Repetitive display of the major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) is the basis for two WHO-recommended vaccines: RTS,S/AS01 and R21/Matrix-M. Recently, however, the CIS43 monoclonal antibody that preferentially targets the junctional region of PfCSP has been shown to be highly protective in humans, highlighting its junctional epitope as a key vaccine target. Here, we develop a vaccine based on tandem repeats of the junctional epitope displayed on a self-assembling nanoparticle and compare this CIS43-based junctional vaccine alone or in combination with the benchmark R21 vaccine, using both B cell analysis and monoclonal antibody isolation to define targeting of the immune response. Comparable reduction in liver burden was observed following vaccination with the best junctional vaccine and R21 at a dose of 1 μg. At a dose of 0.25 μg, a modest reduction of malaria liver burden with the junctional vaccine was observed compared to R21. Further, combining the junctional and R21 vaccines did not yield substantial improvement, although a modest trend was observed. While the R21 vaccine elicited antibodies primarily against the major repeats, the junctional vaccine elicited antibodies against both junctional and major repeat regions. In vivo B cell analysis and isolation of monoclonal antibodies confirmed differences in vaccine-induced antibody specificities. Altogether, these data suggest the nanoparticle-formatted tandem-repeated CIS43-junctional vaccine to be a promising approach to broaden immunity against malaria, either as a standalone intervention or in combination with R21.

Understanding the mechanistic interplay between antibodies and invading pathogens is essential for vaccine development. Current methods are labour and time intensive and limited by sample preparation bottlenecks. Here we present microfluidic electron microscopy-based polyclonal epitope mapping (mEM), which combines microfluidics with single-particle electron microscopy for the structural characterization of immune complexes using small volumes of sera (<4 µl). First, we used mEM to map polyclonal antibodies present in sera from infected and vaccinated individuals against five viral glycoproteins using negative-stain electron microscopy. The mEM detected a greater number of epitopes compared with conventional polyclonal epitope structural mapping methods. Second, we used mEM and cryo-electron microscopy to characterize two coronavirus spikes and one HA glycoprotein with and without polyclonal antibodies. Finally, we mapped individual antibody responses over time in mice vaccinated with human immunodeficiency virus envelope N332-GT5. mEM enables the rapid, high-throughput mapping of antibodies targeting a broad range of glycoproteins, facilitating a better understanding of infection and guiding structure-based vaccine design. A microfluidics- and electron-microscopy-based method enables the characterization of polyclonal antibody responses to infection and vaccination.

In the twenty years since extrafollicular B cell responses were originally described, much has been learned about B cell biology. With this progress, the term "extrafollicular" has expanded beyond its initial use to describe a variety of B cell processes, resulting in ambiguity over the term. Extrafollicular responses are often not identified by location, convoluting the criteria being used to define the pathway. Here, we discuss the current understanding of B cell responses as relevant to the current uses of the term "extrafollicular." In this context, we propose a framework to classify evolving concepts in B cell biology. The use of this framework moving forward is expected to help harmonize and clarify the discussion in the field.

To explore the use of human B cell receptor (BCR) knock-in mice for broadening antibody responses, we diversified CR3022, a monoclonal antibody (mAb) originally identified in a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) convalescent patient. This mAb targets a conserved epitope on the coronavirus receptor-binding domain (RBD). We took advantage of high- and low-affinity CR3022 BCR knock-in mice and immunized them with SARS-CoV-2 Wuhan RBD trimers to expand the breadth of these antibodies toward this virus. The resulting antibodies retained the ability to neutralize SARS-CoV and exhibited enhanced affinity and neutralization against the SARS-CoV-2 WA1/2020 strain, as well as the Delta (B.1.617.2) and Omicron KP.3 variants. They also showed broadened reactivity to two bat coronaviruses: WIV1 and, to a lesser potency, BtKY72. Structural analysis revealed key mutations that enhanced binding and neutralization, highlighting the importance of epitope accessibility and variant-specific conformations in antibody diversification. These findings demonstrate that human BCR-expressing mouse models can generate effective antibodies with broad neutralizing activity against viral epitopes.

Invariant natural killer T (iNKT) cells are activated by glycolipids presented on CD1d. When iNKT cells interact with and activate B cells, they can differentiate into iNKT follicular helper (iNKTfh) cells, and here, we investigate how this, in turn, regulates conventional T follicular helper (Tfh) cells. This is done in an autoimmune model where antibodies are produced against self-antigens relevant to the autoimmune disease systemic lupus erythematosus (SLE). We find a balance between iNKTfh and Tfh cells that directs the B cell response and influences Tfh cell generation. This altered balance also affects the specificities and increases the autoantibody response. We also show that CD1d expression by B cells is essential for iNKTfh cell generation. In conclusion, our data shed light on how T cell help for B cells is divided between conventional and unconventional helper cell populations and how this has an impact on autoreactive B cell responses.

Broadly neutralizing antibodies (bNAbs) are rarely elicited during HIV-1 infection. To identify obstacles to bNAb development, we longitudinally studied 122 rhesus macaques infected by one of 16 different simian-human immunodeficiency viruses (SHIVs). We identified V2 apex as the most common bNAb target and a subset of Envs that preferentially elicited these antibodies. In 10 macaques, we delineated Env-antibody coevolution from B cell priming to bNAb development. Antibody phylogenies revealed permissive developmental pathways guided by evolving Envs that contained few mutations in or near the V2 apex C-strand, which were a sensitive indicator of apex-targeted responses. The absence of such mutations reflected a failure in bNAb priming. These results indicate that efficiency of B cell priming, and not complexities in Env-guided affinity maturation, is the primary obstacle to V2 apex bNAb elicitation in SHIV-infected macaques and identify specific HIV-1 Envs to advance as novel vaccine platforms. One sentence summary:B cell priming is the primary bottleneck to HIV-1 V2 apex bNAb elicitation.

Systemic lupus erythematosus is characterized by activation of many self-reactive B cell clones that produce autoantibodies. This can be modeled using mixed bone marrow chimeras, where autoreactive 564Igi B cells initiate autoimmunity that spreads to wild-type (WT) B cells. The mechanisms controlling the inclusion of new B cell clones into spontaneous germinal centers (GCs) remain unclear. Using CRISPR-Cas9, we generated 2 autoreactive B cell receptor knock-in strains, M05 and G55, based on B cell receptors from WT GC B cells in WT:564Igi chimeras. M05 and G55 mice lacked spontaneous GCs and overt autoantibody production, with receptor editing (λ light chain expression) contributing to tolerance. However, autoreactivity was not purged from the B cell compartment since presence of 564Igi clone allowed M05 and G55 B cells to join GCs and produce autoantibodies. These findings reveal that GCs can override peripheral tolerance, recruiting previously silent autoreactive clones and facilitating diversification of autoantibodies.

Germline-targeting is a promising approach to HIV vaccine development that begins with the elicitation of precursors to broadly neutralizing antibodies (bnAbs), but it remains unclear whether simultaneous elicitation of precursors to multiple epitopes on the HIV envelope (Env) would be inhibited by competition. This study used preclinical mouse models with physiologically relevant frequencies of bnAb precursor-bearing B cells to compare precursor elicitation by coadministration of multiple protein or mRNA lipid nanoparticle (mRNA-LNP) germline-targeting immunogens. These immunogens activate multiple bnAb precursor classes targeting distinct epitopes on Env but with evidence of potential competition. Simultaneous delivery of immunogens encoded by mRNA-LNPs, however, drove maturation across different precursor frequencies and immunogen doses. Furthermore, administration of a cocktail of mRNA-LNP immunogens (N332-GT5 gp151, ApexGT5 gp151, eOD-GT8 60mer, and 10E8-GT12 24mer) led to balanced activation of four distinct bnAb precursor classes, indicating that multiepitope HIV bnAb precursor priming might be successfully implemented in humans but might be immunogen dependent.

Antibodies are crucial therapeutics, comprising a substantial portion of approved drugs due to their safety and clinical efficacy. Traditional antibody discovery methods are labor-intensive, limiting scalability and high-throughput analysis. Here, we improved upon our streamlined approach combining structural analysis and bioinformatics to infer heavy and light chain sequences from cryo-EM (cryo-electron microscopy) maps of serum-derived polyclonal antibodies (pAbs) bound to antigens. Using ModelAngelo, an automated structure-building tool, we accelerated pAb sequence determination and identified sequence matches in B cell repertoires via ModelAngelo-derived hidden Markov models (HMMs) associated with pAb structures. Benchmarking against results from a nonhuman primate HIV vaccine trial, our pipeline reduced analysis time from weeks to under a day with higher precision. Validation with murine immune sera from influenza vaccination revealed multiple protective antibodies. This workflow enhances antibody discovery, enabling faster, more accurate mapping of polyclonal responses with broad applications in vaccine development and therapeutic antibody discovery.

mRNA vaccines have emerged as an important platform for vaccine development. Unlike protein subunit vaccines, mRNA-expressed antigens can be expressed in either secreted or transmembrane (TM) forms mimicking a viral envelope (Env) protein. Here, we investigated the impact of antigen expression format on the antigenicity profile, glycosylation, and immunogenicity of stabilized HIV Env trimer immunogens expressed from self-replicating RNA (replicon) vaccines. Replicon-encoded trimers in both forms exhibited proper folding, and replicon-expressed secreted trimers exhibited glycosylation patterns largely consistent with recombinant trimer protein, though with enrichment of complex glycans over high mannose at some sites. Both formats were highly immunogenic in mice, eliciting comparable serum antibody and T cell responses. Interestingly, the TM format initiated smaller germinal center (GC) responses, but these GCs were enriched for trimer-binding B cells compared to secreted trimer vaccines. In a B cell receptor knock-in adoptive transfer model for assessing germline targeting, replicon-encoded TM trimer elicited a greater frequency of epitope-targeting antibodies and recruited broadly neutralizing antibody precursor B cells to the GC response more efficiently compared to replicon-encoded secreted trimer or protein trimer combined with adjuvant. These results indicate that the form of immunogen expression can impact key elements of immune responses to RNA vaccines.

mRNA vaccines have emerged as an important platform for vaccine development. Unlike protein subunit vaccines, mRNA-expressed antigens can be expressed in either secreted or transmembrane (TM) forms mimicking a viral envelope (Env) protein. Here, we investigated the impact of antigen expression format on the antigenicity profile, glycosylation, and immunogenicity of stabilized HIV Env trimer immunogens expressed from self-replicating RNA (replicon) vaccines. Replicon-encoded trimers in both forms exhibited proper folding, and replicon-expressed secreted trimers exhibited glycosylation patterns largely consistent with recombinant trimer protein, although with enrichment of complex glycans over high mannose at some sites. Both formats were highly immunogenic in mice, eliciting comparable serum antibody and T cell responses. Interestingly, the TM format initiated smaller germinal center (GC) responses, but these GCs were enriched for trimer-binding B cells compared to secreted trimer vaccines. In a B cell receptor knockin adoptive transfer model for assessing germline targeting, the replicon-encoded TM trimer elicited a greater frequency of epitope-targeting antibodies and recruited broadly neutralizing antibody precursor B cells to the GC response more efficiently compared to the replicon-encoded secreted trimer or protein trimer combined with adjuvant. These results indicate that the form of immunogen expression can impact key elements of immune responses to RNA vaccines.

Current vaccine strategies to elicit broadly neutralizing antibodies (bnAbs) against HIV-1 generally propose complex, multi-boost immunization regimens. In rhesus macaques, SHIV infection has been observed to rapidly drive the development of some classes of bnAbs that share structural similarities with those in humans. Here, we generated a knockin mouse model with B cells bearing the unmutated common ancestor (UCA) of the V2 apex-targeted bnAb lineage, V033-a. A single immunization of mice with a germline-targeting native-like trimer was sufficient to recapitulate the ontogeny of the mature rhesus bnAb in knockin mice-including rare, disfavored somatic mutations-leading to the induction of antibodies that exhibited potent neutralization against both autologous and heterologous tier 2 viruses. A boost with Env escape mutant trimers further improved breadth and potency, and cryo-EM structure revealed the structural basis for heterologous neutralization breadth. Non-human primate and mouse models can thus combine with structure to serve as a platform for identifying and confirming immunogens that streamline HIV-vaccination regimens.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.

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.

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.