Assembly of flaviviruses such as Zika virus (ZIKV), dengue virus, and West Nile virus in the host cell endoplasmic reticulum is driven by the structural envelope (E) and premembrane (prM) proteins. The formation of an infectious virion requires cleavage of prM by the host furin protease during a maturation step that is dependent on a conformational change in virion structure. Here, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM protein or proteolytic activation. The expression of E protein preceded by a truncated version of prM (M-E) was sufficient for the formation of noninfectious ZIKV subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response in macaques that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher-quality and more broadly applicable approach for vaccination against flaviviruses.

Background: Vaccines that stimulate systemic and mucosal immunity to a level required to prevent SARS-CoV-2 infection and transmission are an unmet need. Highly protective hepatitis B and human papillomavirus nanoparticle vaccines highlight the potential of multivalent nanoparticle vaccine platforms to provide enhanced immunity. Here, we report the construction and characterization of self-assembling 60-subunit icosahedral nanoparticle SARS-CoV-2 vaccines using the bacterial enzyme lumazine synthase (LuS). Methods and Results: Nanoparticles displaying prefusion-stabilized SARS-CoV-2 spike ectodomains fused to the surface-exposed amino terminus of LuS were designed using structure-guided approaches. Negative stain-electron microscopy studies of purified nanoparticles were consistent with self assembly into 60-mer nanoparticles displaying 20 spike trimers. After two intramuscular doses, these purified spike-LuS nanoparticles elicited significantly higher SARS-CoV-2 neutralizing activity than spike trimers in vaccinated mice. Furthermore, intramuscular DNA priming and intranasal boosting with a SARS-CoV-2 LuS nanoparticle vaccine stimulated mucosal IgA responses. Conclusion: These data identify LuS nanoparticles as highly immunogenic SARS-CoV-2 vaccine candidates and support the further development of this platform against SARS-CoV-2 and its emerging variants.

SARS-CoV-2 initiates infection of host cells by fusing its envelope lipid bilayer with the cell membrane. To overcome kinetic barriers for membrane fusion, the virus-encoded spike (S) protein refolds from a metastable prefusion state to a lower energy, stable postfusion conformation. The protein is first split into S1 and S2 fragments at a proteolytic site after synthesis, and presumably further cleaved at a second site, known as the S2' site, before membrane fusion can occur. Here, we report a cryo-EM structure of S2 fragment after the S2' cleavage, possibly representing a late fusion intermediate conformation, in which the fusion peptide and transmembrane segment have yet to pack together, distinct from the final, postfusion state. Functional assays demonstrate that the S2' cleavage accelerates membrane fusion, probably by stabilizing membrane fusion intermediates. These results advance our understanding of SARS-CoV-2 entry and may guide intervention strategies against pathogenetic coronaviruses.

Immunofocusing on conserved, subdominant epitopes is critical for vaccines against highly diverse viruses such as HIV-1, influenza, and SARS-CoV-2. The eight-residue N-terminus of the HIV-1 fusion peptide (FP) is one such example of a promising yet small target. We developed new FP immunogens using three alphavirus-like particles (VLPs) and introduced additional glycans to mask shared carrier-specific epitopes. In two independent guinea pig studies, sequential immunization with heterologous carriers enhanced FP-directed antibody titers, which were further improved with glycan engineering. Separately, using diverse FP variants sharing the same N-terminal six amino acids increased neutralizing antibody titers. When combined, these two strategies led to higher FP-directed titers and, after Env trimer boosting, induced FP-directed neutralizing antibodies against multi-clade wild-type HIV-1 in nearly all animals. These findings established the importance of minimizing recurrent off-target epitopes across immunizations and support the engineered VLPs as a promising platform for peptide immunization.

Glycoprotein B (gB) refolds between prefusion and postfusion conformations to facilitate herpesvirus entry into host cells. However, the isolation of prefusion-specific neutralizing antibodies, effective against other viral entry machines, has been challenging. Here we describe stabilization of the prefusion gB ectodomain from herpes simplex virus 1 (HSV-1), determine ectodomain structures at 2.9- to 4.1-Å resolution using cryogenic electron microscopy (cryo-EM) and isolate a prefusion-specific gB-neutralizing antibody termed WS.HSV-1.24. Murine immunization with gB stabilized in the prefusion conformation induced high titres of antibodies binding to both prefusion and postfusion gB, but-most notably-without measurable serum neutralization. Accessibility analysis revealed iso-surface exposure, with accessible surfaces on prefusion HSV-1 gB also exposed on postfusion gB. Structural analysis suggested substantial plasticity, with regions that refolded between pre- and postfusion conformations relegated to domain interfaces with limited accessibility; indeed, WS.HSV-1.24 recognized a domain-interface refolding region to facilitate neutralization. We propose that prefusion HSV-1 gB evades neutralization by most antibodies through an iso-surface display that is coupled to structural plasticity.

Endemic human coronaviruses (HCoVs), like HCoV-HKU1, account for ~30% of common cold/year and can cause serious upper and lower respiratory infections, yet no licensed vaccines target HCoVs. In fact, little is known about HCoV-HKU1's antigenic landscape. Thus, we characterized key interactions between HCoV-HKU1 spike (S) with monoclonal antibodies (mAbs) isolated from pre-pandemic HCoV-HKU1 convalescent PBMCs. We isolated 14 mAbs, which bound distinct S regions: receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Structural and functional studies revealed three groups of RBD-specific mAbs targeting diverse footprints within and around the TMPRSS2 receptor binding site, exemplified by: (1) The most potently neutralizing mAb, H501-022 (IC50 = 0.01 μg/mL), which recognizes the TMPRSS2 binding motif, thereby blocking receptor engagement; (2) mAb H501-008 (IC50 = 0.05 μg/mL) that binds a conserved, cross-reactive epitope outside of the TMPRSS2 binding site that is shared with HCoV-OC43; and (3) H501-018 (IC50 = 0.28 μg/mL) that recognizes both "up" and "down" RBD conformations at a distinct, non-overlapping site outside of the TMPRSS2 binding motif, distinguishing itself from H501-022 and H501-008, which bind exclusively to the "up" RBD conformation. These mAbs represent the first type-specific HCoV-HKU1 mAbs isolated from a convalescent donor. Our findings provide molecular insight into HCoV-HKU1 antibody recognition and neutralization mechanisms, importantly highlighting antigenic differences comparing HCoVs and pandemic CoVs - a critical step towards advancing universal CoV vaccine design.

AbstractAnti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 μg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of β-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

Emerging research has demonstrated that genomic alterations disrupting topologically associated domains (TADs) and chromatin interactions underlie the pathogenic mechanisms of specific copy number variants (CNVs) in neurodevelopmental disorders. We report two patients with a de novo deletion and a duplication in chromosome 4q31, potentially causing FBX-related neurodevelopmental syndrome by affecting the regulatory region of FBXW7. High-throughput chromosome conformation capture (Hi-C) analysis using available capture data in neural progenitor cells revealed the rewiring of the TAD boundary close to FBXW7. Both patients exhibited facial dysmorphisms, cardiac and limb abnormalities, and neurodevelopmental delays, showing significant clinical overlap with previously reported FBXW7-related features. We also included an additional 10 patients with CNVs in the 4q31 region from the literature and the DECIPHER database for Hi-C analysis, which confirmed that disruption of the regulatory region of FBXW7 likely contributes to the developmental defects observed in these patients.

Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

The number of mutations in the omicron (B.1.1.529) BA.1 variant of concern led to an unprecedented evasion of vaccine induced immunity. However, despite rise in global infections, severe disease did not increase proportionally and is likely linked to persistent recognition of BA.1 by T cells and non-neutralizing opsonophagocytic antibodies. Yet, the emergence of new sublineage BA.2, which is more transmissible than BA.1 despite relatively preserved neutralizing antibody responses, has raised the possibility that BA.2 may evade other vaccine-induced responses. Here, we comprehensively profiled the BNT162b2 vaccine-induced response to several VOCs, including omicron BA.1 and BA.2. While vaccine-induced immune responses were compromised against both omicron sublineages, vaccine-induced antibody isotype titers, and non-neutralizing Fc effector functions were attenuated to the omicron BA.2 spike compared to BA.1. Conversely, FcγR2a and FcγR2b binding was elevated to BA.2, albeit lower than BA.1 responses, potentially contributing to persistent protection against severity of disease.

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit–DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4+ T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.

As the COVID-19 pandemic continues to pose a great challenge to global health, more effective and durable vaccines are needed owing to the waning protection offered by the current available vaccines. Here we report highly immunogenic self-assembling hepatitis B surface antigen (HBsAg) nanoparticles displaying the ectodomain of SARS-CoV-2 WA1 S6P, the prefusion spike stabilized by six proline substitutions. Three self-assembling nanoparticle constructs with different linkers named CL1, CL2 and CL3 expressed well-formed nanoparticles in mammalian cells. These purified nanoparticles can bind diverse monoclonal antibodies (mAbs) specific to different domains of SARS-CoV-2 spike. Immunization by DNA electroporation of CL2 and CL3 in mice elicited significantly more potent and durable neutralizing antibody responses against diverse SARS-CoV-2 strains than non-nanoparticle forms of stabilized spikes, including SARS-CoV-2 S2P and S6P ectodomain and full-length S2P with its coding sequence matching mRNA-1273. Our data showed that genetic delivery of HBsAg based nanoparticle vaccines displaying SARS-CoV-2 spike can elicit potent and durable neutralizing antibody responses and highlight their potential as next generation genetic vaccine candidates against SARS-CoV-2. NIH Intramural Funding

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity1. This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial ( NCT03374202 ). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1–3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml−1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases. Results from a phase 1 clinical trial show that a broadly neutralizing, HIV-specific antibody is durably expressed in vivo from a viral vector, highlighting an alternate approach for the delivery of antibodies in humans.