Malaria pathology is driven by the accumulation of Plasmodium falciparum-infected erythrocytes in microvessels1. This process is mediated by the polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins of the parasite. A subset of PfEMP1 variants that bind to human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis2. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here we describe two broadly reactive and inhibitory human monoclonal antibodies to CIDRα1. The antibodies isolated from two different individuals exhibited similar and consistent EPCR-binding inhibition of diverse 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 human brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with three 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 are likely to 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. Two broadly reactive and inhibitory human monoclonal antibodies against the malaria parasite Plasmodium falciparum have been characterized, providing insights into immunity, prevention and treatment of severe malaria.

HIV-1 infection leads to chronic disease requiring life-long treatment and therefore alternative therapeutics, a cure and/or a protective vaccine are needed. Antibody-mediated effector functions could have a role in the fight against HIV-1. However, the properties underlying the potential beneficial effects of antibodies during HIV-1 infection are poorly understood. To identify a specific profile of antibody features associated with delayed disease progression, we studied antibody polyfunctionality during untreated HIV-1 infection in the well-documented Amsterdam Cohort Studies. Serum samples were analyzed from untreated individuals with HIV-1 at approximately 6 months (n = 166) and 3 years (n = 382) post-seroconversion (post-SC). A Luminex antibody Fc array was used to profile 15 different Fc features for serum antibodies against 20 different HIV-1 envelope glycoprotein antigens and the resulting data was also compared with data on neutralization breadth. We found that high HIV-1 specific IgG1 levels and low IgG2 and IgG4 levels at 3 years post-SC were associated with delayed disease progression. Moreover, delayed disease progression was associated with a broad and polyfunctional antibody response. Specifically, the capacity to interact with all Fc γ receptors (FcγRs) and C1q, and in particular with FcγRIIa, correlated positively with delayed disease progression. There were strong correlations between antibody Fc features and neutralization breadth and several antibody features that were associated with delayed disease progression were also associated with the development of broad and potent antibody neutralization. In summary, we identified a strong association between broad, polyfunctional antibodies and delayed disease progression. These findings contribute new information for the fight against HIV-1, especially for new antibody-based therapy and cure strategies.

The function of the spike protein N terminal domain (NTD) in coronavirus (CoV) infections is poorly understood. However, some rare antibodies that target the SARS-CoV-2 NTD potently neutralize the virus. This finding suggests the NTD may contribute in part to protective immunity. Pan-sarbecovirus antibodies are desirable for broad protection, but the NTD region of SARS-CoV and SARS-CoV-2 exhibit a high level of sequence divergence, and therefore, cross-reactive NTD-specific antibodies are unexpected, and there is no structure of a SARS-CoV NTD-specific antibody in complex with NTD. Here we report a monoclonal antibody COV1-65 encoded by the IGHV1-69 gene that recognizes the NTD of SARS-CoV S protein. A prophylaxis study showed the MAb COV1-65 prevented disease when administered before SARS-CoV challenge of BALB/c mice, an effect that requires intact Fc effector functions for optimal protection in vivo. The footprint on the S protein of COV1-65 is near to functional components of the S2 fusion machinery, and the selection of COV1-65 escape mutant viruses identified critical residues Y886H and Q974H, which likely affect the epitope through allosteric effects. Structural features of the mAb COV1-65-SARS-CoV antigen interaction suggest critical antigenic determinants that should be considered in the rational design of sarbecovirus vaccine candidates.

Cosolvent molecular dynamics (MDs) are an increasingly popular form of simulations where small molecule cosolvents are added towater-solvated protein systems. These simulations can perform diverse target characterization tasks, including cryptic and allosteric pocket identification and pharmacophore profiling and supplement suites of enhanced sampling methods to explore protein conformational landscapes. The behavior of these systems is tied to the cosolvents used, so the ability to define diverse and complex mixtures is critical in dictating the outcome of the simulations.However, existing methods for preparing cosolvent simulations only support a limited number of predefined cosolvents and concentrations. Here, we present CosolvKit, a tool for the preparation and analysis of systems composed of user-defined cosolvents and concentrations. This tool is modular, supporting the creation of files for multiple MD engines, as well as direct access to OpenMM simulations, and offering access to a variety of generalizable small-molecule force fields. To the best of our knowledge, CosolvKit represents the first generalized approach for the construction of these simulations.

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined. Additionally, molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction and compare the obtained structures with ensembles obtained from MD simulations and NMR. We provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. Significant minima of the free energy surfaces remain undetected.

The humanization of camelid‐derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity‐determining regions (CDRs), Vernier and Hallmark residues, shaping the three‐dimensional fold and influencing VHH structure and function. Additionally, the presence of non‐canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid‐derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next‐generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non‐canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Launching an AI/ML benchmarking competition: AIntibody.org

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, and placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, and B cell and CD4+ T cell responses emerged after vaccination. Five vaccinees developed serum autologous tier 2 nAbs (ID50 titer, 1:28-1:8647) after two to three 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.

The continuous emergence of SARS-CoV-2 variants of concern has rendered many therapeutic monoclonal antibodies (mAbs) ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the recently circulating Omicron sub-lineages BA.2.86 and JN.1. Here, we report the isolation of broad and potent neutralizing human mAbs (HuMabs) from a healthcare worker infected with SARS-CoV-2 early in the pandemic. These include a genetically unique HuMab, named K501SP6, which can neutralize different Omicron sub-lineages, including BQ.1, XBB.1, BA.2.86 and JN.1, by targeting a highly conserved epitope on the N terminal domain, as well as an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants that was escaped by the more recent Omicron sub-lineages through spike F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies. Isolation of a broad and potent neutralizing human monoclonal antibody which targets a conserved N-terminal epitope and neutralizes Omicron sub-lineages, including BA.2.86 and JN.1, offers insights into immune evasion mechanisms.

An effective human immunodeficiency virus 1 (HIV-1) vaccine will most likely have to elicit broadly neutralizing antibodies (bNAbs) to overcome the sequence diversity of the envelope glycoprotein (Env). So far, stabilized versions of Env, such as SOSIP trimers, have been able to induce neutralizing antibody (NAb) responses, but those responses are mainly strain-specific. Here we attempted to broaden NAb responses by using a multivalent vaccine and applying a number of design improvements. First, we used highly stabilized SOSIP.v9 trimers. Second, we removed any holes in the glycan shields and optimized glycan occupancy to avoid strain-specific glycan hole responses. Third, we selected five sequences from the same clade (B), as we observed previously that combining Env trimers from clade A, B and C did not improve cross-reactive responses, as they might have been too diverse. Fourth, to improve antibody (Ab) responses, the Env trimers were displayed on two-component I53-50 nanoparticles (NPs). Fifth, to favor activation of cross-reactive B cells, the five Env trimers were co-displayed on mosaic NPs. Sixth, we immunized rabbits four times with long intervals between vaccinations. These efforts led to the induction of cross-reactive B cells and cross-reactive binding Ab responses, but we only sporadically detected cross-neutralizing responses. We conclude that stabilized HIV-1 Env trimers that are not modified specifically for priming naive B cells are unable to elicit strong bNAb responses, and infer that sequential immunization regimens, most likely starting with specific germline-targeting immunogens, will be necessary to overcome Env’s defenses against the induction of NAbs. The antigens described here could be excellent boosting immunogens in a sequential immunization regimen, as responses to bNAb epitopes were induced.

Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.

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 volunteers challenged with ETEC, diarrheal severity is significantly increased 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 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 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.