Biology subjects

Bjorkman, P. J.

Publications and source records attributed to Bjorkman, P. J..

At least 19 recordsLinked to original sources

bioRxiv2026

A mosaic RBD-nanoparticle pan-sarbecovirus vaccine elicits nanobodies that target highly conserved epitopes

SARS-CoV-2, the virus that causes COVID-19, continually mutates, producing new variants of concern (VOCs). Substitutions in the SARS-CoV-2 spike protein receptor-binding domain (RBD) have resulted in reduced efficacies of vaccines and therapeutic monoclonal antibodies. Indeed, since the emergence of Omicron and earlier VOCs, most previously approved anti-RBD therapeutic monoclonal antibodies are no longer authorized as therapeutics. To address VOC substitutions effects on therapeutic antibody efficacy, we sought to identify single-domain nanobodies (VHHs) targeting RBD regions (class 4, class 1/4, portions of class 3 and class 5 RBD epitopes) that are more conserved across VOCs, reasoning that the smaller size of camelid nanobodies compared with conventional two-domain antibody combining sites would facilitate access to occluded RBD epitopes. We used a Beacon optofluidic platform to isolate and characterize single memory B cells from a llama immunized with a mosaic RBD-nanoparticle immunogen that elicits broad antibody responses across sarbecoviruses. Using multiplex binding assays, we identified nanobodies that bind to spike RBDs from diverse sarbecovirus strains. Competition ELISAs and crystal structures of VHH-RBD complexes showed that isolated nanobodies recognize broadly conserved epitopes, including those on the RBD that are inaccessible to analogous conventional two-domain antibodies. Promising VHH-Fcs neutralized SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, demonstrating potential therapeutic utility for current and future outbreaks.

immunology↗

bioRxiv2026

Stabilized gp120-specific CD4 for next-generation HIV-1 inhibitors

HIV-1 Envs gp120 subunit uses the T-cell coreceptor CD4 to enter host cells in a manner that prevents the evolution of host resistance by sharing the binding epitope with the footprint of CD4s natural ligands, class II MHC proteins1,2. Consequently, CD4-containing biologics, such as CD4-Ig3,4 and derivatives5-9, benefit from this conserved relationship and are promising broad-acting anti-HIV-1 agents that are resistant to viral mutational escape10. However, these biologics suffer from short serum half-lives in humans11,12 and animals3,13, likely due to CD4s poor thermostability14 and/or off-target class II MHC binding15. This latter property also warrants caution for CD4-containing biologics that could indiscriminately recruit Fc-dependent effector functions against uninfected cells and/or compete with host CD4 for class II MHC during T cell interactions with antigen-presenting cells. Here, we describe gp120-specific CD4 (gCD4), which exhibits enhanced thermostability and retains Env, but not class II MHC, binding. CD4-Ig variants incorporating gCD4 did not bind class II MHC on human B cells, displayed greater longevity in human tonsil organoid cultures, showed half-lives equivalent to therapeutic IgG antibodies in mice, and neutralized HIV-1 more broadly and potently compared to the original CD4-Ig molecules. Encouragingly, one variant neutralized 100% of a panel of clinically-relevant HIV-1 strains at titers correlating to infection prevention in humans, outperforming known broadly neutralizing antibodies16,17. Thus, gCD4 holds promise for the development of new CD4-containing biologics with best-in-class specificity, pharmacokinetic properties, and neutralization breadth and potency.

bioengineering↗

bioRxiv2025

Development of a Recombinant Single-Cycle Influenza Viral Vector as an Intranasal Vaccine against SARS-CoV-2 and SARS-like Betacoronaviruses

The COVID-19 pandemic has demonstrated the detrimental potential of zoonotic coronavirus transmission to human populations. Effective vaccines capable of eliciting immunity to SARS-CoV-2 have been pivotal in mitigating the spread of the virus. In this study, we describe the generation of a non-replicating pseudotyped influenza A virus (S-FLU), where the native haemagglutinin (HA) sequence is replaced with the coding sequence of either a membrane-anchored form (TM) or secretory form (Sec) of the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 Wuhan (S-RBD Wuhan). We showed that both S-RBD-TM and S-RBD-Sec viruses can be generated via reverse genetics and grown to high titre. Intranasal immunisation in mice with S-RBD-TM elicits robust serum binding and neutralisation activity against SARS-CoV-2, superior to S-RBD-Sec. Furthermore, we demonstrate that a heterologous prime-boost immunisation regime in mice with S-RBD-TM Wuhan and S-RBD-TM BM48-31 (a distant Clade 3 SARS-like betacoronavirus (sarbecovirus)) increases antibody breadth against mismatched sarbecoviruses compared to homologous prime-boost with S-RBD-TM Wuhan. These results suggest that S-RBD-TM is a promising intranasal vaccine candidate against SARS-CoV-2 and may offer potential as a broadly protective sarbecovirus vaccine.

immunology↗

bioRxiv2025

mRNA delivery of genetically encoded mosaic-8 pan-sarbecovirus RBD vaccines

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 Fc{gamma} 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. SummaryWe 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.

immunology↗

bioRxiv2025

Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice

mRNA vaccines emerged as a leading vaccine technology during the COVID-19 pandemic. However, their sustained protective efficacies were limited by relatively short-lived antibody responses and the emergence of SARS-CoV-2 variants, necessitating frequent and variant-updated boosters. We recently developed the ESCRT- and ALIX-binding region (EABR) mRNA vaccine platform, which encodes engineered immunogens that induce budding of enveloped virus-like particles (eVLPs) from the plasma membrane, thereby resulting in presentation of immunogens on cell surfaces and eVLPs. Prior studies showed that spike (S)-EABR mRNA-LNP immunizations elicited enhanced neutralizing antibody responses against ancestral and variant SARS-CoV-2 compared with conventional S mRNA-LNP in naive mice, but the effectiveness of S-EABR mRNA-LNP boosters in the context of pre-existing immunity has not been investigated. Here, we evaluated monovalent Wuhan-Hu-1 (Wu1) and bivalent (Wu1/BA.5) S-EABR mRNA-LNP boosters in mice pre-vaccinated with conventional Wu1 S mRNA-LNP. Compared to conventional S mRNA-LNP boosters, the EABR approach enhanced monovalent and bivalent mRNA-LNP booster-induced neutralizing responses against Omicron subvariants BA.1, BA.5, BQ.1.1, and XBB.1, with bivalent S-EABR mRNA-LNP consistently eliciting the highest titers. Epitope mapping of polyclonal antisera by deep mutational scanning revealed that bivalent S-EABR mRNA-LNP boosted diverse "polyclass" anti-receptor-binding domain (RBD) responses, suggesting balanced targeting of multiple RBD epitope classes. In contrast, monovalent S, bivalent S, and monovalent S-EABR mRNA-LNP boosters elicited less diverse polyclonal serum responses primarily targeting immunodominant RBD epitopes. Cryo-EM structures demonstrated that bivalent mRNA immunizations promote S heterotrimer formation, potentially enhancing bivalent S-EABR mRNA-LNP booster-induced antibody breadth and polyclass epitope targeting by activating cross-reactive B cells through intra-S crosslinking. These findings support the future design of bivalent or multivalent S-EABR mRNA-LNP boosters as a promising strategy to confer broader, and therefore potentially more durable, protection against emerging SARS-CoV-2 variants and other rapidly evolving viruses.

immunology↗

bioRxiv2025

Single administration of mosaic-8b RBD-nanoparticle vaccine prepared with atomic layer deposition technology elicits broadly neutralizing anti-sarbecovirus responses

Atomic layer deposition (ALD), a new vaccine technology, permits multiple dosing with a single administration by pulsatile release of one or more immunogens. We evaluated ALD delivery of mosaic-8b [60-mer nanoparticles presenting 8 different SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] that elicits broadly cross-reactive antibodies and protects against mismatched sarbecoviruses not represented by RBDs on mosaic-8b. Compared with conventional prime-boost immunizations, ALD-delivered mosaic-8b RBD-nanoparticles elicited antibodies in both naive and pre-vaccinated mice with improved mismatched binding and neutralization. Results of RBD epitope mapping of serum antibodies from ALD-delivered mosaic-8b were consistent with broader coverage of RBD epitopes compared to conventional immunizations, and systems serology revealed distinct IgG subclass and Fc{gamma}R-binding IgG distributions. These results suggest that ALD is a promising technology for use with mosaic-8b RBD-nanoparticle vaccines to protect against future sarbecovirus spillovers and support applications for ALD vaccine delivery to elicit cross-reactive antibodies against rapidly mutating or diverse pathogens.

immunology↗

bioRxiv2025

Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

A primary goal in the development of an AIDS vaccine is the elicitation of broadly neutralizing antibodies (bNAbs) that protect against diverse HIV-1 strains. To this aim, germline-targeting immunogens have been developed to activate bNAb precursors and initiate the induction of bNAbs. While most pre-clinical germline-targeting HIV-1 vaccine candidates only include a single bNAb precursor epitope, an effective HIV-1 vaccine will likely require bNAbs that target multiple epitopes on Env. Here, we report a newly designed germline-targeting Env SOSIP trimer, named 3nv.2, that presents three bNAb epitopes on Env: the CD4bs, V3, and V2 epitopes. 3nv.2 forms a stable trimeric Env and binds to bNAb precursors from each of the desired epitopes. Immunization experiments in rhesus macaques and mice demonstrate 3nv.2 elicits the combined effects of its parent immunogens. Our results provide proof-of-concept for using a germline-targeting immunogen presenting three or more bNAb epitopes and a framework to develop improved next-generation HIV-1 vaccine candidates.

bioengineering↗

bioRxiv2025

Cross-reactive sarbecovirus antibodies induced by mosaic RBD-nanoparticles

Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD-nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD-nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs. Significance StatementSARS-CoV-2 variants and potential zoonotic sarbecovirus infections continue to threaten human health. Anti-SARS-CoV-2 mAbs that recognize conserved epitopes could be used prophylactically or therapeutically. We present approaches to elicit and identify cross-reactive mAbs using immunizations in animals with mosaic RBD-nanoparticles. We show that human and other mammalian immune systems can utilize different antibody gene segments to arrive at similar modes of antigen recognition, underscoring the flexibility of mammalian antibody repertoires and suggesting that experimental animals can be used to generate therapeutically-useful cross-reactive anti-RBD mAbs. The combination of mosaic-8b RBD-nanoparticles to focus the immune response and a multiplexed assay to select cross-reactive mAbs can be applied at larger scale, or against other pathogens, to identify mAbs of therapeutic and scientific potential.

immunology↗

bioRxiv2025

An ultra-long heavy chain bovine antibody neutralizes SARS-CoV-2 and reacts broadly with sarbecoviruses.

The ongoing threat of new SARS-CoV-2 variants and other sarbecoviruses has driven efforts to develop broadly neutralizing monoclonal antibodies (mAbs). This study used immunized cattle, known for producing antibodies with ultra-long CDRH3 domains, to generate 33 mAbs, ten of which had ultra-long CDRH3s (>50 amino acids). Of these, mAbs P7 and 99, demonstrated broad and potent neutralization. Notably, mAb P7 neutralized all tested variants, including SARS-CoV-1, with IC50 values between 0.05 and 9.2 {micro}g/mL, and showed cross-reactivity with RBDs from various sarbecoviruses. Structural analysis revealed that mAb 99 binds the spike proteins RBD at the ACE2 binding site. Although the exact binding of P7 wasnt resolved, evidence suggests it targets a hidden epitope, promoting spike trimer dissociation via its extended CDRH3. In Syrian hamsters, both mAbs significantly reduced lung viral loads. These results support the potential of bovine-derived mAbs, particularly those with ultra-long CDRH3s, for future antiviral therapies.

immunology↗

bioRxiv2024

Mapping essential somatic hypermutations in a CD4-binding site bNAb informs HIV-1 vaccine design

HIV-1 broadly-neutralizing antibodies (bNAbs) targeting the CD4-binding site (CD4bs) contain rare features that pose challenges to elicit these bNAbs through vaccination. The IOMA-class of CD4bs bNAbs includes fewer rare features and somatic hypermutations (SHMs) to achieve broad neutralization, thus presenting a potentially accessible pathway for vaccine-induced bNAb development. Here, we created a library of IOMA variants in which each SHM was individually reverted to the inferred germline (iGL) counterpart to investigate the roles of SHMs in conferring IOMAs neutralization potency and breadth. Impacts on neutralization for each variant were evaluated, and this information was used to design minimally-mutated IOMA-class variants (IOMAmin) that incorporated the fewest SHMs required for achieving IOMAs neutralization breadth. A cryo-EM structure of an IOMAmin variant bound to Env was used to further interpret characteristics of IOMA variants to elucidate how IOMAs structural features correlate with its neutralization mechanism, informing the design of IOMA-targeting immunogens. HighlightsIOMAmin variants with reduced SHMs retain neutralization potency and breadth. Cryo-EM structure reveals IOMAmin preserves key Env interactions despite fewer SHMs. IOMA variants with mutations in CDRH3 and CDRL1 fail to improve neutralization.

immunology↗

bioRxiv2024

Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by EM. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by prehairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow, liver, thymus (BLT) mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study. IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor treated tissues of humanized mice. The finding of trapped HIV-1 virions in all tissues examined demonstrates a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues.

microbiology↗

bioRxiv2024

Neutralizing antibodies elicited in sequentially immunized macaques recognize V3 residues on altered conformations of HIV-1 Env trimer

Eliciting broadly neutralizing antibodies that protect against diverse HIV-1 strains is a primary goal of AIDS vaccine research. We characterized Ab1456 and Ab1271, two heterologously-neutralizing antibodies elicited in non-human primates by priming with an engineered V3-targeting SOSIP Env immunogen and boosting with increasingly native-like SOSIP Envs derived from different strain backgrounds. Structures of Env trimers in complex with these antibodies revealed V3 targeting, but on conformational states of Env distinct from the typical closed, prefusion trimeric SOSIP structure. Env trimers bound by Ab1456 adopted conformations resembling CD4-bound open Env states in the absence of soluble CD4, whereas trimers bound by Ab1271 exhibited a trimer apex-altered conformation to accommodate antibody binding. The finding that elicited antibodies cross-neutralized by targeting altered, non-closed, prefusion Env trimer conformations provides important information about Env dynamics that is relevant for HIV-1 vaccine design aimed at raising antibodies to desired epitopes on closed pre-fusion Env trimers. Highlights- Sequential immunization regimen elicits V3 antibodies targeting non-closed Envs - Cryo-EM structures reveal recognition of multiple Env conformational states - Neutralization by elicited antibody does not require antibody-virus preincubation Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/605918v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@6210b1org.highwire.dtl.DTLVardef@35f683org.highwire.dtl.DTLVardef@cb2d3corg.highwire.dtl.DTLVardef@106e066_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

microbiology↗

bioRxiv2024

Designed mosaic nanoparticles enhance cross-reactive immune responses in mice

Using computational methods, we designed 60-mer nanoparticles displaying SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) by (i) creating RBD sequences with 6 mutations in the SARS-COV-2 WA1 RBD that were predicted to retain proper folding and abrogate antibody responses to variable epitopes (mosaic-2COMs; mosaic-5COM), and (ii) selecting 7 natural sarbecovirus RBDs (mosaic-7COM). These antigens were compared with mosaic-8b, which elicits cross-reactive antibodies and protects from sarbecovirus challenges in animals. Immunizations in naive and COVID-19 pre-vaccinated mice revealed that mosaic-7COM elicited higher binding and neutralization titers than mosaic-8b and related antigens. Deep mutational scanning showed that mosaic-7COM targeted conserved RBD epitopes. Mosaic-2COMs and mosaic-5COM elicited higher titers than homotypic SARS-CoV-2 Beta RBD-nanoparticles and increased potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons. These results support using mosaic-7COM to protect against highly mutated SARS-CoV-2 variants and zoonotic sarbecoviruses with spillover potential.

immunology↗

bioRxiv2024

Mosaic sarbecovirus vaccination elicits cross-reactive responses in pre-immunized animals

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

immunology↗

bioRxiv2023

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection. One Sentence SummaryA vaccine candidate with polyprotein antigens displayed on nanocages induces neutralizing antibodies to multiple SARS-like coronaviruses.

immunology↗

bioRxiv2023

Structural characterization of HIV-1 Env heterotrimers bound to one or two CD4 receptors reveals intermediate Env conformations

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120/gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops, and in fully-saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops. To investigate changes resulting from sub-stoichiometric CD4 binding, we solved 3.4[A] and 3.9[A] single-particle cryo-EM structures of soluble, native-like Envs bound to one or two CD4 molecules. Env trimer bound to one CD4 adopted the closed, prefusion Env state. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state that included gp120 outward rotation but maintained the prefusion, three-stranded gp120 bridging sheet and showed only partial V1V2 displacement and V3 exposure. We conclude that engagement of one CD4 molecule was insufficient to stimulate CD4-induced conformational changes, while binding two CD4 molecules led to Env opening in CD4-bound protomers only. Together, these results illuminate HIV-1 Env intermediate conformations and illustrate the structural plasticity of HIV-1 Env.

molecular biology↗

bioRxiv2022

ESCRT recruitment to mRNA-encoded SARS-CoV-2 spike induces virus-like particles and enhanced antibody responses

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

immunology↗