow rate of 30 μL/min). Two-fold serial dilutions of RBD proteins were injected across the surface under the following concentration ranges: SARS-CoV-1, 0 to 100 nM; SARS-CoV-2, 0 to 2000 nM; RsSH014, 0 to 400 nM; GX-P5L, 0 to 400 nM; RsYN04, 0 to 250 nM; Khosta-2, 0 to 800 nM. Each analyte was injected at a flow rate of 30 μL/min, with a contact time of 120 s and a dissociation time of 200 s. The sensor surface was regenerated after analyte step with 10 mM glycine HCl (pH 1.5) for 60 s at 30 μL/min. Multicycle kinetics analysis was performed under same conditions to characterize all RBS-Fc-tagged ACE2 interactions.

Phylogenetic analysis

RBD coding DNA sequence (CDS) sequences for sarbecovirus or merbecovirus and glycoprotein CDS sequences for henipavirus were reproduced from genomic sequences retrieved from NCBI or NGDC. Accession numbers are provided in the key resources table. Further analysis was performed in Geneious Prime (version 2025.1.2). CDS sequences were aligned with multiple alignment using fast fourier transform (MAFFT) to construct the phylogenetic tree by the maximum-likelihood method with the general-time reversible (GTR) using 1,000 bootstrap replicates in the PHYML 3.0 software.

Antigenic cartography

An antigenic map was generated using the R package ‘Racmacs’ (version 1.1.18) in R (version 4.1.2) on the basis of the matrix of NT50 of sera on the 15 SARS-CoV-2 variants and SARS-CoV-1 on multiplex sVNT. The number of optimizations was set to 1,000.

Design of pan-sarbecovirus vaccine C1.25, C1.26 and C1.28

The pan-sarbecovirus vaccine candidates, named as C1.25, C1.26, and C1.28, were meticulously designed to elicit broad-spectrum immune responses, leveraging advanced molecular techniques and antigenic diversity. These vaccines employed a cross-clade antigenic boosting strategy aimed at inducing comprehensive humoral and cellular immunity from conserved epitopes from different sarbecovirus clades. To design these antigens, we first retrieved all available sarbecovirus spike protein sequences from NCBI and GISAID and grouped them according to lineages (e.g., clade 1a/1b/1c, clade 2, clade 3) and according to their ability to utilize human or bat ACE2 or even independent of ACE2 as the entry receptor. The C1.25 candidate was engineered to incorporate RBDs derived from clade 1a sarbecoviruses, predominantly utilizing human ACE2. Additionally, the non-RBD spike regions, including the NTD and S2 subunit, were sourced from clade 1a and 3 sarbecoviruses. The design aimed to provide robust protection across clades 1a and 3 by targeting both RBD and non-RBD regions. With our knowledge of the spread of antigenic distance of immune-evasive SARS-CoV-2 Omicron subvariants, we updated the design of C1.26 on the C1.25 framework with 5 RBD mutations from Omicron: S371F, S373P, S375F, D405N and R408S. The C1.28 candidate employed a more mosaic antigen design, merging spike sequences from clades 1c, 2, and 3 and were derived from bat sarbecoviruses with zoonotic potential, to address future spillover risks.

Animal experiments

For the pan-sarbecovirus immunization experiments, mice were immunized with either BNT162b2 mRNA vaccines (Pfizer) at 1 μg diluted with normal saline into a total volume of 50 μl per mouse per dose administered intramuscularly, or C1.25, C1.26 or C1.28 trimeric spike proteins produced commercially (Genscript) or in-house at 25 μg diluted with PBS and S6322 adjuvant (Sigma) into a total volume of 200 μl per mouse per dose and administered intraperitoneally. Immunizations were given on day 1 (dose 1), 21 (dose 2), 140–163 (dose 3) and 210–408 (dose 4). For the pan-merbecovirus immunization experiments, mice were immunized intraperitoneally with 10 μg of spike proteins from MERS-CoV, BatCoV-422 or mjHKU4r-CoV diluted with PBS and S6322 adjuvant for 3 doses at day 0, 21 and 49, then followed by 20 μg of spike proteins from BatCoV-422 or MERS-CoV on day 77 for the fourth dose. 20 μg of BtHKU5-CoV-2 spike protein was injected on day 253 for the fifth dose. Blood was collected from the submandibular vein of mice before immunization and weekly after immunization for 21 days after each immunization. Blood was allowed to stand at room temperature to clot, spun down at 10,000g for 15 minutes and serum was collected and stored in −20°C until analysis. All animal experiments were performed with approval from Singhealth IACUC 2022/SHS/1753.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical details for individual experiments are provided in the figures and figure legends. Neutralization titers were analyzed as NT50 values, often after log10 transformation where indicated. Geometric mean titers are shown for neutralization datasets. Statistical analysis was performed using GraphPad Prism 10 software. Differences between groups were analyzed using two-tailed unpaired t tests. Correlations between sVNT and pVNT or PRNT were analyzed using Pearson correlation coefficients.

Supplementary Material

NIHMS2187404-supplement-Supplementary_Material.pdf^{(2.7MB, pdf)}

Highlights.

Multiplex sVNT profiles neutralizing antibodies across two zoonotic viral families
Pre-pandemic serosurveillance identifies human exposure to ACE2-using MERS-like viruses
Pathogen-specific vaccination leaves major neutralization gaps against related viral clades
Cross-clade prime-and-boost vaccination elicits broad-spectrum neutralizing antibodies

ACKNOWLEDGMENTS

This work was supported in part by grants from the Singapore National Medical Research Council (OFLCG19May-0034 and MOH-000696) awarded to L.F.W., the Singapore Programme for Research in Epidemic Preparedness and Response (PREPARE) (PREPARE-CS1-2023-011, PREPARE-UKRI-SF-2025-004, and PREPARE-SF-2024-012), the National University Hospital (NUHS/2023/018/Startup/10), and the Wellcome Trust (grant no. 226120) awarded to C.W.T. Sample collection in Laos was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract no. U01AI15180 and the Armed Forces Health Surveillance Branch (AFHSB) Global Emerging Infections Surveillance program (GEIS) work unit number PROMIS ID P00034_17_N2. We would like to thank Prof. Paul Newton, Prof. Matthew Robinson, and Dr. Danoy Chommanam and the patients in LOMWRU and Laos who coordinated and participated in the study, respectively. Sample collection in Cambodia was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases. Sample collection in Myanmar was supported by the National Institute of Health and the National Institute of Allergy and Infectious Diseases, under contract nos. U01Al151814, K01TW010279, and R21AI175905. The project and sample collection in Indonesia was supported by the US CDC for the project detecting emerging vector-borne zoonotic pathogens in Indonesia (5R18CK000278-03, 5R18CK000278-03, 5U18CK000443-04, and 6U18CK 00043-05-01). Sample collection in Tanzania was funded by the US National Institutes of Health National Institute of Allergy and Infectious Diseases (grant no. R01AI121378) that also provided support to J.A.C., V.P.M., and M.P.R. This project was partly supported by the Collaborative Research Fund #C2103-20G from the Hong Kong Research Grant Council to C.H.H.H. and C.W.T. Nipah convalescent samples were provided as part of the WHO collaborative study for the development of International Standard for anti-Nipah antibody coordinated by the MHRA (NIBSC) in partnership with CEPI. We thank Mr. Leo CH Tsang from Hong Kong University for providing assistance in study. A.L. (CAPT, USN, MC) is a military service member. This work was prepared as part of his official duties. Title 17, USC, §105 provides that copyright protection under this title is not available for any work of the US government. Title 17, USC, §101 defines a US government work as a work prepared by a military service member or employee of the US government as part of that person’s official duties. The views expressed in the article are those of the authors and do not necessarily express the official policy and position of the US Navy, the Department of Defense, or the US government.

Footnotes

DECLARATION OF INTERESTS

L.-F.W., W.N.C., C.W.T., and F.Z. have filed a patent on pan-sarbecovirus vaccine candidate (patent nos. WO2025021681 and WO2022245287).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material

NIHMS2187404-supplement-Supplementary_Material.pdf^{(2.7MB, pdf)}

Data Availability Statement

This paper analyzes existing, publicly available data. Accession numbers are provided in the key resources table.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
This paper does not report original code.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
E7 monoclonal antibody	This paper	PDB ID: 7Y71
S309 monoclonal antibody	This paper	PDB ID: 6WPS
CR3022 monoclonal antibody	This paper	PDB ID: 6W7Y
S2H97 monoclonal antibody	This paper	PDB ID: 7M7W
S2X324 monoclonal antibody	This paper	PDB ID: 8ERR
SA55 monoclonal antibody	This paper	PDB ID: 7Y0W
M102.4 monoclonal antibody	This paper	PDB ID: 6CMG
HENV-26 monoclonal antibody	This paper	PDB ID: 6VY5
MERS-27 monoclonal antibody	This paper	PDB ID: 4ZS6
M336 monoclonal antibody	This paper	PDB ID: 4XAK
LCA60 monoclonal antibody	This paper	PDB ID: 6NB5
JC57-11 monoclonal antibody	This paper	PDB ID: 8SAK
Anti-VSV-G [8G5F11] Antibody	Kerafast	UniProt: P03522; Cat# EB0010; RRID: AB_2811223
Bacterial and virus strains
MERS-CoV EMC/2012	Dr. R Fouchier (Erasmus University Medical Center, Rotterdam, Netherlands)	GenBank: JX869059
VSV–Nipah G/F pseudovirus	This paper	GenBank: AY988601
Chemicals, peptides, and recombinant proteins
Pierce^™ Protein A/G Agarose	Thermo Scientific	Cat# 20422
Pierce^™ Avidin	Thermo Scientific	Cat# 21121
SARS-CoV-2 Wuhan Hu-1 receptor-binding domain	This paper	GenBank: NC_045512
Bat CoV BANAL-20-52 receptor-binding domain	This paper	GenBank: MZ937000
Pangolin CoV GD/1/2019 receptor-binding domain	This paper	GISAID: EPI_ISL_410721
Pangolin CoV GX-P5L receptor-binding domain	This paper	GISAID: EPI_ISL_410540
Bat CoV RaTG13 receptor-binding domain	This paper	GenBank: MN996532
Bat CoV RsSHC014 receptor-binding domain receptor-binding domain	This paper	GenBank: KC881005
Bat CoV WIV1 receptor-binding domain	This paper	GenBank: KF367457
SARS-CoV BJ01 receptor-binding domain	This paper	GenBank: AY278488
Bat CoV RsYN04 receptor-binding domain	This paper	GenBank: MZ081380
Bat CoV Khosta-2 receptor-binding domain	This paper	GenBank: MZ190138
MERS-CoV EMC/2012 receptor-binding domain	This paper	GenBank: NC_019843
Camel MERSr-CoV Nigeria/NV1405/2016 (MERSr-CoV NV1405) receptor-binding domain	This paper	GenBank: MG923474
Bat CoV NL140422 (BatCoV 422) receptor-binding domain	This paper	GenBank: MG021452
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) receptor-binding domain	This paper	GenBank: OM009282
Neoromicia/PML-PHE1/RSA/2011 (NeoCoV) receptor-binding domain	This paper	GenBank: KC869678
Nipah virus isolate UMMC1 (NiV) soluble G glycoprotein	This paper	GenBank: AY029767
Hendra virus (HeV) soluble G glycoprotein	This paper	GenBank: NC_001906
Cedar virus isolate CG1a (CedV) soluble G glycoprotein	This paper	GenBank: NC_025351
Ni Sepharose^™ High Performance histidine-tagged protein purification resin	Cytiva	Cat# 17526802
Recombinant human ACE2	GenScript	Cat# Z03516
Recombinant human DPP4	GenScript	Cat# Z03324
Recombinant human ephrin B2	GenScript	Cat# Z05259
MagPlex-C Microspheres, Region 12	Luminex	Cat# MC10012-01
MagPlex-C Microspheres, Region 13	Luminex	Cat# MC10013-01
MagPlex-C Microspheres, Region 15	Luminex	Cat# MC10015-01
MagPlex-C Microspheres, Region 19	Luminex	Cat# MC10019-01
MagPlex-C Microspheres, Region 20	Luminex	Cat# MC10020-01
MagPlex-C Microspheres, Region 26	Luminex	Cat# MC10026-01
MagPlex-C Microspheres, Region 27	Luminex	Cat# MC10027-01
MagPlex-C Microspheres, Region 30	Luminex	Cat# MC10030-01
MagPlex-C Microspheres, Region 34	Luminex	Cat# MC10034-01
MagPlex-C Microspheres, Region 36	Luminex	Cat# MC10036-01
MagPlex-C Microspheres, Region 46	Luminex	Cat# MC10046-01
MagPlex-C Microspheres, Region 56	Luminex	Cat# MC10056-01
MagPlex-C Microspheres, Region 64	Luminex	Cat# MC10064-01
MagPlex-C Microspheres, Region 72	Luminex	Cat# MC10072-01
MagPlex-C Microspheres, Region 76	Luminex	Cat# MC10076-01
MagPlex-C Microspheres, Region 37	Luminex	Cat# MC10037-01
MagPlex-C Microspheres, Region 66	Luminex	Cat# MC10066-01
MagPlex-C Microspheres, Region 44	Luminex	Cat# MC10044-01
MagPlex-C Microspheres, Region 28	Luminex	Cat# MC10028-01
FuGene 6	Promega	Cat# E5911
Series S Sensor Chip protein A	Cytiva Biacore	Cat# 29127555
C1.25 trimeric spike proteins	Genscript	This study
C1.26 trimeric spike proteins	Genscript	This study
C1.28 trimeric spike proteins	Genscript	This study
MERS-CoV EMC/2012 spike	This paper	GenBank: NC_019843
Bat CoV NL140422 (BatCoV 422) spike	This paper	GenBank: MG021452
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) spike	This paper	GenBank: OM009282
Sigma Adjuvant System	Sigma aldrich	Cat# S6322
BNT162b2 mRNA vaccine	Pfizer-BioNTech	BNT162b2 mRNA vaccine (Pfizer-BioNTech)
Recombinant human ACE-2, hFc	Genscript	Cat# Z03484
Critical commercial assays
Enzymatic Protein Biotinylation Kit	Sigma-Aldrich	Cat# CS0008
PE / R-Phycoerythrin Conjugation Kit - Lightning-Link^®	abcam	Cat# ab102918
ONE-Glo^™ Luciferase Assay System	Promega	Cat# E6110
Deposited data
SARS-CoV BJ01	NCBI	GenBank: AY278488
Bat CoV Rs4231	NCBI	GenBank: KY417146
Bat CoV RsSHC014	NCBI	GenBank: KC881005
Bat CoV Rs3367	NCBI	GenBank: KC881006
Bat CoV WIV1	NCBI	GenBank: KF367457
Bat CoV LYRa11	NCBI	GenBank: KF569996
SARS-CoV-2 Wuhan Hu-1	NCBI	GenBank: NC_045512
Bat CoV RaTG13	NCBI	GenBank: MN996532
Pangolin CoV GD/1/2019	GISAID	EPI_ISL_410721
Pangolin CoV GX-P5L	GISAID	EPI_ISL_410540
Bat CoV BANAL-20-52	NCBI	GenBank: MZ937000
Bat CoV BANAL-20-236	NCBI	GenBank: MZ937003
Bat CoV RsYN04	NCBI	GenBank: MZ081380
Bat CoV RaTG15	NGDC	CNGB: GWHBAUP01000001
Bat CoV RacCS203	NCBI	GenBank: MW251308
Bat CoV RmYN02	GISAID	EPI_ISL_412977
Bat CoV Anlong-103	NCBI	GenBank: KY770858
Bat CoV Rp3	NCBI	GenBank: DQ071615
Bat CoV JTMC15	NCBI	GenBank: KU182964
Bat CoV Rf1	NCBI	GenBank: DQ412042
Bat CoV SL-ZXC21	NCBI	GenBank: MG772934
Bat CoV SL-ZC45	NCBI	GenBank: MG772933
Bat CoV BM48-31	NCBI	GenBank: GU190215
Bat CoV BtKY72	NCBI	GenBank: KY352407
Bat CoV Khosta-1	NCBI	GenBank: MZ190137
Bat CoV Khosta-2	NCBI	GenBank: MZ190138
MERS-CoV EMC/2012	NCBI	GenBank: NC_019843
MERS-CoV England 1	NCBI	GenBank: KC164505
MERS-CoV England-Qatar/2012	NCBI	GenBank: KC667074
MERS-CoV THA/CU/17/06/2015	NCBI	GenBank: KT225476
Camel MERSr-CoV Burkina Faso/CIRAD-HKU697/2015	NCBI	GenBank: MG923473
Camel MERSr-CoV Nigeria/NV1405/2016 (MERSr-CoV NV1405)	NCBI	GenBank: MG923474
Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV)	NCBI	GenBank: OM009282
Pangolin GX/HKU4-GX/2020	NCBI	GenBank: OQ297693
Pangolin HKU4/GX-2020	NCBI	GenBank: OP288969
Bat CoV NL140422 (BatCoV 422)	NCBI	GenBank: MG021452
Erinaceus hedgehog CoV HKU31 strain Rs13	NCBI	GenBank: MK907287
Neoromicia/PML-PHE1/RSA/2011 (NeoCoV)	NCBI	GenBank: KC869678
Bat CoV PREDICT/PDF-2180	NCBI	GenBank: KX574227
BtVs-BetaCoV/SC2013	NCBI	GenBank: KJ473821
BtPa-BetaCoV/GD2013	NCBI	GenBank: KJ473820
BtTp betaCoV/Gx2012	NCBI	GenBank: KJ473822
Pipistrellus bat CoV HKU5	NCBI	GenBank: EF065509
Pipistrellus bat CoV HKU5 BY140562	NCBI	GenBank: MH002341
Pipistrellus bat CoV HKU5 BY140535	NCBI	GenBank: MH002340
Pipistrellus bat CoV HKU5 YD13403	NCBI	GenBank: MH002342
Tylonycteris bat CoV HKU4 CZ07	NCBI	GenBank: MH002338
Tylonycteris bat CoV HKU4 CZ01	NCBI	GenBank: MH002337
Bat CoV JPDB144	NCBI	GenBank: KU182965
Tylonycteris bat CoV HKU4 GZ131656	NCBI	GenBank: MN611519
Tylonycteris bat CoV HKU4 SZ140324	NCBI	GenBank: MH002339
Bat coronavirus HKU4-4	NCBI	GenBank: EF065508
Tylonycteris bat CoV HKU4 SM3A	NCBI	GenBank: MW218395
Bat coronavirus HKU4-3	NCBI	GenBank: EF065507
Bat coronavirus HKU4-2	NCBI	GenBank: EF065506
Tylonycteris bat CoV HKU4	NCBI	GenBank: EF065505
HKU5-CoV-2 441	NGDC	CNGB: C_AA085194.1
HKU5-CoV-2 023	NGDC	CNGB: C_AA085189.1
HKU5-CoV-2 381	NGDC	CNGB: C_AA085193.1
Measles virus (out goup)	NCBI	GenBank: NC_001498
Melian virus strain GN/Meliandou/Cg/1/2018	NCBI	GenBank: OK623353
Denwin virus strain BE/Ninove/Cr/1/2019	NCBI	GenBank: OK623354
Henipavirus sp. strain Cs17-65	NCBI	GenBank: MZ574409
Jingmen Crocidura shantungensis henipavirus 2	NCBI	GenBank: OM030315
Langya virus	NCBI	GenBank: OM101125
Mojiang virus isolate Tongguan1	NCBI	GenBank: NC_025352
Wenzhou Apodemus agrarius henipavirus 1	NCBI	GenBank: MZ328275
Henipavirus sp. strain Cl17-46	NCBI	GenBank: MZ574408
Henipavirus sp. strain Cl17-32	NCBI	GenBank: MZ574407
Jingmen Crocidura shantungensis henipavirus 1	NCBI	GenBank: OM030314
Wufeng Crocidura attenuata henipavirus 1	NCBI	GenBank: OM030317
Wufeng Chodsigoa smithii henipavirus 1	NCBI	GenBank: OM030316
Hendra henipavirus HeV-G2	NCBI	GenBank: MZ229746
Hendra virus (HeV)	NCBI	GenBank: NC_001906
Nipah virus isolate UMMC1 (NiV)	NCBI	GenBank: AY029767
Cedar virus isolate CG1a (CedV)	NCBI	GenBank: NC_025351
Bat Paramyxovirus Eid_hel/GH-M74a/GHA/2009 (GhV)	NCBI	GenBank: NC_025256
Experimental models: Cell lines
Homo sapiens: HEK293T	ATCC	RRID: CVCL_0063; Cat# CRL-3216
Mesocricetus auratus: BHK-21	ATCC	RRID: CVCL_1915;Cat# CCL-10
Homo sapiens: Expi293T	Thermo Fisher	RRID: CVCL_D615
Cercopithecus aethiops: Vero	ATCC	RRID: CVCL_0059
Chlorocebus sabaeus: Vero E6	ATCC	RRID: CVCL_0574
Experimental models: Organisms/strains
Mus musculus strain Balb/c	InVivos	BALB/cJInv
Recombinant DNA
pTRIOZ-hIgG1 E7	This paper	PDB ID: 7Y71
pTRIOZ-hIgG1S309	This paper	PDB ID: 6WPS
pTRIOZ-hIgG1CR3022	This paper	PDB ID: 6W7Y
pTRIOZ-hIgG1S2H97	This paper	PDB ID: 7M7W
pTRIOZ-hIgG1 S2X324	This paper	PDB ID: 8ERR
pTRIOZ-hIgG1SA55	This paper	PDB ID: 7Y0W
pTRIOZ-hIgG1M102.4	This paper	PDB ID: 6CMG
pTRIOZ-hIgG1HENV-26	This paper	PDB ID: 6VY5
pTRIOZ-hIgG1MERS-27	This paper	PDB ID: 4ZS6
pTRIOZ-hIgG1M336	This paper	PDB ID: 4XAK
pTRIOZ-hIgG1LCA60	This paper	PDB ID: 6NB5
pTRIOZ-hIgG1 JC57-11	This paper	PDB ID: 8SAK
pCAGGS NiV-G	This paper	GenBank: AY988601
pCAGGS-NiV-F	This paper	GenBank: AY988601
pCAGGS MERS-CoV EMC/2012 spike	This paper	GenBank: NC_019843
pCAGGS Bat CoV NL140422 (BatCoV 422) spike	This paper	GenBank: MG021452
pCAGGS Pangolin HKU4/P251T/Pangolin/2018 (mjHKU4r-CoV) spike	This paper	GenBank: OM009282
Software and algorithms
GraphPad Prism	Dotmatics	RRID: SCR_002798
R	https://www.r-project.org/	RRID: SCR_001905
Geneious Prime	Dotmatics	RRID: SCR_010519
MAFFT	https://mafft.cbrc.jp/alignment/server/	RRID: SCR_011811
Racmacs	GitHub/CRAN	https://acorg.github.io/Racmacs/

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PERMALINK

Cross-clade vaccination to overcome sarbecovirus or merbecovirus neutralization gaps

Wan Ni Chia

Feng Zhu

Samuel Mo Sheng Cheng

Wee Chee Yap

Abeer N Alshukairi

Yun Yan Mah

Mayfong Mayxay

Vilada Chansamouth

Andrew Letizia

Beng Lee Lim

Yidan Wang

Madeline Sheng Si Kwek

Tierra Smiley Evans

Ohnmar Aung

Hlaing Myat Thu

Christine K Johnson

Yanqun Wang

Jincun Zhao

Jessica E Manning

Christina Yek

Chanthap Lon

Seng Heng

Sovann Ly

Rithea Leang

Rekol Huy

Aghnianditya Kresno Dewantari

Frilasita Aisyah Yudhaputri

Khin Saw Aye Myint

Matthew P Rubach

John A Crump

Venance P Maro

Nicole J Moreland

Aimee Paterson

Richard Charlewood

Jingjing Song

Shiwei Chen

Wan Yi Tan

Chris Ka Pun Mok

Catherine Hong Huan Hor

David Lye

David SC Hui

Barnaby E Young

Malik Peiris

Lin-Fa Wang

Chee Wah Tan

SUMMARY

Graphical Abstract

In brief

INTRODUCTION

RESULTS

Multi-family multiplex surrogate virus neutralization test

Figure 1. Multi-family multiplex surrogate virus neutralization test.

Limited cross-neutralizing antibodies against pre-emergent related viruses

Figure 2. Limited cross-neutralization against antigenically distinct viruses.

Serosurveillance revealed low to no pre-existing immunity against the three virus groups

Figure 3. Lack of cross-neutralizing antibodies against sarbecoviruses, merbecoviruses, and henipaviruses in samples collected prior to the COVID-19 pandemic.

Cross-clade prime-and-boost with antigenically distinct vaccine candidates induces broadly neutralizing antibodies in mice

Figure 4. Cross-clade prime-and-boost regime induces broad-spectrum neutralizing antibodies.

DISCUSSION

Limitations of the study

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Human and animal serum panels

Cell line

Ethical approval

METHOD DETAILS

Monoclonal antibodies

Protein expression, purification and enzymatic biotinylation

Multiplex sVNT

MERS-CoV plaque reduction neutralization test

Pseudotyped neutralization test

Surface plasmon resonance

Phylogenetic analysis

Antigenic cartography