B cells mature into antibody-secreting cells by antigen processing and presentation and undergo productive germinal center (GC) reactions in secondary lymphoid organs. In solid tumors, antigen presentation and local anti-tumor humoral immunity can arise through the formation of GC-like tertiary lymphoid structures (TLS). Here, we show that cathepsin S activity is essential to form mature GC and TLS by regulating the communication of B cells with CD4+ Tfh cells and follicular dendritic cells. The absence of cathepsin S activity impairs B cells' affinity maturation and high-affinity antibody production, and it limits the formation of TLS in lung and colorectal cancers, accelerating tumor progression. Conversely, the expression of an overactive form of cathepsin S, CTSSY132D, which was identified in follicular lymphoma patients, induces an accumulation of proliferating GC B cells, predisposing to lymphoma development. Overall, cathepsin S plays a nonredundant role in B cells, where maintaining its balanced activity is crucial to prevent lymphomagenesis and develop effective humoral immune responses.

Germinal centers (GCs) are a complex and important aspect of humoral immunity. How GCs deal with changing antigens remains unclear, yet this biology could be central to next-generation vaccine strategies such as germline targeting. Here we demonstrate, in a mouse model with human immunodeficiency virus envelope surface protein immunogens, that rapid delivery of homologous or heterologous boosts results in highly positive outcomes. Rapid reimmunization expands on-target GC B cell (BGC) populations, which emerge almost exclusively from existing BGC cells. Early homologous boosting avoids prohibitive antibody titers and utilizes off-target antibodies to maximize the BGC response. Heterologous rapid boosting shifts affinity maturation towards the new antigen. The 'refueled' GCs are sustained, developing large affinity gains and evolving rapidly to bind wildtype HIV Env trimer within 56 days, even when using as few as two distinct antigens. These findings provide insights into GC biology and translatable paths to leveraging accelerated GC function.

The high antigenic diversity of HIV has been a major obstacle to development of a broadly protective vaccine. Nevertheless, protective HIV broadly neutralizing antibodies (bnAbs) exist and have been proposed as templates for vaccine development1-6. Germline-targeting is a conceptually radical vaccine design approach to elicit bnAbs, aiming to prime rare bnAb-precursor B cells possessing pre-determined human genetic and structural features shared with template bnAbs, and then guide B cell affinity maturation to potent bnAb evolution with heterologous boosters7-11. Although the approach has shown promise in clinical12-17 and pre-clinical18-34 studies, it faces many immunological challenges and, to date, has not succeeded in generating bnAbs in humans or nontransgenic animals. Here, we report an adjuvanted protein germline-targeting vaccine tested in outbred nonhuman primates that generated bnAb-class memory B cells and sera capable of neutralizing diverse HIV clinical isolates. bnAb lineages were generated in ≥50% of animals, achieving up to 67% neutralization breadth compared to the reference bnAb. Vaccine-induced bnAbs exhibited precise structural mimicry of human bnAb interactions with HIV envelope (Env), matching the germline-targeting predictions. Furthermore, serum bnAb activity developed in 44% of animals and in the most striking instance reached titers expected to confer protection against diverse HIV isolates. These results demonstrate proof of principle that germline-targeting vaccines can reproducibly elicit prespecified classes of bnAbs to prespecified epitopes under endogenous conditions, supporting further optimization of this approach for HIV vaccine development.

Multiple broadly neutralizing antibody lineages can co-exist and mature in the same germinal centers.

Precise, reversible control of gene expression from self‑amplifying RNA (saRNA) remains difficult, limiting the therapeutic flexibility of this otherwise potent platform. Although alphavirus‑derived saRNAs encode non‑structural proteins that drive RNA replication and offer an intrinsic regulatory point, no existing approach has enabled direct, drug‑dependent control of this machinery for high‑fidelity modulation of expression. Here we engineer saRNA constructs whose replication is activated by the approved small‑molecule drug trimethoprim, using drug‑responsive degradation domains fused to individual non‑structural proteins to regulate self‑amplification. As each replication protein contributes differently to RNA copying, we systematically screened fusion configurations and identified an optimal design combining modified replication proteins with a regulated payload. This construct achieved more than a 104‑fold difference between on and off states with negligible background expression. In mice, oral trimethoprim enabled tunable, reversible and temporally programmed expression patterns. When encoding a human immunodeficiency virus antigen, an escalating trimethoprim regimen enhanced germinal centre responses, a key determinant of antibody affinity maturation. This drug‑regulated saRNA platform provides a controllable and clinically compatible strategy for vaccines, immunotherapies and gene therapies.

Autoimmune diseases are characterized by the immune system's breakdown of self-tolerance, and manifest as either organ-specific or systemic conditions. Within the target tissues of diverse autoimmune diseases, tertiary lymphoid structures (TLSs) emerge under persistent inflammatory conditions. These pathological, ectopic lymphoid formations serve as sites for sustained antigen presentation, affinity maturation of antibodies, and proliferation /differentiation of B cells, thereby exacerbating local immune-mediated damage. Given their functional significance, TLSs represent promising candidates as diagnostic biomarkers and novel therapeutic targets for autoimmune pathologies. This review synthesizes the current knowledge on the pathological relevance of TLSs, the mechanisms driving their formation and the therapeutic targeting potential, aiming to deepen the understanding of how TLSs influence the immune microenvironment in autoimmune disease pathology.

A cholinergic checkpoint controls autoantibody selection via somatic hypermutation in lupus germinal centers.

Background and ObjectivesAntineuronal autoantibodies targeting surface membrane proteins are the hallmark of an increasing number of autoimmune encephalitides. The autoantibodies can be directly pathogenic and cause various symptoms ranging from epilepsy and psychosis to amnesia and autonomic dysfunction. It is largely unclear how the humoral autoimmune response is triggered and propagated.MethodsWe analyzed whether affinity maturation leads to increasing affinity of encephalitis-related autoantibodies to their receptors by determining the binding strength of patient-derived monoclonal autoantibodies and their germline ancestors from 11 patients with NMDAR, LGI1, mGluR5, CASPR2, and GABAAR encephalitis. In addition, binding to foreign and self-antigens was assessed.ResultsFor most autoantibodies, affinity maturation generated or increased binding to neuronal surface antigens. However, high numbers of somatic hypermutations were not necessarily needed, as half of germline-encoded variants already recognized the respective antigen with strong binders in all groups. 10%–15% of reverted and mutated monoclonal autoantibodies recognized nuclear antigens, and one-third were reactive to mammalian tissues other than brain. Immunoprecipitation combined with mass spectrometry identified the nuclear antigen proteins major vault protein and translocated promotor region as targets of selected germline ancestors.DiscussionThe findings suggest analogous immunologic mechanisms across 5 different encephalitides, with high-affinity autoantibodies already in the germline pool and frequent reactivity to nuclear antigens. Future studies should determine the contribution of immunologic checkpoint deficiencies, as seen in one patient with a pathogenic immune checkpoint mutation.

Phased variability in cell fate determination orchestrates antibody evolution.

Norovirus (NoV) is a leading cause of acute gastroenteritis. We expressed GI.1 and GII.4 VP1 proteins and generated genotype-specific monoclonal antibodies (1C1 for GI.1; 7B5 and 10B8 for GII.4). Variable heavy chains and variable light chains were obtained, and full-length heavy/light chain vectors were constructed. Recombinant antibodies (rAbs) expressed in Expi293F and ExpiCHO cells showed an affinity comparable to that of ascite-derived antibodies. Using these rAbs, we developed a dual-antibody sandwich ELISA (DAS-ELISA) and a gold immunochromatographic strip (GICS). In oyster samples, DAS-ELISA limits of detection (LOD) were 3.23 ng/g (GI.1) and 2.16 ng/g (GII.4); visual limits of detection of GICS were 10 ng/g and 5 ng/g, respectively. Validation with 60 clinical samples showed good concordance with PCR, confirming the field-screening suitability. Molecular docking and molecular dynamics simulations identified 12 key interacting residues at the antibody-VP1 interface. Site-directed mutagenesis confirmed that mutating these CDR-localized residues reduced binding affinity by 60-90%, whereas mutation of a framework residue (GLN1) had only a minor effect. These findings provide structural insights into future antibody humanization or affinity maturation.

Acute respiratory distress syndrome (ARDS) is a devastating complication of respiratory infections; however, the biological mechanisms that initiate its onset are poorly defined. Here we show that TNFRSF13B polymorphisms increase the risk of ARDS following SARS-CoV-2 infection up to 7.4-fold compared to the WT genotype. The increased risk was not due to immune-deficiency or impaired virus neutralization. On the contrary, TNFRSF13B mutant subjects mounted better antibody neutralization compared to subjects with WT TNFRSF13B. However, IgG from subjects expressing TNFRSF13B variants had less sialic acid, terminal galactose, and fucose than IgG from subjects with a WT genotype. Moreover, IgG from TNFRSF13B mutant subjects exhibited increased recruitment of complement factors. Thus, besides well-known actions governing plasma cell differentiation, TNFRSF13B impacts both affinity maturation and effector functions of IgG in ways that independently govern complement activation controlling inflammatory responses known to trigger ARDS.

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway.

Activation-induced cytidine deaminase (AID) is essential for B cell affinity maturation. We investigated why AID deficiency gives rise to giant germinal centers (GCs) using the AIDR112H mouse model that is devoid of AID activity. The increased GC response was associated with accumulation of GC B cells in the light zone in immunized AIDR112H mice. AIDR112H GC B cells had reduced capacity to upregulate IRF4 to initiate plasma cell differentiation, leading to accumulation of a transitional GC population with reduced GL7 expression. Genetic introduction of a high-affinity B cell receptor was unable to restore plasma cell differentiation of AIDR112H B cells, while ectopic expression of catalytically active AID rescued plasma cell generation. AID and ten-eleven translocation 2 (TET2) synergistically facilitated demethylation of the Irf4 promoter/enhancer, and this was impeded in AIDR112H cells. These data reveal a B cell-intrinsic mechanism that governs the plasma cell fate decision through epigenetic remodeling mediated by AID in cooperation with TET2.

Beyond antibody titers: Corticosteroid effects on IgG avidity dynamics in experimental strongyloidiasis.

Replaying germinal center evolution on a quantified affinity landscape.

The role of antibodies in the host response against Mycobacterium tuberculosis (M. tb) bacteria is still poorly understood. We previously isolated two monoclonal antibodies (mAbs), p4-36 and p4-163, from an M. tb infected donor that target two non-overlapping epitopes on PstS1, a subunit of the M. tb phosphate transporter. Although these antibodies reduced lung bacterial burden in mice (30–40% reduction in CFU), their efficacy remained modest for therapeutic application. Here, we employed a rational antibody engineering approach to further enhance their anti-M. tb potency. Affinity maturation of p4-163 yielded p4-163LR, a variant with superior binding to PstS1 and improved recognition of live, attenuated M. tb. Surprisingly, p4-163LR alone did not confer enhanced protection against virulent M. tb in vivo. However, the generation of a bispecific antibody combining p4-36 and p4-163LR (Bi-S 36/163LR) significantly improved bacterial binding and antibody-dependent cellular phagocytosis (ADCP). Notably, prophylactic administration of Bi-S 36/163LR led to a ~ 1 log reduction in lung bacterial burden compared to control animals treated with isotype control. These findings define a novel, structure-guided strategy to amplify the functional capacity of natural anti-M. tb antibodies and highlight bispecific antibody platforms as promising candidates for host-directed tuberculosis immunotherapy.

Common deep learning approaches for antibody engineering focus on modeling the marginal distribution of sequences. By treating sequences as independent samples, however, these methods overlook affinity maturation as a rich and largely untapped source of information about the evolutionary process by which antibodies explore the underlying fitness landscape. In contrast, classical phylogenetic models explicitly represent evolutionary dynamics but lack the expressivity to capture complex epistatic interactions. We bridge this gap with CoSiNE, a continuous-time Markov chain parameterized by a deep neural network. Mathematically, we prove that CoSiNE provides a first-order approximation to the intractable sequential point mutation process, capturing epistatic effects with an error bound that is quadratic in branch length. Empirically, CoSiNE outperforms stateof-the-art language models in zero-shot variant effect prediction by explicitly disentangling selection from context-dependent somatic hypermutation. Finally, we introduce Guided Gillespie, a classifier-guided sampling scheme that steers CoSiNE at inference time, enabling efficient optimization of antibody binding affinity toward specific antigens.

Entrenchment — epistasis that locks in amino acid differences between homologous proteins, so each disfavors substitutions toward the other’s state — has been demonstrated along individual protein lineages over deep evolutionary time. Antibodies offer a unique system for studying entrenchment: multiple homologous germline V gene paralogs provide diverse starting points, and the rapid somatic evolution of affinity maturation generates dense phylogenies from which selection on germline-encoded residues can be inferred. Using DASM, a deep-learning model that separates selection from mutation in antibody repertoire data, we test for entrenchment across immunoglobulin heavy chain variable (IGHV) genes. We detect entrenchment at two levels of germline divergence, driven by different sources of epistasis. Within V gene families (up to ~20% amino acid divergence), entrenched sites cluster at the borders of the complementarity-determining regions (CDRs, the antigen-binding loops) and show high germline diversity. These sites contact antigen, light chain, and the heavy chain CDR3 loop, all of which are encoded independently of the IGHV germline. This pattern is consistent with epistasis from genetically uncoupled partners. Between V gene families, at deeper levels of divergence (25–40%), entrenchment additionally includes positions in the framework scaffold distant from binding interfaces, suggesting a larger contribution from intra-heavy-chain structural constraints. Observed mutation frequencies in human repertoires corroborate these predictions where data are sufficient. Together, these results demonstrate that the rapid somatic evolution of antibodies can serve as a lens for revealing epistatic constraints acting on germline-encoded residues, including constraints imposed by genetically uncoupled partners assembled during B cell development.

In the field of vaccines, adjuvants have been optimized to enhance immune responses while reducing adverse effects. Transferring these advances to antivenom production could improve antibody quality and animal welfare. Here, we evaluated a novel adjuvant platform, CpG-ODN/Coa-ASC16, which combines immunostimulatory CpG-ODN oligodeoxynucleotides with the biodegradable nanostructure Coa-ASC16, co-formulated with a hemorrhagic antigenic model based on Bothrops diporus venom (B.dV). Immunization with B.dV/CpG-ODN/Coa-ASC16 elicited IgG titers equivalent to those induced by Freund's adjuvant, with significantly higher IgG1 levels. Antibodies generated with both formulations exhibited progressively higher avidity, reflecting effective affinity maturation. Sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice recognized major venom proteins and neutralized key toxic activities-proteolytic, coagulant, and indirect hemolytic-at levels comparable to Freund's-induced sera. Functionally, sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice conferred protection against a lethal venom challenge, resulting in survival of a fraction of animals and prolonged time to death in non-survivors. Importantly, histopathological analyses revealed minimal tissue alterations in the mice treated with CpG-ODN/Coa-ASC16, in sharp contrast to the severe abscesses and granulomas caused by Freund's adjuvant. Overall, this study provides new evidence that CpG-ODN/Coa-ASC16 can be effectively combined with hemorrhagic Bothrops venom to generate robust, high-affinity antibodies that efficiently neutralize the major venom toxins while markedly reducing local tissue damage. These findings position CpG-ODN/Coa-ASC16 as a safer and ethical alternative platform for antivenom production.

The germinal center (GC) reaction requires tight regulation of B cell and T follicular helper (Tfh) cell interactions to ensure B cell expansion and antibody affinity maturation, while preventing oncogenesis. However, regulatory mechanisms fine-tuning B-T cell interactions within the GC to prevent aberrant activation and proliferation remain incompletely understood. Here, we identify Siglec-G, the mouse ortholog of human Siglec-10, as an immune checkpoint that restrains the GC by dampening B-T cell interactions. Selective and temporal ablation of Siglec-G on B cells after immunization triggers GC hyperplasia and enhanced plasma cell and antibody output. While Siglec-G is dispensable in B cell receptor (BCR)-mediated processes, it acts as an intrinsic inhibitory receptor of B-T cell interactions in the GC, ultimately limiting Myc and mTORC activation within positively selected GC B cells. Trans interactions of Siglec-G and its glycan ligands on Tfh likely contribute in fine-tuning the strength of bidirectional signaling following contact between GC B cells and Tfh cells. This interaction is further reinforced by glycan remodeling that occurs in the GC, resulting in concurrent decreased in glycan ligands on GC B cells and increased in glycan ligands on Tfh. This augmented binding of Siglec-G/10 on Tfh is mainly due to the upregulation of α2-6 linked sialic acid ligands. Moreover, APEX2-based proximity labeling revealed several candidate Siglec-G/10 binding partners on T cells, including BTLA, CD6, and Slamf6, which are known negative regulators of Tfh cell activation. Taken together, our findings identified that Siglec-G acts as a GC checkpoint receptor, restricting B cell proliferation by tuning T cell help following B-T cell interactions.