HIV BG505 SOSIP.664 trimer with 3M-052-AF/alum induces human autologous tier-2 neutralizing antibodies.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140, formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding and immunogenicity in a first-in-healthy adult (n=17), randomized, placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, B-cell and CD4+ T-cell responses emerged post-vaccination. Five vaccinees developed serum autologous tier-2 nAbs (ID50 titer, 1:28-1:8647) after 2-3 doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B-cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes. KEY TAKEAWAY/TAKE-HOME MESSAGES: HIV BG505 SOSIP.664 trimer with novel 3M-052-AF/alum adjuvant in humans appears safe and induces serum neutralizing antibodies to matched clade A, tier 2 virus, that map to diverse Env epitopes with relatively high titers. The novel adjuvant may be an important mediator of vaccine response.

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design. Author summary: Enterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the atomic structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, allowing us to pinpoint key regions involved in blood-group A antigen recognition and uncover the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected human volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.

Pollen-mediated gene flow from wild carrots (Daucus carota L. subsp. carota) affects the production of commercial carrot seeds (Daucus carota L. subsp. sativus) internationally and in New Zealand in the context of climate change: A systematic review.

Climate change will impact the carrot seed industry globally. One adaptation strategy to limit climatic impacts on the production of commercial carrot seeds is geographical shift. However, production must be shifted to climate-optimal places that are free from weeds such as wild carrots to avoid genetic contamination via hybridization. The process of gene flow between wild and cultivated carrots is critical to enable management of wild carrots in the face of climate change. This review systematically assesses the resilience of wild carrots to climate change and their impact on commercial carrot seed production globally with a focus on New Zealand as a major carrot seed producer. The literature was critically analyzed based on three specific components: i) resilience of wild carrots to climate change ii) genetic contamination between wild and cultivated carrots, and iii) management of wild carrots. The majority of the articles were published between 2013 and 2023 (64.71 %), and most of these studies were conducted in Europe (37.26 %) and North America (27.45 %). Country-wise analysis demonstrated that the majority of the studies were carried out in the United States (23.53 %) and the Netherlands (11.77 %). There was limited research conducted in other regions, especially in Oceania (1.96 %). Spatial distribution analysis revealed that the wild carrot was reported in around 100 countries. In New Zealand the North Island has a higher incidence of wild carrot invasion than the South Island. The findings indicated that the wild carrot is becoming more adaptable to climate change, compromising the genetic purity of cultivated carrots due to pollen flow from wild to cultivated carrots. Therefore, ongoing research will be helpful in developing sustainable weed management strategies and predicting potential geographical invasiveness. This study provides a guide for scientists, policymakers, industrialists, and farmers to control wild carrots and produce genetically pure commercial seeds amid climate change.

Anti-Immune Complex Antibodies are Elicited During Repeated Immunization with HIV Env Immunogens.

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Early antibody responses to easily accessible epitopes on these antigens are directed to non-neutralizing epitopes instead of bnAb epitopes. Autologous neutralizing antibody responses appear upon boosting once immunodominant epitopes are saturated. Here we report another type of antibody response that arises after repeated immunizations with HIV Env immunogens and present the structures of six anti-immune complexes discovered using polyclonal epitope mapping. The anti-immune complex antibodies target idiotopes composed of framework regions of antibodies bound to Env. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens, in which repeated exposure to antigen is required.

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows.

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli.

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses.

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines.

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Effect of verbal instructions on trunk muscle activity during volitional preemptive abdominal contraction.

OBJECTIVE: This study investigated the effect of Verbal Instruction (VI) strategies on trunk muscle contraction among healthy subjects. The effect of three VI Abdominal Drawing-In Maneuver (ADIM) and two VI Abdominal Bracing Maneuver (ABM) strategies on left Internal (LIO) and External Oblique (LEO) and bilateral superficial Multifidi (sMf) activation was examined. DESIGN: Within-subjects, repeated measure design. METHODS: Surface EMG (sEMG) measured LIO, LEO, and sMf activity in 28 subjects (mean age 23.5 ± 5.5 years). Testing included five supine hook-lying and five quiet standing conditions. RESULTS: One-way ANOVAs demonstrated no significant main effect for ADIM or ABM in supine or standing (p > .05). Muscle activation amplitudes during VPAC conditions demonstrated higher mean values for standing versus supine (p < .05) except for two conditions involving LEO. Friedman Tests for dominant strategy demonstrated a significant main effect for ADIM-VI and ABM-VI strategies. Post-hoc testing generally showed the dominant strategy to be significantly higher versus others. CONCLUSION: No single preferred VI cue for ADIM or ABM was observed. Each subject's dominant strategy dictated the most suitable VI. Standing was preferred for LIO and sMf activation, whereas position did not change LEO activation. Non-significant correlations between all muscle pairings during all ADIM and ABM strategies were observed. These findings may suggest the need for healthcare providers who understand the intricacies of trunk stability to teach and monitor VPAC with either ADIM or ABM options.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli.

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension1. Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e., they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). In an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization2. Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

Structure of mechanically activated ion channel OSCA2.3 reveals mobile elements in the transmembrane domain.

Members of the OSCA/TMEM63 family are mechanically activated ion channels and structures of some OSCA members have revealed the architecture of these channels and structural features that are potentially involved in mechanosensation. However, these structures are all in a similar state and information about the motion of different elements of the structure is limited, preventing a deeper understanding of how these channels work. Here, we used cryoelectron microscopy to determine high-resolution structures of Arabidopsis thaliana OSCA1.2 and OSCA2.3 in peptidiscs. The structure of OSCA1.2 matches previous structures of the same protein in different environments. Yet, in OSCA2.3, the TM6a-TM7 linker adopts a different conformation that constricts the pore on its cytoplasmic side. Furthermore, coevolutionary sequence analysis uncovered a conserved interaction between the TM6a-TM7 linker and the beam-like domain (BLD). Our results reveal conformational heterogeneity and differences in conserved interactions between the TMD and BLD among members of the OSCA family.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.