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Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry. Bispecific antibody architecture is often important for function but rarely optimized. Here, authors present a modular approach to assemble bispecifics in varied formats using a SpyTag/SpyCatcher approach called SpyMask, and build anti-HER2 bispecifics whose activities depend on binder orientation and bispecific geometry.

The upsurge of mpox in Africa and the recent global outbreak have stimulated the development of new vaccines and therapeutics. We describe the construction of virus-like particle (VLP) vaccines in which modified M1, A35 and B6 proteins from monkeypox virus (MPXV) clade Ia are conjugated individually or together to a scaffold that accommodates up to 60 ligands using the SpyTag/SpyCatcher nanoparticle system. Immunisation of female mice with VLPs induces higher anti-MPXV and anti-vaccinia virus (VACV) neutralizing antibodies than their soluble protein (SP) counterparts or modified VACV Ankara (MVA). Vaccination with individual single protein VLPs provides partial protection against lethal respiratory infections with VACV or MPXV clade IIa, whereas combinations or a chimeric VLP with all three antigens provide complete protection that is superior to SPs. Additionally, the VLP vaccine reduces the replication and spread of the virus at intranasal and intrarectal sites of inoculation. VLPs induce higher neutralizing activity than the Jynneos vaccine in rhesus macaques, and the VLP-induced antiserum provides better protection against MPXV and VACV than the Jynneos-induced antiserum when passively transferred to female mice. These data demonstrate that an mpox VLP vaccine derived from three MPXV clade Ia proteins protects against clade IIa MPXV and VACV, indicating cross-reactivity for orthopoxviruses. The upsurge of mpox has stimulated the development of new vaccines and therapeutics. Here, the authors describe a VLP vaccine comprised of modified MPXV proteins M1, A35, and B6 and demonstrate induction of protective antibodies in mice and non-human primates.

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-H5 ) displayed multivalently on the mi3 nanocage using the SpyTag003/SpyCatcher003 conjugation system. Mice immunized with DIOSvax-H5 Homotypic Nanocages at low doses demonstrate potent, cross-clade neutralizing antibody and T cell responses against diverse H5 strains. DIOSvax-H5 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.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle’s branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 ‘Kraken’ RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection. The ability to vaccinate against multiple related pathogens is a significant advantage. Here, the authors report on quartets of linked receptor-binding domains attached to designed nanocages using SpyTag/SpyCatcher links, demonstrating effective vaccination against similar viruses as well as the variant of concern.

Global health remains threatened by spillovers of zoonotic SARS-like betacoronaviruses (sarbecoviruses) that could be mitigated by a pan-sarbecovirus vaccine . We described elicitation of potently neutralizing and cross-reactive anti-sarbecovirus antibodies by mosaic-8 nanoparticles (NPs) displaying eight different sarbecovirus spike receptor-binding domains (RBDs) as 60 copies of eight individual RBDs (mosaic-8 RBD-NPs) or 30 copies of two \"quartets,\" each presenting four tandemly-arranged RBDs (dual quartet RBD-NPs). To facilitate manufacture of a broadly protective mosaic-8 vaccine, we generated membrane-bound RBD quartets that can be genetically encoded and delivered via mRNA: dual quartet RBD-mRNA and dual quartet RBD-EABR-mRNA, which utilizes ESCRT- and ALIX-binding region (EABR) technology that promotes immunogen presentation on cell surfaces and circulating enveloped virus-like particles (eVLPs) . Immunization with mRNA immunogens elicited equivalent or improved binding breadths, neutralization potencies, T cell responses, and targeting of conserved RBD epitopes across sarbecoviruses, demonstrating successful conversion of protein-based mosaic-8 RBD vaccines to mRNA formats. Systems serology showed that the mRNA vaccines elicited balanced IgG subclass responses with increased Fcγ receptor-binding IgGs, consistent with potentially superior Fc effector functions. A new technique, Systems Serology-Polyclonal Epitope Mapping (SySPEM), revealed distinct IgG-subclass-specific epitope targeting signatures across mRNA and protein-based vaccine modalities. These results demonstrate successful conversion of mosaic-8 RBD-NPs to mRNA or EABR-mRNA vaccines that provide easy manufacturing and enhanced protection from future pandemic sarbecovirus outbreaks.

The increasing global spread of the highly pathogenic avian influenza (HPAI) A/H5 viruses poses a serious public health 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 immunised with DIOSvax-H5inter Homotypic Nanocages at low doses demonstrate potent, cross-clade neutralising 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 product characteristic is essential for prospective strategic stockpiling in the pre-pandemic phase.Competing Interest StatementJ.L.H., G.W.C., S.V. and S.D.W.F. developed, tested and validated the H5 antigen DIOSvax-H5inter by gene delivery and are inventors of patent applications on computational vaccine development methods (US20220040284A1) and influenza vaccines (US20230149530A1). J.L.H. and S.D.W.F. are co-founders and shareholders of DIOSynVax Ltd. M.R.H. is a co-founder and shareholder of SpyBiotech. M.R.H. is an inventor on a patent on spontaneous amide bond formation (EP2534484) and a patent on SpyTag003:SpyCatcher003 (UK Intellectual Property Office 1706430.4). S.D.W.F. is an employee of Microsoft. All other authors have no competing interests to declare.

Effective pan-sarbecovirus vaccines could prevent future zoonotic spillovers of SARS-like betacoronaviruses. We previously developed protein-based mosaic-8 nanoparticles displaying eight diverse sarbecovirus RBDs, either individually (mosaic-8 RBD-NPs) or as two “quartets” of four tandemly-arranged RBDs (dual-quartet RBD-NPs), which elicited broadly cross-reactive antibodies but require multi-component manufacturing. Here, we address scalability challenges by extending the mosaic-8 concept to mRNA by encoding membrane-bound RBD quartets as dual-quartet RBD-mRNA and dual-quartet RBD-EABR-mRNA, the latter leveraging ESCRT- and ALIX-binding region (EABR) technology for immunogen display on cell surfaces and secreted virus-like particles. Compared with protein-based mosaic-8 immunogens, mRNA-encoded mosaic-8 vaccines induced equivalent or enhanced antibody breadth, neutralization potencies, T-cell responses, and targeting of conserved RBD epitopes. In addition, mRNA-encoded mosaic-8 vaccines elicited more balanced IgG subclass profiles and increased Fcγ receptor–binding IgGs, consistent with potentially superior Fc effector functions. These findings demonstrate successful translation of mosaic-8 RBD-NPs into mRNA/EABR-mRNA vaccines, enabling scalable manufacturing and improving protection against future sarbecovirus outbreaks. Finally, our newly developed technique, Systems Serology–Polyclonal Epitope Mapping (SySPEM), revealed distinct IgG-subclass-specific epitope signatures across mRNA, EABR-mRNA, and protein vaccines, demonstrating that the mode of antigen display can shape epitope recognition. We translated a pan-sarbecovirus RBD vaccine from protein nanoparticles to scalable mRNA and EABR-mRNA platforms encoding RBD quartets. Compared with protein-based immunogens, mRNA-based vaccines matched or improved antibody breadth, T-cell responses, Fc functionality, and conserved epitope targeting. A newly-developed Systems Serology–Polyclonal Epitope Mapping (SySPEM) technique revealed that antigen presentation modality shapes IgG subclass–specific epitope recognition.

Influenza neuraminidase is a crucial target for protective antibodies, yet the development of recombinant neuraminidase protein as a vaccine has been held back by instability and variable expression. We have taken a pragmatic approach to improving expression and stability of neuraminidase by grafting antigenic surface loops from low-expressing neuraminidase proteins onto the scaffold of high-expressing counterparts. The resulting hybrid proteins retained the antigenic properties of the loop donor while benefiting from the high-yield expression, stability, and tetrameric structure of the loop recipient. These hybrid proteins were recognised by a broad set of human monoclonal antibodies elicited by influenza infection or vaccination, with X-ray structures validating the accurate structural conformation of the grafted loops and the enzymatic cavity. Immunisation of mice with neuraminidase hybrids induced inhibitory antibodies to the loop donor and conferred protected against lethal influenza challenge. This pragmatic technique offers a robust solution for improving the expression and stability of influenza neuraminidase proteins for vaccine development.Competing Interest StatementP.R., A.T., and L.W. are inventor on patents filed on Influenza Neuraminidase loop-based design (Pending). M.R.H. is an inventor on patents filed on spontaneous amide bond formation (EP2534484 and UK Intellectual Property Office 1706430.4) and a SpyBiotech co-founder and shareholder.Footnotes* The title and abstract have been amended for clarity. Yellow and green highlights on figures and supplemental data within the text have been removed.

The central nervous system and gastrointestinal tract form the primary targets of chemotherapy-induced toxicities. Symptoms associated with damage to these regions have been clinically termed chemotherapy-induced cognitive impairment and mucositis. Whilst extensive literature outlines the complex etiology of each pathology, to date neither chemotherapy-induced side-effect has considered the potential impact of one on the pathogenesis of the other disorder. This is surprising considering the close bidirectional relationship shared between each organ; the gut-brain axis. There are complex multiple pathways linking the gut to the brain and vice versa in both normal physiological function and disease. For instance, psychological and social factors influence motility and digestive function, symptom perception, and behaviors associated with illness and pathological outcomes. On the other hand, visceral pain affects central nociception pathways, mood and behavior. Recent interest highlights the influence of functional gut disorders, such as inflammatory bowel diseases and irritable bowel syndrome in the development of central comorbidities. Gut-brain axis dysfunction and microbiota dysbiosis have served as key portals in understanding the potential mechanisms associated with these functional gut disorders and their effects on cognition. In this review we will present the role gut-brain axis dysregulation plays in the chemotherapy setting, highlighting peripheral-to-central immune signaling mechanisms and their contribution to neuroimmunological changes associated with chemotherapy exposure. Here, we hypothesize that dysregulation of the gut-brain axis plays a major role in the intestinal, psychological and neurological complications following chemotherapy. We pay particular attention to evidence surrounding microbiota dysbiosis, the role of intestinal permeability, damage to nerves of the enteric and peripheral nervous systems and vagal and humoral mediated changes.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.