Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Congenital human cytomegalovirus (HCMV) infection and HCMV infection of immunosuppressed patients cause significant morbidity and mortality, and vaccine development against HCMV is a major public health priority. HCMV envelope glycoproteins gB, gH, and gL, which constitute the core fusion machinery, play critical roles in HCMV fusion and entry into host cells. HCMV gB and gH/gL have been reported to elicit potent neutralizing antibodies. Recently, the gB/gH/gL complex was identified in the envelope of HCMV virions, and 16-50% of the total gH/gL bound to gB, forming the gB/gH/gL complex. These findings make the gB/gH/gL a unique HCMV vaccine candidate. We previously reported the production of HCMV trimeric gB and gH/gL heterodimers, and immunization with a combination of trimeric gB and gH/gL heterodimers elicited strong synergistic HCMV-neutralizing activity. To further improve the immunogenicity of gH/gL, we produced trimeric gH/gL. Rabbits immunized with HCMV trimeric gH/gL induced up to 38-fold higher serum titers of gH/gL-specific IgG relative to HCMV monomeric gH/gL, and elicited ~10-fold higher titers of complement-dependent and complement-independent HCMV-neutralizing activity for both epithelial cells and fibroblasts. HCMV trimeric gH/gL in combination with HCMV trimeric gB would be a novel promising HCMV vaccine candidate that could induce highly potent neutralizing activities.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

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.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

Human cytomegalovirus (HCMV) core fusion machinery proteins gB and gH/gL, and accessory proteins UL128/UL130/UL131A, are the key envelope proteins that mediate HCMV entry into and infection of host cells. To determine whether these HCMV envelope proteins could elicit neutralizing activities synergistically, we immunized rabbits with individual or various combinations of these proteins adsorbed to aluminum hydroxide mixed with CpG-ODN. We then analyzed serum neutralizing activities with multiple HCMV laboratory strains and clinical isolates. HCMV trimeric gB and gH/gL elicited high and moderate titers of HCMV neutralizing activity, respectively. HCMV gB in combination with gH/gL elicited up to 17-fold higher HCMV neutralizing activities compared to the sum of neutralizing activity elicited by the individual proteins analyzed with both fibroblasts and epithelial cells. HCMV gB+gH/gL+UL128/UL130/UL131A in combination increased the neutralizing activity up to 32-fold compared to the sum of neutralizing activities elicited by the individual proteins analyzed with epithelial cells. Adding UL128/UL130/UL131A to gB and gH/gL combination did not increase further the HCMV neutralizing activity analyzed with fibroblasts. These data suggest that the combination of HCMV core fusion machinery envelope proteins gB+gH/gL or the combination of gB and pentameric complex could be ideal vaccine candidates that would induce optimal immune responses against HCMV infection.

Multidonor antibodies are of interest for vaccine design because they can in principle be elicited in the general population by a common set of immunogens. For influenza, multidonor antibodies have been observed against the hemagglutinin (HA) stem, but not the immunodominant HA head. Here, we identify and characterize a multidonor antibody class (LPAF-a class) targeting the HA head. This class exhibits potent viral entry inhibition against H1N1 A/California/04/2009 (CA09) virus. LPAF-a class antibodies derive from the HV2-70 gene and contain a "Tyr-Gly-Asp"-motif, which occludes the HA-sialic acid binding site as revealed by a co-crystal structure with HA. Both germline-reverted and mature LPAF antibodies potently neutralize CA09 virus and have nanomolar affinities for CA09 HA. Moreover, increased frequencies for LPFA-a class antibodies are observed in humans after a single vaccination. Overall, this work highlights the identification of a multidonor class of head-directed influenza-neutralizing antibodies and delineates the mechanism of their recurrent elicitation in humans.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

Nanobodies can achieve remarkable neutralization of genetically diverse pathogens, including HIV-1. To gain insight into their recognition, we determined crystal structures of four llama nanobodies (J3, A12, C8, and D7), all of which targeted the CD4-binding site, in complex with the HIV-1 envelope (Env) gp120 core, and determined a cryoelectron microscopy (cryo-EM) structure of J3 with the Env trimer. Crystal and cryo-EM structures of J3 complexes revealed this nanobody to mimic binding to the prefusion-closed trimer for the primary site of CD4 recognition as well as a secondary quaternary site. In contrast, crystal structures of A12, C8, and D7 with gp120 revealed epitopes that included portions of the gp120 inner domain, inaccessible on the prefusion-closed trimer. Overall, these structures explain the broad and potent neutralization of J3 and limited neutralization of A12, C8, and D7, which utilized binding modes incompatible with the neutralization-targeted prefusion-closed conformation of Env.

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer - stabilized in the prefusion conformation and fused with SpyCatcher - could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with the SARS-CoV-2 spike-LuS nanoparticles elicited ~25-fold higher neutralizing responses, weight-per-weight relative to spike alone. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

Soluble "SOSIP"-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome.

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

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.