Structural basis for VLDLR recognition by eastern equine encephalitis virus.

Alphaviruses are arthropod-borne enveloped RNA viruses that include several important human pathogens with outbreak potential. Among them, eastern equine encephalitis virus (EEEV) is the most virulent, and many survivors develop neurological sequelae, including paralysis and intellectual disability. The spike proteins of alphaviruses comprise trimers of heterodimers of their envelope glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), two closely related proteins that are expressed in the brain, as cellular receptors for EEEV and a distantly related alphavirus, Semliki forest virus (SFV) 1 . The EEEV and SFV spike glycoproteins have low sequence homology, and how they have evolved to bind the same cellular receptors is unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain. The structures reveal that EEEV and SFV use distinct surfaces to bind VLDLR; EEEV uses a cluster of basic residues on the E2 subunit of its spike glycoprotein, while SFV uses two basic residues at a remote site on its E1 glycoprotein. Our studies reveal that different alphaviruses interact with the same cellular receptor through divergent binding modes. They further suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

Humoral immunity to an endemic coronavirus is associated with postacute sequelae of COVID-19 in individuals with rheumatic diseases.

Beyond the acute illness caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) infection, about one-fifth of infections result in long-term persistence of symptoms despite the apparent clearance of infection. Insights into the mechanisms that underlie postacute sequelae of COVID-19 (PASC) will be critical for the prevention and clinical management of long-term complications of COVID-19. Several hypotheses have been proposed that may account for the development of PASC, including persistence of virus and dysregulation of immune responses. Among the immunological changes noted in PASC, alterations in humoral immunity have been observed in some patient subsets. To begin to determine whether SARS-CoV-2- or other pathogen-specific humoral immune responses evolve uniquely in PASC, we performed comprehensive antibody profiling against SARS-CoV-2, a panel of endemic pathogens, and a panel of routine vaccine antigens using systems serology in two cohorts of patients with preexisting systemic autoimmune rheumatic disease (SARD) who either developed or did not develop PASC. A distinct qualitative shift observed in Fcγ receptor (FcγR) binding was observed in individuals with PASC. Specifically, individuals with PASC harbored weaker FcγR-binding anti-SARS-CoV-2 antibodies and stronger FcγR-binding antibody responses against the endemic coronavirus OC43. Individuals with PASC developed an OC43 S2-specific antibody response with stronger FcγR binding, linked to cross-reactivity across SARS-CoV-2 and common coronaviruses. These findings identify previous coronavirus imprinting as a potential marker for the development of PASC in individuals with SARDs.

A VH1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite.

An oligomannose patch around the V3 base of HIV-1 envelope glycoprotein (Env) is recognized by multiple classes of broadly neutralizing antibodies (bNAbs). Here, we investigated the bNAb response to the V3 glycan supersite in an HIV-1-infected Chinese donor by Env-specific single B cell sorting, structural and functional studies, and longitudinal analysis of antibody and virus repertoires. Monoclonal antibodies 438-B11 and 438-D5 were isolated that potently neutralize HIV-1 with moderate breadth, are encoded by the VH1-69 germline gene, and have a disulfide-linked long HCDR3 loop. Crystal structures of Env-bound and unbound antibodies revealed heavy chain-mediated recognition of the glycan supersite with a unique angle of approach and a critical role of the intra-HCDR3 disulfide. The mechanism of viral escape was examined via single-genome amplification/sequencing and glycan mutations around the N332 supersite. Our findings further emphasize the V3 glycan supersite as a prominent target for Env-based vaccine design.

Proof of concept for rational design of hepatitis C virus E2 core nanoparticle vaccines.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are responsible for cell entry, with E2 being the major target of neutralizing antibodies (NAbs). Here, we present a comprehensive strategy for B cell-based HCV vaccine development through E2 optimization and nanoparticle display. We redesigned variable region 2 in a truncated form (tVR2) on E2 cores derived from genotypes 1a and 6a, resulting in improved stability and antigenicity. Crystal structures of three optimized E2 cores with human cross-genotype NAbs (AR3s) revealed how the modified tVR2 stabilizes E2 without altering key neutralizing epitopes. We then displayed these E2 cores on 24- and 60-meric nanoparticles and achieved substantial yield and purity, as well as enhanced antigenicity. In mice, these nanoparticles elicited more effective NAb responses than soluble E2 cores. Next-generation sequencing (NGS) defined distinct B cell patterns associated with nanoparticle-induced antibody responses, which target the conserved neutralizing epitopes on E2 and cross-neutralize HCV genotypes.

HIV-1 vaccine design through minimizing envelope metastability.

Overcoming envelope metastability is crucial to trimer-based HIV-1 vaccine design. Here, we present a coherent vaccine strategy by minimizing metastability. For 10 strains across five clades, we demonstrate that the gp41 ectodomain (gp41ECTO) is the main source of envelope metastability by replacing wild-type gp41ECTO with BG505 gp41ECTO of the uncleaved prefusion-optimized (UFO) design. These gp41ECTO-swapped trimers can be produced in CHO cells with high yield and high purity. The crystal structure of a gp41ECTO-swapped trimer elucidates how a neutralization-resistant tier 3 virus evades antibody recognition of the V2 apex. UFO trimers of transmitted/founder viruses and UFO trimers containing a consensus-based ancestral gp41ECTO suggest an evolutionary root of metastability. The gp41ECTO-stabilized trimers can be readily displayed on 24- and 60-meric nanoparticles, with incorporation of additional T cell help illustrated for a hyperstable 60-mer, I3-01. In mice and rabbits, these gp140 nanoparticles induced tier 2 neutralizing antibody responses more effectively than soluble trimers.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.IMPORTANCE Both epitope-focused and trimer-based strategies are currently being explored in HIV-1 vaccine development, which aims to elicit broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the viral envelope (Env). However, little is known about the differences in antibody response to these bNAb targets presented by foreign scaffolds and native Env. In this study, a systematic effort was undertaken to design multivalent epitope scaffolds and soluble gp140.681 trimers with a complete antigenic surface, and to comparatively analyze the antibody responses elicited by these antigens to the N332 supersite and MPER in a mouse model. This study will inform both epitope-focused and trimer-based vaccine design and will facilitate integration of the two vaccine strategies.