Cellular & Molecular Immunology

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

Individuals with diabetes are at increased risk for severe outcomes from vaccine-preventable infections and often mount weaker immune responses to vaccination. The factors underlying this impaired immunity remain unclear, and defining them is critical to improve vaccine strategies for this vulnerable population. Here, we focused on insulin deficiency as a contributing factor. Following immunization with an alum-adjuvanted protein subunit vaccine, insulin-deficient mice exhibited reduced antigen-specific IgG antibody responses, decreased lymphocyte numbers, and lower germinal center B-cell counts within the vaccine-draining lymph node. Three-dimensional whole-organ light sheet microscopy combined with virtual reality-assisted analysis revealed significantly smaller germinal center volumes in insulin-deficient mice than controls. These findings indicate that insulin deficiency can significantly constrain germinal center responses and impair antibody production from vaccination. Our results provide foundational evidence that diabetes-associated metabolic changes can significantly and negatively influence the quality of vaccine-induced immunity and highlight insulin deficiency as a potential physiological factor.

Cyclooxygenase-1 (COX-1) is classically regarded as a constitutive enzyme that produces eicosanoids with housekeeping functions, but recent evidence indicates that it may also be involved in the acute phase of severe systemic inflammation. There is evidence indicating that COX-1 is selectively activated in the spleen via post-translational mechanisms early the course of LPS-induced systemic inflammation. However, the mechanistic link between COX-1 and the spleen has not yet been demonstrated in direct experiments. The present study was conducted to fill this gap. The effects of the COX-1 inhibitor SC-560 on the LPS-induced severity triad (hypotension, hypothermia and acidosis) were evaluated in rats subjected to splenectomy or in sham-operated controls. In the sham-operated group, SC-560 significantly attenuated the severity triad independently of changes in plasma cytokines (TNF and IL-1{beta}). In the splenectomized rats, SC-560 completely lost its ability to attenuate the hypotension and the acidosis induced by LPS. The effect of SC-560 on LPS-induced hypothermia was also impaired by splenectomy, though not completely. We then conducted a lipidomic screening to identify which COX-1-derived eicosanoids might be responsible for mediating the severity triad. Based on spleen-blood correlations, the screening identified PGE2 and PGD2 as putative candidates. In conclusion, the present study provides direct evidence for a mechanistic link between the spleen and COX-1 in the mediation of severity in systemic inflammation, and identifies PGE2 and PGD2 as putative candidates involved.

RNA-binding protein LARP4 plays an important role in T cell activation and differentiation, but its role in B cell biology and the pathogenesis of systemic lupus erythematosus (SLE) remains unclear. This study found that LARP4 was specifically highly expressed in B cells of SLE patients and was positively correlated with disease activity. By constructing T cell-specific and B cell-specific conditional knockout mice, we found that deletion of LARP4 in B cells, but not in T cells, significantly alleviated pristane-induced and Bm12-induced lupus nephritis. Further analysis showed that LARP4 deletion selectively inhibited B cell differentiation into plasma cells, but did not affect germinal center B cell formation. Integrated transcriptomic and metabolomics analyses revealed that this effect is due to reduced phosphatidic acid synthesis and decreased mTORC1 activity caused by mitochondrial oxidative phosphorylation dysfunction. Furthermore, we used LIPEP, a LARP4 inhibitory peptide that effectively mimicked the therapeutic effects of LARP4 gene knockout in the MRL/lpr spontaneous lupus model and outperformed cyclophosphamide in reducing glomerular immune complex deposition and improving extrarenal dermatitis. These results indicates that LARP4 is a key metabolic checkpoint regulating B cell differentiation into Plasma cells and suggest that it may be a potential therapeutic target for SLE.

Dimeric immunoglobulin A (dIgA) is generated from IgA monomers (mIgA) via JCHAIN-dependent polymerization. DIgA is transported across epithelial barriers by the poly Ig receptor (PIGR) and confers mucosal protection, while serum contains substantial amounts of IgA monomers. Distinct plasma cell subsets have been proposed to produce either monomeric or dimeric IgA, with bone marrow plasma cells as a primary source of mIgA. Here, we addressed whether IgA plasma cell populations segregate based on mIgA or dIgA production. Flow cytometric analysis of antibody-secreting cells from bone marrow, lymphoid and mucosal tissues revealed universal intracellular JCHAIN expression across isotypes and failed to identify a discrete JCHAIN-negative IgA plasma cell population. To detect polymeric IgA, we generated a recombinant soluble PIGR that selectively bound JCHAIN-containing dIgA in Western blot, ELISA, and flow cytometry. Soluble PIGR binding was detected in all IgA plasma cells irrespective of tissue origin, arguing against a dedicated mIgA-producing plasma cell subset incapable of dIgA formation. Ex vivo cultures and single-cell DropMap secretion assays demonstrated that bone marrow and lamina propria IgA antibody-secreting cells co-secrete mIgA and dIgA. These findings suggest that dIgA assembly and secretion are general properties of IgA plasma cells and disfavor a dedicated mIgA-producing population.

Current SARS-CoV-2 vaccines provide limited breadth of protection, underscoring the need for vaccine strategies that optimize immune responses. Virosomesoffer a modular vaccine platform that enables multivalent antigen display and incorporation of adjuvants which can steer immune responses. We evaluated the immune response in BALB/c mice with virosomes displaying SARS-CoV-2 Wuhan or Delta spike antigens and coupled with various distinct adjuvants. Adjuvant selection differentially influenced both humoral and cellular immune outcomes. The TLR7/8 agonist 3M -052 induced a strong Th1-biased response, characterized by elevated IgG2a/IgG1 ratios and robust type 1 cytokine induction with suppression of Th2-associated cytokines. In contrast, the saponin QS-21 enhanced antibody functional quality, illustrated by improved virus neutralization potency and breadth. Furthermore, the combined incorporation of both 3M-052 and QS-21 induced an elevated Th1-biased response without improving neutralization capacity. In conclusion, different adjuvants added onto our virosome-basedvaccine led to distinct antibody responses and splenic T-cell profiles, reflective of differences in immune programming. This information guides the selection of adjuvants for respiratory virus vaccines.

The rapid evolution of avian influenza A/H5N1, including the recent U.S. clade 2.3.4.4b outbreak, highlights its pandemic potential and the urgent need for durable, broadly protective vaccines. Given the capacity of CD8+ T cells to mediate cross-strain immunity, we investigated whether geographically distinct HLA-A33 allotypes, HLA-A*33:01 in East/Southeast Asia and HLA-A*33:03 in South Asia, differentially shape the influenza immunopeptidome and influence antiviral immunity. Antigen-presenting cells overexpressing HLA-A*33:01 or HLA-A*33:03 were transfected with single A/H5N1 antigens or infected with A/X-31 (H3N2) as a control comparison representing current seasonal influenza virus. We identified novel ligands restricted to HLA-A*33:01 (57 from A/H5N1; 55 from A/X-31) and HLA-A*33:03 (29 from A/H5N1; 45 from A/X-31). Although fewer peptides were recovered for HLA-A*33:03, a larger proportion of A/X-31-derived peptides were predicted as high-affinity binders (74%) compared with HLA-A*33:01 (61%), indicating qualitative differences in antigen presentation. To determine immunogenicity, peripheral blood lymphocytes from HLA-A*33:03-positive, A/H5N1-naive donors were stimulated with four conserved peptides: PB2GTF, PB2KTY, NPSVQ and PB1MTK. All elicited robust CD8 T cell activation despite the absence of prior A/H5N1 exposure, demonstrating cross-recognition by memory T cells primed against seasonal influenza. These findings define HLA-A33-restricted influenza epitopes and reveal allotype-specific presentation features that shape CD8+ T cell immunity. Conserved, immunogenic peptides identified here represent promising candidates for rational design of broadly cross-reactive vaccines to protect HLA-A33-expressing populations against severe A/H5N1 disease. Data are available via ProteomeXchange with identifier PXD078870. Author SummaryAvian influenza A/H5N1 continues to pose a significant pandemic threat because of its ability to infect humans and its potential to acquire sustained human-to-human transmissibility. While current influenza vaccines primarily target rapidly evolving viral surface proteins, CD8+ T cells can recognize more conserved internal viral proteins and may provide broader protection against diverse influenza strains. In this study, we investigated how two common HLA-A33 variants, which are prevalent in South, East, and Southeast Asian populations, present influenza-derived peptides to CD8+ T cells. We identified novel influenza peptides presented by HLA-A*33:01 and HLA-A*33:03. Importantly, several conserved A/H5N1-derived peptides were recognized by memory CD8+ T cells from healthy individuals with no prior exposure to A/H5N1, suggesting that previous infection with seasonal influenza viruses can generate cross-reactive immune responses. Our findings expand the current repository of influenza T cell targets and provide new insights into antiviral immunity in HLA-A33-expressing populations. The conserved and immunogenic peptides identified in this study may help guide the development of broadly protective influenza vaccines and contribute to future pandemic preparedness efforts.

Type 1 diabetes (T1D) is caused by T cell-mediated autoimmune destruction of insulin-producing islet beta-cells. Treatment with T-cell depleting therapies delays the progression of stage 2 and 3 T1D, but these agents exert broad immunosuppressive effects on T cell populations, including T regulatory cells (Tregs), which are key in promoting immune tolerance. We evaluated non-obese diabetic (NOD) mice and recently diagnosed T1D patients and identified CD38 as a marker for pathogenic T cell populations. Using adoptive T-cell transfer in Recombination Activating Gene 1 knockout NOD mice and in a humanized mouse model of autoimmune diabetes, we demonstrated that CD38-expressing autoreactive T cells drive diabetes pathogenesis. Furthermore, we found that selective depletion of CD38+ cells, using an anti-CD38 monoclonal antibody (mAb), prevents insulitis and diabetes onset without depleting CD4+CD25+ Tregs. Administration of anti-CD38 mAb did not adversely affect islet function and may selectively eliminate immunogenic senescent islet beta-cells. These results support the strategy of selectively depleting diabetogenic T cells using an anti-CD38 mAb to treat T1D and restore immune tolerance. Therefore, transient depletion of autoreactive T cells using anti-CD38 mAb may provide a novel strategy to prevent or abrogate autoimmunity in T1D.

Abstract/SummaryHuman variants in renal polycystins (PKD1, PKD2) are responsible for most forms of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder without curative drug treatment. Renal polycystins form ion channels in primary cilia, but our understanding of their molecular dysregulation caused by disease-associated variants is limited. Using cryo-electron microscopy (cryo-EM), primary cilia electrophysiology and super-resolution analysis, we investigated the mechanistic impact and pathogenic potential of the disease-associated PKD2 missense variant (D511V) located within the channels voltage sensor domain (VSD). Our findings define how this mutation neutralizes critical transmembrane charge interactions, which attenuates PKD2 protein stability resulting in abolished ciliary channel trafficking and function in membranes. To assess the pathogenic effect of this variant in vivo, we generated novel mouse strains carrying the analogous PKD2 mutation in combination with a conditional floxed allele (Pkd2D509V/fl) that exhibit renal tubule primary cilia degeneration and develop rapid renal cysts. Our results establish a clear direct correlation between the in vitro molecular dysfunction and phenotypic in vivo consequences while providing a valuable tool to evaluate ADPKD therapeutic interventions. Translational StatementADPKD is a genetic kidney disorder affecting millions of patients globally and is primarily caused by variants in renal polycystin genes (PKD1, PKD2). Polycystins function as ion channel subunits in primary cilia but the mechanistic impact and cystogenic propensity of disease-associated variants remain poorly defined. The authors employ advanced methodologies including cryo-EM to uncover distinct structurally destabilizing effects of a human PKD2 mutation, while generating a new mouse model which genetically expresses the same variant and recapitulates the human disease. The findings define primary cilia degeneration results from PKD2 hypostasis and establish new tools to assess ADPKD therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/734313v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1128cb0org.highwire.dtl.DTLVardef@d2aec1org.highwire.dtl.DTLVardef@1cf3083org.highwire.dtl.DTLVardef@179ee83_HPS_FORMAT_FIGEXP M_FIG C_FIG

Summary Background Severe dengue reflects a temporally regulated interaction between viral burden, NS1 antigenaemia, cytokine and chemokine amplification, endothelial activation, glycocalyx injury, and organ stress. Although individual cytokines, endothelial markers, viral-burden measures, and clinical markers have been widely studied, the integrated pathogen-host evidence base remains fragmented. We synthesised evidence for cytokine, endothelial, and viral-burden signatures associated with severe dengue and assessed whether paired pathogen-host measurement provides a biologically coherent framework for severity assessment. Methods We searched MEDLINE, Embase, Scopus, Web of Science, Cochrane Library, Global Health, WHO Global Index Medicus, and medRxiv from database inception to 30 April 2026, without language restriction, for studies reporting viral burden, NS1 antigenaemia, cytokine, chemokine, endothelial, glycocalyx, inflammatory, or routine host-response markers in laboratory-confirmed dengue with severity outcomes. Eligible designs were prognostic-factor association studies, cross-sectional biomarker studies, and multivariable prediction-model studies. Risk of bias was assessed using QUIPS for prognostic-factor studies, PROBAST for prediction-model studies, and the relevant JBI critical appraisal checklist for cross-sectional biomarker studies, with the Newcastle-Ottawa Scale used selectively for cohort or case-control designs not amenable to QUIPS. Random-effects meta-analysis pooled standardised mean differences using restricted maximum likelihood with Hartung-Knapp adjustment. The protocol was registered with PROSPERO (CRD420261396923) before final extraction and synthesis. Findings Of 4,180 records identified, 79 studies including 47,612 participants met eligibility criteria. Forty-nine studies evaluated paired pathogen-host markers, 14 evaluated viral burden or NS1 antigenaemia alone, nine evaluated host biomarkers alone, and seven reported multivariable prediction models. Pathogen-side markers showed modest pooled severity associations whose magnitude depended on day of illness, immune status, and infecting serotype. Cytokine and chemokine markers, particularly IL-10, IL-6, IL-8, and CXCL10/IP-10, showed larger pooled effects favouring severe disease, while endothelial and glycocalyx markers, including angiopoietin-2 and syndecan-1, provided the most direct mechanistic link to plasma leakage. Routine clinical markers, especially platelet count, AST, ferritin, ALT, and lactate, retained substantial discriminatory value. Prediction models reported areas under the curve of up to 0{middle dot}96 in internal validation and 0{middle dot}97 in discovery analyses, but three had been externally validated, calibration was reported in two, and decision-curve analysis in none. Interpretation Current evidence supports severe dengue as an integrated pathogen-host injury syndrome in which viral burden and NS1 antigenaemia interact with cytokine amplification, endothelial dysfunction, glycocalyx injury, and routine markers of organ stress. The strongest translational direction is not a single biomarker but a parsimonious cytokine-endothelial-pathogen panel requiring prospective external validation across age groups, serotypes, immune-status strata, and endemic regions. Existing evidence supports candidate marker prioritisation and mechanistic synthesis, but not immediate routine clinical deployment.

The tick Ixodes ricinus is the main vector of human and animal pathogens in Europe. Despite its importance in epidemiology and medicine, our understanding of physiological mechanisms controlling blood feeding, osmoregulation, or development are still limited. Here, we identify novel neuropeptide invertebrate parathyroid hormone-like peptide (iPTH) and its two receptors - PTHR1 and PTHR2 in I. ricinus. Functional aequorin-based assay confirmed specific activation of both receptors by iPTH. Using RT-qPCR we detected the PTHR1 transcript in the synganglion, while increased expression levels of PTHR2 were found in the salivary glands, hindgut and female gonads. RNA-mediated knockdown of iPTH receptors in nymphs resulted in delayed blood feeding, and a high incidence of defects in adult ecdysis. Consistent with observed phenotypes, iPTH is expressed in multiple neurons of the synganglion which project arborizing axons to the salivary glands, rectal sack and skeletal muscles. iPTH was colocalized with orcokinin-immunoreactivity (OK-IR) in all neurons that innervate these peripheral tissues. iPTH is further colocalized with tachykinin (TK) in Pd1DL1 neurons, suggesting coordinated action with other neuropeptides. Our findings indicate that iPTH signaling is required for normal feeding, development and successful ecdysis.

Tuberculosis (TB) remains a leading global cause of infectious mortality due, in part, to the limited efficacy of the Mycobacterium bovis BCG vaccine against pulmonary TB. Previous studies in mice have shown that stimulating type I interferon (IFN) signaling during BCG vaccination can bolster protection against Mycobacterium tuberculosis, yet clinically feasible delivery strategies for this approach are lacking. Adenoviral vectors, which induce potent type I IFN responses and are utilized in approved vaccine platforms, represent a promising adjuvant strategy. To evaluate the host immune response to this combination, bone marrow-derived murine macrophages were co-infected with replication-deficient adenovirus and BCG. Adenovirus-infected macrophages elicited a robust type I IFN response via the cGAS/STING pathway. Compared to BCG infection alone, co-infected macrophages exhibited additive expression of genes with known host-protective roles against M. tuberculosis. Conversely, co-infection with BCG suppressed adenovirus-induced type I IFN signaling and diminished the production of IFN-stimulated genes compared to adenovirus infection alone. Together, these findings reveal a complex regulatory interplay during adenovirus and BCG co-infection. While BCG partially restricts adenoviral IFN induction, the co-infection still drives an enhanced host-protective gene profile, suggesting that adenoviral vectors could serve as a viable platform to modulate innate immunity and improve BCG vaccine efficacy. IMPORTANCETuberculosis (TB) remains the leading cause of death by a single infectious organism with approximately 1.25 million deaths annually. M. bovis BCG remains the only approved vaccine for TB; however, its efficacy against the contagious and most common pulmonary form of the disease is limited. There have been numerous attempts to improve BCG efficacy, but these approaches have not resulted in any clinically approved vaccine. We propose that BCG combined with a replication-deficient adenovirus presents a way to bolster vaccine-conferred protection as the combination may elicit a robust innate immune response and drive a more protective T cell response. Moreover, BCG and replication-deficient adenoviruses have well-assessed safety profiles and decades of studies regarding their use in patients. The significance of our work is in leveraging their complementary immunology to function as a combined vaccine platform. This approach presents a novel and clinically feasible approach to improve the BCG vaccine.

The development of effective mucosal vaccination strategies against human immunodeficiency virus type 1 (HIV-1) remains a major challenge. This study investigated whether oral administration of hyperimmune anti-HIV-1 gp120 immunoglobulin Y (IgY) could induce mucosal and systemic immune responses in outbred felines through an anti-idiotypic network mechanism. A controlled immunization study involving 42 cats (18 immunized and 24 controls) was conducted to evaluate mucosal anti-gp120 IgA responses. In addition, a proof-of-concept cohort was used to investigate anti-idiotypic antibody (Ab-3) induction, competitive inhibition, and HIV-1 neutralization. Anti-gp120 IgA antibodies were detected in saliva from immunized animals but were absent or present at low levels in controls, indicating activation of mucosal immunity. All immunized cats developed detectable Ab-3 responses against HIV-1 gp120. Competitive inhibition assays demonstrated specific in hibition of gp120-related interactions, supporting the presence of biologically relevant anti-idiotypic antibodies. Furthermore, sera from immunized animals significantly reduced HIV-1 infectivity in a TZM-bl luciferase-based neutralization assay, with viral inhibition exceeding 60% at selected dilutions. Collectively, these findings demonstrate that oral administration of hyperimmune anti-gp120 IgY can induce mucosal IgA responses, systemic anti-idiotypic antibodies, and functional HIV-1 neutralizing activity. This preclinical proof-of-concept study supports further investigation of IgY-based oral immunization as a potential platform for HIV vaccine development. However, the Ab3, competitive inhibition assay using Ab3, and HIV-1 neutralization studies should be regarded as exploratory proof-of-concept investigations designed to establish biological plausibility rather than definitive efficacy.

SIGLECs remain poorly defined in human B-cell biology beyond SIGLEC-2/CD22 and SIGLEC-10. Here, we identify a previously unrecognized regulatory pathway involving the paired receptors SIGLEC-5 and SIGLEC-14 at the human B-T-cell interface. We show that activated B-cells differentially regulate these receptors: SIGLEC-5 is predominantly surface-expressed and induced by CD40 engagement, whereas SIGLEC-14 is primarily secreted and upregulated after both CD40 and TLR9 stimulation. We further identify EBP (elastin binding protein) and CTSA (cathepsin A) components of the elastin receptor complex (ERC), expressed by activated T-cells, as a novel ligand for both SIGLEC-5 and SIGLEC-14. Functionally, ERC-associated engagement of SIGLEC-5 on B-cells suppresses T-cell IFN-g; and IL-17 expression, establishing SIGLEC-5 as a B-cell-expressed inhibitory SIGLEC that restrains inflammatory T-cell cytokine responses. SIGLEC-14 does not alter this suppression, as SIGLEC-5+ B-cells from SIGLEC-14-sufficient and -null individuals show comparable inhibitory activity. These findings broaden SIGLEC-mediated adaptive immune regulation, with relevance to inflammatory and autoimmune disease.

Fc receptor-like protein 5 (FCRL5) is a low-affinity IgG receptor expressed on B cells, with emerging therapeutic relevance due to its expression on multiple myeloma cells, and a potential role in regulating B cell responses. Previous reports on the FCRL5-IgG interaction vary widely in reported affinities, binding differences across IgG subclasses, and molecular requirements for maximal binding. Furthermore, the impact of Fc-engineering strategies, as used in (therapeutic) monoclonal antibodies, remains poorly understood. Here, we provide a comprehensive biochemical analysis of the FCRL5-IgG interaction. We demonstrate that FCRL5 is a true IgG Fc-receptor, binding with very low affinity (60-80 M). FCRL5 binds IgG in a manner involving primarily the two N-terminal domains of FCRL5, and the third domain for maximal binding, but with distinct essential residues in the IgG Fc-tail. Surface plasmon resonance analysis of the binding of FCRL5 to the various IgG subclasses revealed a preference for IgG1 and IgG4. Interestingly, various Fc-engineered IgG variants commonly used for silencing or enhancing of Fc receptor binding do not impact FCRL5 binding. Screening the binding of a set of IgG antibodies carrying defined sets of Fc-mutations to FCRL5 revealed E293 as a key binding determinant and led to the discovery of E293R as a mutation that selectively abrogates FCRL5 binding while preserving binding to other classical Fc{gamma}Rs. Lastly, we show that FCRL5 has considerable preference for binding afucosylated IgG. Together, our results define the essential characteristics of the IgG-FCRL5 interaction and demonstrate the potential of both naturally occurring IgG variants as well as therapeutically explored bioengineered IgG formats to differentially engage FCRL5.

Multiple sclerosis (MS) is a chronic T cell-mediated autoimmune disease characterized by blood-brain barrier (BBB) disruption, neuroinflammation, and demyelination of the central nervous system (CNS). Emerging evidence links gut microbiota to disease pathogenesis, but the microbial factors that regulate pathogenic microRNA (miRNA) programs are largely unknown. Here, using experimental autoimmune encephalomyelitis (EAE, a MS mouse model), we investigated whether gut microbiota exacerbate EAE pathogenesis by modulating host miRNA expression. Antibiotic-induced depletion of the gut microbiota markedly attenuated EAE scores and reduced circulating inflammatory miRNAs, with miR-21 emerging as the dominant pathogenic candidate. Functional inhibition of miR-21 significantly ameliorated disease severity and reduced CNS T-cell infiltration. Mechanistically, miR-21 enhanced IL-17 and GM-CSF production by CD4 T cells and promoted immune-cell entry into the CNS through endothelial activation and blood-brain barrier dysfunction. We identified a transient expansion of Akkermansia muciniphila during the prodromal phase of EAE that positively correlated with circulating miR-21 levels. Colonization of antibiotic-treated mice with A. muciniphila exacerbated EAE and increased serum miR-21, whereas monocolonization of germ-free mice was insufficient to induce systemic miR-21, indicating a requirement for an inflammatory host environment. Further analyses revealed that atypical lipopolysaccharides (LPS) derived from A. muciniphila induce epithelial miR-21 production through coordinated TLR2/TLR4 signaling. Circulating miR-21 subsequently promoted endothelial dysfunction through the TIMP3-ADAM17 pathway, facilitating pathogenic T-cell migration into the CNS. Importantly, circulating miR-21 was also elevated in patients with MS. Collectively, these findings identify a previously unrecognized A. muciniphila-LPS-miR-21 axis linking gut dysbiosis to neuroinflammation and suggest that host-derived miRNAs function as systemic mediators through which microbial signals influence CNS autoimmunity.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

The IgG Fc chain carries a single N-linked glycan which may undergo changes. Increased agalactosylated N-glycans are associated with rheumatoid arthritis (RA) and regarded as pro-inflammatory. Dysregulated neutrophils can make important contributions to host tissue damage. In RA, immune complexes (ICs) that have precipitated onto synovial joint surfaces activate neutrophils via Fc receptors, promoting localised inflammation. We engineered recombinant human monoclonal IgG with agalactosylated or galactosylated N-glycans, generated immobilised ICs and stimulated healthy donor and RA patient blood-derived neutrophils, comparing reactive oxygen species (ROS) production as read-out of neutrophilic inflammation. Both healthy donor and RA patient neutrophils generated less ROS when stimulated with ICs made from agalactosylated IgG. Mechanistically this was due to poorer binding of agalactosylated ICs to neutrophil FcgammaRs, causing lower activation of Akt and p38 MAPK. Both are required for immobilised IC-mediated stimulation of the neutrophil NADPH oxidase. Taken together, this suggests that disease-associated, agalactosylated IgG does not in fact promote inflammation and host tissue injury, at least not by acting on neutrophils. We propose that rather than promoting inflammation, agalactosylated IgG N-glycans that accompany inflammatory disease may arise as part of a compensatory mechanism that is aimed at reducing excessive inflammation and host tissue injury.

BackgroundPeople with cystic fibrosis (pwCF) are at increased risk of severe disease following respiratory viral infections including influenza and respiratory syncytial virus, and were therefore given priority for COVID-19 vaccination. Retrospective epidemiological data have revealed a lower incidence of SARS-CoV-2 infection and a reduced fatality rate among pwCF than in the general population, possibly from the stringent infection control measures and early adoption of protective behaviors. Despite several reports of adequate binding and neutralizing titers after vaccination, the molecular features of vaccine-elicited serum antibody repertoires in pwCF remain unknown. MethodsWe performed high-resolution proteomic analysis of serum IgG, combined with next-generation sequencing of B cells, to quantitatively profile Spike (S)-reactive serological repertoires from nine infection-naive pwCF after two doses of COVID-19 mRNA vaccine. We recombinantly expressed 20 IgG clonotypes from 6 pwCF as monoclonal antibodies (mAbs) and measured their binding and neutralization against vaccine-strain and variant viruses. ResultsAll donors mounted strong binding and neutralizing titers to vaccine-strain virus. Ig-Seq revealed diverse serum repertoires in all vaccinees, with antibodies targeting the receptor-binding domain (RBD) comprising 64% of each circulating repertoire by abundance. Several serum IgG clonotypes identified across our cohort shared identical or similar IGHV and CDRH3 amino acid sequences with serum IgG from other pwCF or S-reactive mAbs isolated from non-CF individuals. These convergent antibodies made up 14.6% of the anti-S repertoires in our cohort, reaching 24.8% in one pwCF. Convergent antibodies tended to be RBD-reactive and enriched in specific IGHV; surprisingly, a higher abundance of convergent antibodies in serum correlated with a lower breadth of serum neutralization. All 20 mAbs bound Wuhan S with high affinity (EC50 < 10 nM), and only RBD-reactive mAbs conferred neutralization to vaccine or variant pseudovirus. While most mAbs bound both the B.1.617.2 (Delta, 17/20) and B.1.1.529 (Omicron, 12/20) strains, convergent mAbs were less likely to bind either variant. ConclusionsPost-vaccine serum IgG repertoires in pwCF are dominated by RBD-focused, high-affinity antibodies and include a substantial convergent component shared with non-CF vaccinees. These findings demonstrate that pwCF mount antibody responses comparable to the general population, and a large group of convergent antibodies may contribute to strain-specific rather than cross-variant immunity.

Vaccination remains the most successful preventative measure against viral infection, but methods to stably deter rapidly-evolving pathogens have remained elusive. Vaccines capable of incorporating and anticipating viral evolution could address current challenges in seasonal vaccination efforts against SARS-CoV-2 and influenza where economic and disease burdens remain high despite decades of combined study. Rare epitope suppression (RES) is an underutilized concept within vaccine design, where humoral epitope targeting can be molded using complex antigen pools. Based in mRNA vaccine technology, 'wobble vaccines' represent the novel application of RES to human pathogens designed to anticipate and resist viral evolution. To establish this platform, public SARS-CoV-2 sequencing data was compiled from the first two years of the COVID-19 pandemic to identify high-diversity sites across the receptor binding domain (RBD) of the spike protein. Wobble RBD (WobbRBD) libraries reflecting that entropy were synthesized and incorporated into established self-amplifying (SA) vaccine constructs. Animals immunized with these complex antigen pools showed no obvious adverse effects. By three days-post vaccination, WobbRBD stimulated robust primary immune activation with distinctive characteristics compared to traditional single-strain vaccine modalities. By day 14, germinal centers, class switching, and antibody-secreting cells were induced, creating potent SARS-CoV-2 spike-binding IgG antibodies. Despite similar overall activation profiles, WobbRBD generated significantly increased breadth against SARS-CoV-2 variant spikes in comparison to single-strain controls -- even against future-emerging strains. Taken together, wobble vaccines represent a novel method for anticipating and preventing viral escape with promising applications in SARS-CoV-2, influenza, HIV, and beyond.