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Abstract Introduction Diffuse midline gliomas (DMG) are universally lethal central nervous system tumors that carry almost unanimously the clonal driver mutation histone-3 K27M (H3K27M). The single amino acid substitution of lysine to methionine harbors a neoantigen that is presented in tumor tissue. The long peptide vaccine H3K27M-vac targeting this major histocompatibility complex class II (MHC class II)-restricted neoantigen induces mutation-specific immune responses that suppress the growth of H3K27M+ flank tumors in an MHC-humanized rodent model. Methods INTERCEPT H3 is a non-controlled open label, single arm, multicenter national phase 1 trial to assess safety, tolerability and immunogenicity of H3K27M-vac in combination with standard radiotherapy and the immune checkpoint inhibitor atezolizumab (ATE). 15 adult patients with newly diagnosed K27M-mutant histone-3.1 (H3.1K27M) or histone-3.3 (H3.3K27M) DMG will be enrolled in this trial. The 27mer peptide vaccine H3K27M-vac will be administered concomitantly to standard radiotherapy (RT) followed by combinatorial treatment with the programmed death‐ligand 1 (PD-L1) targeting antibody ATE. The first three vaccines will be administered bi-weekly (q2w) followed by a dose at the beginning of recovery after RT and six-weekly administrations of doses 5 to 11 thereafter. In a safety lead-in, the first three patients (pts. 1–3) will be enrolled sequentially. Perspective H3K27M-vac is a neoepitope targeting long peptide vaccine derived from the clonal driver mutation H3K27M in DMG. The INTERCEPT H3 trial aims at demonstrating (1) safety and (2) immunogenicity of repeated fixed dose vaccinations of H3K27M-vac administered with RT and ATE in adult patients with newly diagnosed H3K27M-mutant DMG. Trial registration NCT04808245.

PurposeTisagenlecleucel is a CD19-specific chimeric antigen receptor T-cell therapy, US Food and Drug Administration-approved for children, adolescents, and young adults (CAYA) with relapsed and/or refractory (RR) B-cell acute lymphoblastic leukemia (B-ALL). The US Food and Drug Administration registration for tisagenlecleucel was based on a complete response (CR) rate of 81%, 12-month overall survival (OS) of 76%, and event-free survival (EFS) of 50%. We report clinical outcomes and analyze covariates of outcomes after commercial tisagenlecleucel.MethodsWe conducted a retrospective, multi-institutional study of CAYA with RR B-ALL across 15 US institutions, who underwent leukapheresis shipment to Novartis for commercial tisagenlecleucel. A total of 200 patients were included in an intent-to-treat response analysis, and 185 infused patients were analyzed for survival and toxicity.ResultsIntent-to-treat analysis demonstrates a 79% morphologic CR rate (95% CI, 72 to 84). The infused cohort had an 85% CR (95% CI, 79 to 89) and 12-month OS of 72% and EFS of 50%, with 335 days of median follow-up. Notably, 48% of patients had low-disease burden (< 5% bone marrow lymphoblasts, no CNS3, or other extramedullary disease), or undetectable disease, pretisagenlecleucel. Univariate and multivariate analyses associate high-disease burden (HB, ≥ 5% bone marrow lymphoblasts, CNS3, or non-CNS extramedullary) with inferior outcomes, with a 12-month OS of 58% and EFS of 31% compared with low-disease burden (OS; 85%, EFS; 70%) and undetectable disease (OS; 95%, EFS; 72%; P < .0001 for OS and EFS). Grade ≥ 3 cytokine release syndrome and neurotoxicity rates were 21% and 7% overall and 35% and 9% in patients with HB, respectively.ConclusionCommercial tisagenlecleucel in CAYA RR B-ALL demonstrates efficacy and tolerability. This first analysis of commercial tisagenlecleucel stratified by disease burden identifies HB preinfusion to associate with inferior OS and EFS and increased toxicity.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRβ-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRβ-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous β-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

The T cell receptor (TCR) endows T cells with antigen specificity and is central to nearly all aspects of T cell function. Each naïve T cell has a unique TCR sequence that is stably maintained during cell division. In this way, the TCR serves as a molecular barcode that tracks processes such as migration, differentiation, and proliferation of T cells. Recent technological advances have enabled sequencing of the TCR from single cells alongside deep molecular phenotypes on an unprecedented scale. In this review, we discuss strengths and limitations of TCR sequences as molecular barcodes and their application to study immune responses following Programmed Death-1 (PD-1) blockade in cancer. Additionally, we consider applications of TCR data beyond use as a barcode.

γδ T cells are important immunoregulatory cells in experimental autoimmune uveitis (EAU), and the activation status of γδ T cells determines their disease-enhancing or inhibitory effects. Because γδ T cells can be activated via various pathways, we questioned whether the nature of their activation might impact their function. In this study, we show that γδ T cells activated under different inflammatory conditions differ greatly in their functions. Whereas anti-CD3 treatment activated both IFN-γ+ and IL-17+ γδ T cells, cytokines preferentially activated IL-17+ γδ T cells. γδ T cells continued to express high levels of surface CD73 after exposure to inflammatory cytokines, but they downregulated surface CD73 after exposure to dendritic cells. Although both CD73high and CD73low cells have a disease-enhancing effect, the CD73low γδ T cells are less inhibitory. We also show that polarized activation not only applies to αβ T cells and myeloid cells, but also to γδ T cells. After activation under Th17-polarizing conditions, γδ T cells predominantly expressed IL-17 (gdT17), but after activation under Th1 polarizing conditions (gdT1) they mainly expressed IFN-γ. The pro-Th17 activity of γδ T cells was associated with gdT17, but not gdT1. Our results demonstrate that the functional activity of γδ T cells is strikingly modulated by their activation level, as well as the pathway through which they were activated.

BackgroundDevelopment of a diverse T-cell receptor β (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution.ObjectivesWe investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID.MethodsWe used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements.ResultsTRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention.ConclusionsTRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

BACKGROUND: Covalent (irreversible) Brutons tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

Chimeric antigen receptor (CAR) T cells have transformed the therapeutic options for relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia. Data for CAR therapy in extramedullary (EM) involvement are limited. Retrospective data were abstracted from the Pediatric Real World CAR Consortium (PRWCC) of 184 infused patients from 15 US institutions. Response (complete response) rate, overall survival (OS), relapse-free survival (RFS), and duration of B-cell aplasia (BCA) in patients referred for tisagenlecleucel with EM disease (both central nervous system (CNS)3 and non-CNS EM) were compared with bone marrow (BM) only. Patients with CNS disease were further stratified for comparison. Outcomes are reported on 55 patients with EM disease before CAR therapy (CNS3, n = 40; non-CNS EM, n = 15). The median age at infusion in the CNS cohort was 10 years (range,

Innate-like T cells, including invariant natural killer T cells, mucosal-associated invariant T cells, and γδ T cells, are present in various barrier tissues, including the lung, where they carry out protective responses during infections. Here, we investigate their roles during pulmonary pneumococcal infection. Following infection, innate-like T cells rapidly increase in lung tissue, in part through recruitment, but T cell antigen receptor activation and cytokine production occur mostly in interleukin-17-producing NKT17 and γδ T cells. NKT17 cells are preferentially located within lung tissue prior to infection, as are CD103+ dendritic cells, which are important both for antigen presentation to NKT17 cells and γδ T cell activation. Whereas interleukin-17-producing γδ T cells are numerous, granulocyte-macrophage colony-stimulating factor is exclusive to NKT17 cells and is required for optimal protection. These studies demonstrate how particular cellular interactions and responses of functional subsets of innate-like T cells contribute to protection from pathogenic lung infection.

Human T-lymphotropic virus 1 (HTLV-1) infection causes two serious diseases: adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Immunological studies have revealed that HTLV-1 Tax-specific CD8+ cytotoxic T-cells (Tax-CTLs) in asymptomatic carriers (ACs) and ATL patients play an important role in the elimination of HTLV-1-infected host cells, whereas Tax-CTLs in HAM patients trigger an excessive immune response against HTLV-1-infected host cells infiltrating the central nervous system (CNS), leading to local inflammation. Our previous evaluation of HTLV-1 Tax301-309 (SFHSLHLLF)-specific Tax-CTLs (Tax301-309-CTLs) revealed that a unique T-cell receptor (TCR) containing amino acid (AA)-sequence motif PDR, was shared among HLA-A*24:02+ ACs and ATL patients and behaved as an eliminator by strong activity against HTLV-1. However, it remains unclear whether PDR+Tax301-309-CTLs also exist in HLA-A*24:02+ HAM patients and are involved in the pathogenesis of HAM. In the present study, by high-throughput TCR repertoire analysis technology, we revealed TCR repertoires of Tax301-309-CTLs in peripheral blood (PB) of HLA-A*24:02+ HAM patients were skewed, and a unique TCR-motif PDR was conserved in HAM patients (10 of 11 cases). The remaining case dominantly expressed (-DR, P-R, and PD-), which differed by one AA from PDR. Overall, TCRs with unique AA-sequence motifs PDR, or (-DR, P-R, and PD-) accounted for a total of 0.3-98.1% of Tax301-309-CTLs repertoires of HLA-A*24:02+ HAM patients. Moreover, TCR repertoire analysis of T-cells in the cerebrospinal fluid (CSF) from four HAM patients demonstrated the possibility that PDR+Tax301-309-CTLs and (-DR, P-R, and PD-)+Tax301-309-CTLs efficiently migrated and accumulated in the CSF of HAM patients fostering increased inflammation, although we observed no clear significant correlation between the frequencies of them in PB and the levels of CSF neopterin, a known disease activity biomarker of HAM. Furthermore, to better understand the potential function of PDR+Tax301-309-CTLs, we performed immune profiling by single-cell RNA-sequencing of Tax301-309-CTLs, and the result showed that PDR+Tax301-309-CTLs up-regulated the gene expression of natural killer cell marker KLRB1 (CD161), which may be associated with T-cell activation and highly cytotoxic potential of memory T-cells. These findings indicated that unique and shared PDR+Tax301-309-CTLs have a potential role in promoting local inflammation within the CNS of HAM patients.

Certain CD8 T cell responses are particularly effective at controlling infection, as exemplified by elite control of HIV in individuals harboring HLA-B57. To understand the structural features that contribute to CD8 T cell elite control, we focused on a strongly protective CD8 T cell response directed against a parasite-derived peptide (HF10) presented by an atypical MHC-I molecule, H-2Ld. This response exhibits a focused TCR repertoire dominated by Vβ2, and a representative TCR (TG6) in complex with Ld-HF10 reveals an unusual structure in which both MHC and TCR contribute extensively to peptide specificity, along with a parallel footprint of TCR on its pMHC ligand. The parallel footprint is a common feature of Vβ2-containing TCRs and correlates with an unusual Vα-Vβ interface, CDR loop conformations, and Vβ2-specific germline contacts with peptides. Vβ2 and Ld may represent specialist components for antigen recognition that allows for particularly strong and focused T cell responses.

CD4+ and CD8+ double-positive (DP) thymocytes play a crucial role in T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, regulatory T cells, or invariant nature kill T cells (iNKT) in response to TCR signaling. Chromatin organizer SATB1 is highly expressed in DP cells and is essential in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing shows that Satb1 deletion changes the cell identity of DP thymocytes and down-regulates genes specifically and highly expressed in DP cells. Super-enhancers regulate the expressions of DP-specific genes, and our Hi-C data show that SATB1 deficiency in thymocytes reduces super-enhancer activity by specifically decreasing interactions among super-enhancers and between super-enhancers and promoters. Our results reveal that SATB1 plays a critical role in thymocyte development to promote the establishment of DP cell identity by globally regulating super-enhancers of DP cells at the chromatin architectural level.

Immunotherapy has been the fifth pillar of cancer treatment in the past decade. Chimeric antigen receptor (CAR) T-cell therapy is a newly designed adoptive immunotherapy that is able to target and further eliminate cancer cells by engaging with MHC-independent tumor-antigens. CAR T-cell therapy has exhibited conspicuous clinical efficacy in hematological malignancies, but more than half of patients will relapse. Of note, the efficacy of CAR T-cell therapy has been even more disappointing in solid tumors. These challenges mainly include (1) the failures of CAR T-cells to treat highly heterogeneous solid tumors due to the difficulty in identifying unique tumor antigen targets, (2) the expression of target antigens in non-cancer cells, (3) the inability of CAR T-cells to effectively infiltrate solid tumors, (4) the short lifespan and lack of persistence of CAR T-cells, and (5) cytokine release syndrome and neurotoxicity. In combination with these characteristics, the ideal CAR T-cell therapy for solid tumors should maintain adequate T-cell response over a long term while sparing healthy tissues. This article reviewed the status, clinical application, efficacy, safety, and challenges of CAR T-cell therapies, as well as the latest progress of CAR T-cell therapies for solid tumors. In addition, the potential strategies to improve the efficacy of CAR T-cells and prevent side effects in solid tumors were also explored.

TCR stimulation triggers Ca2+ signals that are critical for T cell function and immunity. Several pore-forming α and auxiliary β subunits of voltage-gated Ca2+ channels (VGCC) were reported in T cells, but their mechanism of activation remains elusive and their contribution to Ca2+ signaling in T cells is controversial. We here identify CaVβ1, encoded by Cacnb1, as a regulator of T cell function. Cacnb1 deletion enhances apoptosis and impairs the clonal expansion of T cells after lymphocytic choriomeningitis virus (LCMV) infection. By contrast, Cacnb1 is dispensable for T cell proliferation, cytokine production and Ca2+ signaling. Using patch clamp electrophysiology and Ca2+ recordings, we are unable to detect voltage-gated Ca2+ currents or Ca2+ influx in human and mouse T cells upon depolarization with or without prior TCR stimulation. mRNAs of several VGCC α1 subunits are detectable in human (CaV3.3, CaV3.2) and mouse (CaV2.1) T cells, but they lack transcription of many 5' exons, likely resulting in N-terminally truncated and non-functional proteins. Our findings demonstrate that although CaVβ1 regulates T cell function, these effects are independent of VGCC channel activity.

T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen-binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tiseher and Weiner, 2019). Here, we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. Leveraging the ability of our system to rapidly disengage ligand binding, we also measure slower reset rates for downstream signaling events. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at slower resetting downstream signaling complexes that could help balance antigen sensitivity and discrimination.

There is an urgent need for an effective TB vaccine capable of controlling both acute and chronic Mycobacterium tuberculosis infection in populations with diverse genetic backgrounds. In this study, we characterised the immunogenicity and protective efficacy of a novel protein-in-adjuvant subunit vaccine. The protein component is a fusion protein of three different M. tuberculosis antigens, which we termed CysVac5: CysD, a major component of the M. tuberculosis sulfate activation pathway that is highly expressed during the chronic stage of M. tuberculosis infection, is fused with two major secreted mycobacterial antigens, Ag85B and MPT83. Vaccination of C57BL/6 mice with CysVac5, formulated in a monophosphoryl lipid A (MPLA) and dimethyldioctadecylammonium (DDA) adjuvant combination, resulted in the potent generation of polyfunctional CD4+ T cells secreting multiple cytokines, including IFN-γ, IL-2, TNF and IL-17, against each of the three components of the fusion protein. Furthermore, vaccination with CysVac5-MPLA/DDA conferred significant protection against infection in mouse lungs, which was greater than that afforded by BCG at extended time points post-challenge. The generation of antigen-specific and protective immunity was also observed in CysVac5 vaccinated BALB/c mice, indicating the vaccine could display efficacy across multiple genetic backgrounds. These results indicate that the CysVac5 vaccine has broad immunogenicity, is effective in controlling both acute and chronic phases of M. tuberculosis infection in mice, and warrants further investigation to assess its potential to control pulmonary TB.

The need for rapid and cheap synthesis of large numbers of chemical compounds has contributed to the emergence of combinatorial chemistry (simultaneous synthesis of different compounds, in contrast to traditional synthesis, in which each substance is produced individually). Combinatorial library methods were initially applied only to peptides and oligonucleotides. By now, the scope of these libraries has expanded considerably to include proteins, synthetic oligomers, small molecules, and oligosaccharides. The enormous variety of antibodies (Abs) makes it possible to detect clones able to interact highly specifically with almost any natural or synthetic antigen (Ag). Phage Abs are an excellent alternative to mono- and polyclonal Abs, because they are highly stable, have no disulfide bonds, and are much cheaper to make. Monitoring of various substances, including proteins, in a living organism is much in demand. Despite the vast amount of literature available on Ab phage display, the use of phage display to determine diagnostically important Ags has not been sufficiently covered. Many studies have confirmed that unlike other types of Abs, phage Abs ensure highly sensitive Ag detection. Therefore, this review focuses on the use of phage display to prepare Abs specific to diagnostically important Ags (allergens, disease and cancer biomarkers, toxins) and on their application in analytical systems, including biosensors. The use of phage Abs in Ag diagnostics is compared with the use of classical Abs, and the prospects are shown for the use of phage Abs as biosensor sensing elements. This review analyzes the recent advances in the detection of diagnostically important Ags by using phage display–based biosensors. Systematic information is presented about allergens, disease and cancer biomarkers, and toxins detected by using phage Abs. Phage display Abs for sensor-based Ag detection are presented as an affordable alternative to classic tests.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and β-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.

Lymphocytes within the intestinal epithelial layer (IEL) in mammals have unique composition compared with their counterparts in the lamina propria. Little is known about the role of some of the key colonic IEL subsets, such as TCRαβ+CD8+ T cells, in inflammation. We have recently described liver-enriched innate-like TCRαβ+CD8αα regulatory T cells, partly controlled by the non-classical MHC molecule, Qa-1b, that upon adoptive transfer protect from T cell-induced colitis. In this study, we found that TCRαβ+CD8αα T cells are reduced among the colonic IEL during inflammation, and that their activation with an agonistic peptide leads to significant Qa-1b-dependent protection in an acute model of colitis. Cellular expression of Qa-1b during inflammation and corresponding dependency in peptide-mediated protection suggest that Batf3-dependent CD103+CD11b- type 1 conventional dendritic cells control the protective function of TCRαβ+CD8αα T cells in the colonic epithelium. In the colitis model, expression of the potential barrier-protective gene, Muc2, is enhanced upon administration of a Qa-1b agonistic peptide. Notably, in steady state, the mucin metabolizing Akkermansia muciniphila was found in significantly lower abundance amid a dramatic change in overall microbiome and metabolome, increased IL-6 in explant culture, and enhanced sensitivity to dextran sulfate sodium in Qa-1b deficiency. Finally, in patients with inflammatory bowel disease, we found upregulation of HLA-E, a Qa-1b analog with inflammation and biologic non-response, in silico, suggesting the importance of this regulatory mechanism across species.

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαβ sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαβ constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR β-chain (TCR-β) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-β and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-β clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL.

IL-27 controls a diverse range of immune responses in many disease settings. Here, we identify intestinal epithelial cells (IECs) as one of the major IL-27 cellular sources in the gut-associated tissue. Unlike IL-27 secreted by innate immune cells, gut epithelial IL-27 is dispensable for T-bet+ regulatory T cell (T reg cell) differentiation or IL-10 induction. Rather, IEC-derived IL-27 specifically promotes a distinct CD8αα+CD4+ intraepithelial lymphocyte (IEL) population that acquires their functional differentiation at the intestinal epithelium. Loss of IL-27 in IECs leads to a selective defect in CD8αα+CD4+ IELs over time. Consequently, mice with IEC-specific IL-27 ablation exhibited elevated pathogen burden during parasitic infection, and this could be rescued by transfer of exogenous CD8αα+CD4+ IELs. Collectively, our data reveal that in addition to its known regulatory properties in preventing immune hyperactivity, gut epithelial IL-27 confers barrier immunity by inducing a specific IEL subset and further suggest that IL-27 produced by different cell types plays distinct roles in maintaining intestinal homeostasis.

B cell signaling for activation via the BCR occurs as an isolated event only in vitro; in real life, BCR signaling takes place within a complex milieu that involves interactions with agents that trigger additional receptors. Chief among these is IL-4. We have shown that BCR signaling is reprogrammed by IL-4 receptor engagement and that this reprogramming involves creation of a new, signalosome-independent, Lyn-dependent alternate signaling pathway in B cells isolated from BALB/cByJ mice. A unique aspect of the alternate pathway is protein kinase Cδ (PKCδ) phosphorylation. In dissecting this pathway, we unexpectedly found that Lyn is associated with IL-4Rα, that IL-4 induces Lyn activation, and that Lyn immunoprecipitated from IL-4-treated B cells capably phosphorylates PKCδ in a cell-free system. However, PKCδ phosphorylation does not occur in the absence of BCR triggering in vivo. This raised the question of why IL-4 alone failed to produce PKCδ phosphorylation. We considered the possibility that Lyn and PKCδ may be spatially separated. As expected, before any treatment, Lyn is located primarily in the membrane fraction, whereas PKCδ is located mainly in the cytosol fraction. However, when anti-Ig follows IL-4 treatment, PKCδ is found in the membrane fraction and phosphorylated. This translocation of PKCδ to the membrane fraction is not affected by loss of Lyn, although PKCδ phosphorylation requires Lyn. Thus, PKCδ phosphorylation through the alternate pathway represents the result of signal integration, whereby neither IL-4 nor anti-Ig working alone produces this outcome, but together they achieve this result by Lyn activation (IL-4) and PKCδ translocation (IL-4 followed by anti-Ig).

BackgroundT cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements.ObjectiveDevelop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats.AnimalsCats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31).MethodsMolecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity.ResultsThe new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers.Conclusions and clinical importanceThe assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.

Special AT-rich sequence binding protein-1 (SATB1) is localized to the nucleus and remodels chromatin structure in T cells. SATB1-deficient CD4 T cells cannot respond to TCR stimulation; however, the cause of this unresponsiveness is to be clarified. Here, we demonstrate that SATB1 is indispensable to proper mitochondrial functioning and necessary for the activation of signal cascades via the TCR in CD4 T cells. Naïve SATB1-deficient CD4 T cells contain fewer mitochondria than WT T cells, as the former do not express mitochondrial transcription factor A (TFAM). Impaired mitochondrial function in SATB1-deficient T cells subverts mitochondrial ROS production and SHP-1 inactivation by constitutive oxidization. Ectopic TFAM expression increases mitochondrial mass and mitochondrial ROS production and rescues defects in the antigen-specific response in the SATB1-deficient T cells. Thus, SATB1 is vital for maintaining mitochondrial mass and function by regulating TFAM expression, which is necessary for TCR signaling.

Malaria, caused by parasites of the Plasmodium genus, is responsible for significant morbidity and mortality globally. Chronic Plasmodium falciparum exposure affects the B cell compartment, leading to the accumulation of atypical memory B cells (atMBCs). IgM-positive (IgM+) and IgG+ atMBCs have not been compared in-depth in the context of malaria, nor is it known if atMBCs in malaria-experienced individuals are different from phenotypically similar B cells in individuals with no known history of Plasmodium exposure. To address these questions, we characterized the B cell receptor (BCR) repertoire of naive B cells (NBCs), IgM+ and IgG+ classical MBCs (cMBCs), and IgM+ and IgG+ atMBCs from 13 malaria-naive American adults and 7 malaria-experienced Ugandan adults. Our results demonstrate that P. falciparum exposure mainly drives changes in atMBCs. In comparison to malaria-naive adults, the BCR repertoire of Plasmodium-exposed adults showed increased levels of somatic hypermutation in the heavy chain V region in IgM+ and IgG+ atMBCs, shorter heavy chain complementarity-determining region 3 (HCDR3) in IgG+ atMBCs, and increased usage of IGHV3-73 in IgG+ cMBCs and both IgM+ and IgG+ atMBCs. Irrespective of Plasmodium exposure, IgM+ atMBCs closely resembled NBCs, while IgG+ atMBCs resembled IgG+ cMBCs. Physicochemical properties of the HCDR3 seemed to be intrinsic to cell type and independent of malaria experience. The resemblance between atMBCs from Plasmodium-exposed and naive adults suggests similar differentiation pathways regardless of chronic antigen exposure. Moreover, these data demonstrate that IgM+ and IgG+ atMBCs are distinct populations that should be considered separately in future analyses. IMPORTANCE Malaria, caused by Plasmodium parasites, still contributes to a high global burden of disease, mainly in children under 5 years of age. Chronic and recurrent Plasmodium infections affect the development of B cell memory against the parasite and promote the accumulation of atypical memory B cells (atMBCs), which have an unclear function in the immune response. Understanding where these cells originate from and whether they are beneficial in the immune response to Plasmodium will help inform vaccination development efforts. We found differences in B cell receptor (BCR) properties of atMBCs between malaria-naive and malaria-experienced adults that are suggestive of divergent selection processes, resulting in more somatic hypermutation and differential immunoglobulin heavy chain V (IGHV) gene usage. Despite these differences, atMBCs from malaria-naive and malaria-experienced adults also showed many similarities in BCR characteristics, such as physicochemical properties of the HCDR3 region, suggesting that atMBCs undergo similar differentiation pathways in response to different pathogens. Our study provides new insights into the effects of malaria experience on the B cell compartment and the relationships between atMBCs and other B cell populations.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Intracerebral hemorrhage (ICH) and perihematomal edema (PHE) volumes are major determinants of ICH outcomes as is the immune system which plays a significant role in damage and repair. Thus, we performed whole-transcriptome analyses of 18 ICH patients to delineate peripheral blood genes and networks associated with ICH volume, absolute perihematomal edema (aPHE) volume, and relative PHE (aPHE/ICH; rPHE). We found 440, 266, and 391 genes correlated with ICH and aPHE volumes and rPHE, respectively (p  |0.6|). These mainly represented inflammatory pathways including NF-κB, TREM1, and Neuroinflammation Signaling-most activated with larger volumes. Weighted Gene Co-Expression Network Analysis identified seven modules significantly correlated with these measures (p

Chimeric antigen receptor (CAR)-T cell therapies have improved the outcome for many patients with relapsed or refractory aggressive B-cell lymphomas. In 2017, axicabtagene ciloleucel and soon after tisagenlecleucel became the first approved CAR-T cell products for patients with high-grade B-cell lymphomas or diffuse large B-cell lymphoma (DLBCL) who are relapsed or refractory to ≥ 2 prior lines of therapy; lisocabtagene maraleucel was approved in 2021. Safety and efficacy outcomes from the pivotal trials of each CAR-T cell therapy have been reported. Despite addressing a common unmet need in the large B-cell lymphoma population and utilizing similar CAR technologies, there are differences between CAR-T cell products in manufacturing, pivotal clinical trial designs, and data reporting. Early reports of commercial use of axicabtagene ciloleucel and tisagenlecleucel provide the first opportunities to validate the impact of patient characteristics on the efficacy and safety of these CAR-T cell therapies in the real world. Going forward, caring for patients after CAR-T cell therapy will require strategies to monitor patients for sustained responses and potential long-term side effects. In this review, product attributes, protocol designs, and clinical outcomes of the key clinical trials are presented. We discuss recent data on patient characteristics, efficacy, and safety of patients treated with axicabtagene ciloleucel or tisagenlecleucel in the real world. Finally, we discuss postinfusion management and preview upcoming clinical trials of CAR-T cell therapies.

Anthrax is a highly dangerous disease of animals and humans caused by the spore-forming bacterium Bacillus anthracis. Currently, the disease is widespread in many countries of the world. Many regions of the Russian Federation are anthrax-endemic. A large number of anthrax treatment, diagnosis and prevention tools are developed using hyperimmune serum. Currently known commercial hyperimmune sera areproduced by 2-month long immunization of horses, which is a long and expensive process. This suggests the need to develop faster and cheaper ways to produce anti-anthrax hyperimmune sera; such possible ways became the objective of this study. A live culture of Bacillus anthracis 55-VNIIVViM vaccine strain, used to produce live vaccines against animal anthrax, was used in the experiments. Rabbits were used as animal models. Based on the findings the method of rabbit immunization was selected. The optimal method included intravenous injection of the antigen in increasing amounts according to the following scheme: injection I – 0.5 cm3 ; injection II – 1 cm3 ; injection III – 2 cm3 at a dose of 100 million mc/animal in 1 cm3 , with 4-day interval between injections. This scheme made it possible to produce the serum with a high antibody titer equal to 14 log2 . For long-term storage of the serum produced, the freeze-drying modes were optimized, giving 2% residual moisture content of the finished product. The analysis of the freeze-dried serum storage terms showed that the initial activity and physico-chemical properties of the product are maintained for 30 months.

Severe acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019 pandemic disease is an enveloped virus, showing genome similarity with bat coronavirus. This virus initially infects the upper respiratory tract, with subsequent spread to the lower respiratory tract. Despite the availability of antigen and antibody detection methods, reverse transcription-polymerase chain reaction (RT-PCR) is the diagnostic test of choice for this novel coronaviral infection. Care must be taken while interpreting the RT-PCR results, as single RT-PCR, especially in early days of infection, maybe false negative. The availability of cartridge-based nucleic acid amplification test has improved the diagnostic facilities in a peripheral setting of developing countries.

Abstract Background ETRAMP11.2 (PVX_003565) is a well-characterized protein with antigenic potential. It is considered to be a serological marker for diagnostic tools, and it has been suggested as a potential vaccine candidate. Despite its immunological relevance, the polymorphism of the P. vivax ETRAMP11.2 gene (pvetramp11.2) remains undefined. The genetic variability of an antigen may limit the effectiveness of its application as a serological surveillance tool and in vaccine development and, therefore, the aim of this study was to investigate the genetic diversity of pvetramp11.2 in parasite populations from Amazonian regions and worldwide. We also evaluated amino acid polymorphism on predicted B-cell epitopes. The low variability of the sequence encoding PvETRAMP11.2 protein suggests that it would be a suitable marker in prospective serodiagnostic assays for surveillance strategies or in vaccine design against P. vivax malaria. Methods The pvetramp11.2 of P. vivax isolates collected from Brazil (n = 68) and Peru (n = 36) were sequenced and analyzed to assess nucleotide polymorphisms, allele distributions, population differentiation, genetic diversity and signature of selection. In addition, sequences (n = 104) of seven populations from different geographical regions were retrieved from the PlasmoDB database and included in the analysis to study the worldwide allele distribution. Potential linear B-cell epitopes and their polymorphisms were also explored. Results The multiple alignments of 208 pvetramp11.2 sequences revealed a low polymorphism and a marked geographical variation in allele diversity. Seven polymorphic sites and 11 alleles were identified. All of the alleles were detected in isolates from the Latin American region and five alleles were detected in isolates from the Southeast Asia/Papua New Guinea (SEA/PNG) region. Three alleles were shared by all Latin American populations (H1, H6 and H7). The H1 allele (reference allele from Salvador-1 strain), which was absent in the SEA/PNG populations, was the most represented allele in populations from Brazil (54%) and was also detected at high frequencies in populations from all other Latin America countries (range: 13.0% to 33.3%). The H2 allele was the major allele in SEA/PNG populations, but was poorly represented in Latin America populations (only in Brazil: 7.3%). Plasmodium vivax populations from Latin America showed a marked inter-population genetic differentiation (fixation index [Fst]) in contrast to SEA/PNG populations. Codon bias measures (effective number of codons [ENC] and Codon bias index [CBI]) indicated preferential use of synonymous codons, suggesting selective pressure at the translation level. Only three amino acid substitutions, located in the C-terminus, were detected. Linear B-cell epitope mapping predicted two epitopes in the Sal-1 PvETRAMP11.2 protein, one of which was fully conserved in all of the parasite populations analyzed. Conclusions We provide an overview of the allele distribution and genetic differentiation of ETRAMP11.2 antigen in P. vivax populations from different endemic areas of the world. The reduced polymorphism and the high degree of protein conservation supports the application of PvETRAMP11.2 protein as a reliable antigen for application in serological assays or vaccine design. Our findings provide useful information that can be used to inform future study designs. Graphical abstract

Abstract The COVID‐19 pandemic highlighted the clear risk that zoonotic viruses pose to global health and economies. The scientific community responded by developing several efficacious vaccines which were expedited by the global need for vaccines. The emergence of SARS‐CoV‐2 breakthrough infections highlights the need for additional vaccine modalities to provide stronger, long‐lived protective immunity. Here we report the design and preclinical testing of small extracellular vesicles (sEVs) as a multi‐subunit vaccine. Cell lines were engineered to produce sEVs containing either the SARS‐CoV‐2 Spike receptor‐binding domain, or an antigenic region from SARS‐CoV‐2 Nucleocapsid, or both in combination, and we tested their ability to evoke immune responses in vitro and in vivo. B cells incubated with bioengineered sEVs were potent activators of antigen‐specific T cell clones. Mice immunised with sEVs containing both sRBD and Nucleocapsid antigens generated sRBD‐specific IgGs, nucleocapsid‐specific IgGs, which neutralised SARS‐CoV‐2 infection. sEV‐based vaccines allow multiple antigens to be delivered simultaneously resulting in potent, broad immunity, and provide a quick, cheap, and reliable method to test vaccine candidates.