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Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices.

Vaccination against the most common oncogenic human papillomavirus (HPV) types, HPV-16 and HPV-18, could prevent development of up to 70% of cervical cancers worldwide. We did a randomised, double-blind, controlled trial to assess the efficacy, safety, and immunogenicity of a bivalent HPV-16/18 L1 virus-like particle vaccine for the prevention of incident and persistent infection with these two virus types, associated cervical cytological abnormalities, and precancerous lesions. We randomised 1113 women between 15–25 years of age to receive three doses of either the vaccine formulated with AS04 adjuvant or placebo on a 0 month, 1 month, and 6 month schedule in North America and Brazil. Women were assessed for HPV infection by cervical cytology and self-obtained cervicovaginal samples for up to 27 months, and for vaccine safety and immunogenicity. In the according-to-protocol analyses, vaccine efficacy was 91·6% (95% CI 64·5–98·0) against incident infection and 100% against persistent infection (47·0–100) with HPV-16/18. In the intention-to-treat analyses, vaccine efficacy was 95·1% (63·5–99·3) against persistent cervical infection with HPV-16/18 and 92·9% (70·0–98·3) against cytological abnormalities associated with HPV-16/18 infection. The vaccine was generally safe, well tolerated, and highly immunogenic. The bivalent HPV vaccine was efficacious in prevention of incident and persistent cervical infections with HPV-16 and HPV-18, and associated cytological abnormalities and lesions. Vaccination against such infections could substantially reduce incidence of cervical cancer.

Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD. Foot-and-mouth disease vaccine efficacy is reduced when full virion 146S dissociates into pentameric 12S subunit. Here, the authors elucidate the molecular basis of this compromised immunogenicity by comparing antigenicity, humoral immunogenicity and structures of 146S and 12S particles.

Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of livestock that is controlled in endemic areas by vaccines containing intact inactivated FMDV particles. In this study, a novel monoclonal antibody named 5B6 has been identified and characterised, that permits the detection of all serotypes of FMDV via a conserved epitope near the N-terminus of the VP4 capsid protein. The antibody recognises intact virus particles known as 146S (the protective antigen) which contain VP4 and not dissociated capsids known as 12S (poorly protective antigen) which lack VP4. This allowed the development of a universal assay to specifically detect the protective antigen in vaccine samples using a simple ELISA. Such a test could be used to assess the quality of formulated vaccine following manufacture or prior to administration, or to assess unformulated vaccine antigen, and would be of great utility to enhance the effectiveness of FMD vaccination programmes.

Monoclonal antibodies are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids that are used as gene therapy delivery vectors. The presence of pre-existing neutralizing antibodies in large portions of the human population poses a significant challenge for AAV-mediated gene therapy, primarily targeting the capsid leading to vector inactivation and loss of treatment efficacy. This study structurally characterizes the interactions of 21 human-derived neutralizing antibodies from three patients treated with the AAV9 vector, Zolgensma®, utilizing high-resolution cryo-electron microscopy. The antibodies bound to the 2-fold depression or the 3-fold protrusions do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with many antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with antibody escape phenotypes. These AAV9 capsid variants have the potential to expand the patient cohort to include those that were previously excluded due to their pre-existing neutralizing antibodies against the wtAAV9 capsid, and the possibly of further treatment to those requiring redosing. Here, the authors have mapped the antibody response of the adeno-associated virus 9 (AAV9) gene therapy vector Zolgensma. AAV9 capsid variants were designed to escape this response while preserving manufacturing and biodistribution properties.

Poliovirus remains a serious threat to human health. Complete eradication of wild-type poliovirus has not yet succeeded, making the development of successful antivirals critical. Microneutralization assays against all three poliovirus serotypes identified a panel of human monoclonal IgGs, which are either serotype-specific or cross-neutralizing. Here, through cryoEM single particle analysis, we solved high resolution structures of four distinct poliovirus-FAb complexes. These antibodies bind to capsids at the circular depression (canyon) surrounding the icosahedral five-fold symmetry axis, which is also the binding site of the poliovirus receptor (PVR). Analysis of these structures confirms overlap of FAb contacts on the viral capsid with those of PVR. For three of the FAbs, the capsid residues are identified that dictate serotype-specific recognition. Contacts for the cross-neutralizing mAb 10D2 are located deep in the capsid canyon. These structural analyses indicate that antibody competition with the receptor likely leads to neutralization of virus particles and inhibition of poliovirus entry into host cells. Thus, the human IgGs studied here may facilitate development of therapeutics for the ongoing efforts in global eradication of poliovirus. The authors detail human antibodies that neutralize all three serotypes of poliovirus and mimic poliovirus receptor binding, accelerating antiviral development to further efforts to eradicate poliovirus.

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae , including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi’s sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid. MxB is an interferon-induced GTPase that inhibits HIV replication. Here, Crameri et al. show that MxB restricts replication of herpesviruses by inhibiting delivery of incoming viral DNA into the nucleus, and this antiviral activity depends on MxB’s GTPase activity.

Multi-layered antiviral activity of TRIM5 TRIM5 is an E3 ubiquitin ligase with known antiretroviral restriction factor activity, although the mechanisms involved are poorly understood. Luban and colleagues now demonstrate that TRIM5 activates innate immune signalling pathways and acts as a pattern recognition receptor specific for the retrovirus capsid lattice. TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm 1 , 2 . Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice 3 , 4 and several reports indicate that TRIM5 has a role in signal transduction 5 , 6 , 7 , but the precise mechanism of restriction is unknown 8 . Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13–UEV1A (also known as UBE2N–UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13–UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13–UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, but their immunogenicity remains a significant challenge, limiting long-term efficacy and the feasibility of repeated administration. In this study, we combine computational prediction with experimental validation to engineer AAV9 capsids with reduced immunogenicity. To facilitate this, we developed the Epitope Modification and MHC Prediction (EMMP) pipeline, which systematically automates the evaluation of amino acid substitutions for their predicted effects on major histocompatibility complex (MHC) presentability. Using this pipeline, we modify a CD4⁺ T-cell epitope in the AAV9 capsid that is identified and characterized as a proof-of-concept. Two mutant variants, R312H and R312Q, are selected and evaluated for transduction efficiency in vitro and immune response modulation in vivo. Notably, R312Q shows a significant reduction in T-cell activation and anti-AAV9 antibody production, albeit with a slight reduction in transduction at low multiplicities of infection (MOI). These results demonstrate a rational approach for optimizing AAV vector design, with potential applications for improving the safety and efficacy of gene therapy. Adeno-associated virus (AAV) vectors are widely used in gene therapy but can trigger immune responses. Here, the authors combine computational prediction with experimental validation to engineer AAV9 capsids with reduced T-cell immunogenicity while preserving vector function.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.