Journal of Immunological Methods

The accurate measurement of Ebola virus (EBOV)-specific antibody responses is crucial to assessing immunity induced by EBOV infection or vaccination. For this purpose, the Filovirus Animal Nonclinical Group (FANG) anti-EBOV glycoprotein (GP1,2) ELISA is considered the "gold-standard". However, it has limitations such as high repeat-rates and variability, and low throughput. Here, we describe two new alternative assays: a Single-Molecule Assay Planar EBOV GP1,2 ELISA and a multiplexed EBOV GP1,2, EBOV nucleoprotein, and EBOV Viral Protein 40 Luminex assay, and compare these with two versions of the FANG ELISA. Samples were selected from participants receiving vaccine or placebo in a randomized, placebo-controlled, double-blinded study of two EBOV vaccines (PREVAIL 1), and a longitudinal cohort study of Ebola virus disease (EVD) survivors and their close contacts (PREVAIL 3). All four assays were concordant in their measurements of anti-EBOV GP1,2-specific immunoglobulin G responses, allowing for the determination of conversion equations for antibody measurements across assays. In addition, all four showed a similar ability to distinguish vaccine recipients from placebo recipients and EVD survivors from their close contacts. Compared to the FANG assays, the Quanterix and Luminex assays had lower variability, lower repeat rates, and higher throughput, making them good alternatives for future studies.

BackgroundCompanion canines need advances in therapeutic options for solid tumor malignancies. Prior studies established feasibility of autologous natural killer (NK) cell infusions in canines with solid tumors; however, autologous products are limited by dysfunctional immunity and a manufacturing process that delays care. Allogeneic NK cells offer the possibility of "off-the-shelf" therapy to be administered from healthy donors. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from healthy canine donors via density gradient separation. NK cells were expanded with recombinant human IL-2 and canine IL-21 with the addition of K562 feeder cells transfected with CD137 ligand and membrane bound human IL-15. Additional experiments included IL-12 in the expansions. In vitro potency was assessed via co-culture with the D17-mKate2 canine osteosarcoma cell line. Three canines were enrolled in a phase 1 trial infusing ex vivo expanded allogeneic NK cells after lymphodepletion. ResultsFlow cytometric analysis confirmed successful expansion of canine NK cells with up to 50% of cells demonstrating NKp46+ after 14 days of expansion. Residual T cell numbers varied based on donor. The addition of IL-12 led to increased NK cell expansion. Incucyte demonstrated potency with increasing osteosarcoma cell death at higher effector to target ratios. Three canines with metastatic/refractory solid tumors were successfully lymphodepleted and infused with allogeneic NK cell products. The canines tolerated the infusions well. ConclusionsCanine allogeneic NK cells were successfully expanded and activated ex vivo, demonstrated potency in vitro, and safety in vivo. Further studies will optimize the NK cell product and escalate dosing to reach the maximal tolerable dose.

BackgroundHigh-content assays (HCAs) have problems distinguishing biologically significant effects from the incidental effects of non-repeatable technical factors. Non-repeatable results are attributed to variations in the cell culture environment and the numerous, heterogeneous descriptors evaluated. The aim here was to determine whether preprocessing operations impacted the reproducibility of class assignments of experimental data. MethodsBatch effects that could affect reproducibility, i.e., signal/noise ratio, instrumental conditions, and segmentation, were controlled variables. The remaining batch effects, variations in materials, personnel, and culture environment could not be controlled. Descriptors values were measured directly from images. Exploratory factor analysis was used to solve the identifiable and interpretable feature, factor 4. In each of five trials, one sample was treated with the same chemical mixture (EXP) and another with the solvent vehicle alone (CON). ResultsRepeated CON and EXP samples showed significant differences among factor 4 means in data regularized within each trial. The mean of Trial 3 CON differed significantly from all other CON samples. These differences disappeared upon regularization to comprehensive databases. Among repeated EXPs, the Trial 2 mean differed from three other EXPs, but regularization to comprehensive databases had little effect. However, classification patterns were unchanged after regularization to any comprehensive database derived by the same protocol. After regularization to datasets derived by two different protocols, the classification pattern differed but only reflected elevation of differences that had been marginal to statistical significance. Outlier removal was deleterious. Even with the most sparing definition of outliers, over 3% of a single samples contents were removed from most trials. Elimination based on the overall within-trial distributions caused type I and type II errors. ConclusionsNon-repeatable factor 4 means in repeated trials had negligible influence on classification outcomes, so repeatability may not be a good indicator of assay quality. Irreducible batch effects, combined with small sample sizes and skewed distributions of descriptors values, may account for non-repeatability. As the current results are based on real-world data, they suggest that non-repeatability is an uncorrectable feature of these assays. Classification patterns are not affected by several irreducible technical factors, namely materials, personnel, and non-repeatable environmental variables.

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

Introduction: Herpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap Statement: The comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. Aim: This study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. Methodology: Four hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. Results: HSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. Conclusion: CLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification.

BackgroundKidney cell lines are widely used to model kidney physiology and disease; however, their gene expression profiles may differ from primary cells due to immortalization, culture conditions, or experimental treatments. Determining whether a cell line resembles its native cell type is critical for interpreting in vitro findings. We developed a transcriptome-based approach that matches bulk RNA-seq data from kidney cell lines, primary cells, or tissues to reference cell types derived from single-cell RNA-seq (scRNA-seq) datasets. MethodsReference transcriptomic profiles were generated from two human and two murine kidney scRNA-seq datasets by pseudobulk aggregation. Bulk RNA-seq data from microdissected kidney tissue, non-kidney negative controls, and kidney cell lines were matched to these references using three statistical similarity measures (Spearman correlation, Euclidean distance, Poisson distance) and three machine learning classifiers (Random Forest, XGBoost, TabPFN). Each was assessed with global gene expression, curated kidney marker gene lists, and the most variable genes. Matching accuracy was evaluated through a three-step validation strategy: within-dataset matching, cross-reference comparison, and validation against primary kidney tissue and negative controls. ResultsGene expression rank-based Spearman correlation and TabPFN, a foundation model for tabular data, emerged as the most accurate and specific approaches, particularly with curated kidney marker gene lists. Both methods correctly identified microdissected kidney tubule segments and were robust against non-kidney negative controls. Applied to commonly used kidney cell lines, OK cells retained proximal tubule identity, particularly under shear stress, while other proximal tubule lines (HK-2, HKC-8, HKC-11) showed inconsistent matching. Collecting duct-derived mIMCD-3 maintained stable similarity across passages, culture conditions, and genetic modifications. ConclusionWe provide two complementary implementations: CellMatchR, an accessible web-based tool using Spearman correlation for routine use, and comprehensive scripts for TabPFN-based matching (link will be added after peer reviewed publication). Together, these resources enable researchers to make informed decisions about kidney cell culture model selection, interpretation, and stability. Translational StatementKidney cell lines are fundamental tools in nephrology research, yet their transcriptomic similarity to native cell types is rarely validated systematically. We demonstrate that combining bulk RNA-seq data with single-cell reference datasets enables robust assessment of cell line identity using gene expression-rank-based correlation and machine learning approaches. By providing a comprehensive evaluation of matching methods, curated kidney marker gene lists, and reference datasets, our study serves as both a practical resource and a methodological framework for the kidney research community, facilitating informed selection of cell culture models, quality control of experimental conditions, developing new experimental cell culture models, and more reliable translation of in vitro findings to kidney physiology and disease.

BackgroundDNA polymerase activity assays are required for enzyme quality control in biotechnology and diagnostics, but standard methods rely on specialist reagents, radioactivity and other hazardous materials, or real-time PCR instruments that are not widely accessible in resource-limited settings. This constrains local production of high quality, validated reagents and increases dependence on imported enzymes. MethodsBased on experiences derived from partnerships with scientists in several low and middle-income countries (LMICs) and stakeholder consultations, we adapted a commercial EvaGreen-based fluorometric DNA polymerase activity assay for isothermal operation using minimal equipment. Assay conditions were optimized using Design of Experiments (DOE) methodology, varying temperature, reaction volume, and MgCl2 concentration. To address reagent cost and supply-chain constraints, we developed detailed protocols for in-house synthesis of the off-patent AOAO-12 DNA dye (sold commercially as EvaGreen) and generation of single-stranded DNA templates via asymmetric PCR. ResultsOptimized isothermal assay conditions (40{degrees}C, 7.75 mM MgCl2) reliably quantified activity across multiple DNA polymerase families. In-house synthesized AOAO-12 dye exhibited comparable DNA-binding performance to commercial alternatives (R{superscript 2} = 0.95), reducing costs by more than an order of magnitude when normalized to working concentrations, enabling assay costs of approximately {pound}0.001 per reaction. The assay is effective across multiple polymerases (Bst-LF, OpenVent, Taq, Q5) and is compatible with both plate readers and qByte, a low-cost, open-source fluorometric device. ConclusionsThis stakeholder-informed assay provides an accessible, cost-effective solution for DNA polymerase quality control in resource-limited settings. The combination of optimized commercial protocols and in-house reagent synthesis offers flexibility for different resource contexts, potentially improving access to molecular biology tools globally.

Background: External Quality Assurance (EQA) is an essential component of modern laboratory medicine. Current scientific evidence on EQA focuses primarily on the analyses carried out by EQA providers while relatively little research has been conducted in individual clinical laboratories. Methods: In this retrospective single-center observational study in a clinical laboratory, EQA results were analyzed over a period of four years (2021-2024). The evaluation was based on EQA action reports documented in the institutes internal quality management system. Deviations were classified according to department, type of discrepancy, root cause category (analytical, preanalytical, systemic, unidentifiable), and measures taken. Results: A total of 7226 EQA participations were evaluated during the observation period. The overall error rate remained consistently low, ranging between 0.8% and 1.6%, with no significant change over time (p = 0.87). Most deviations occurred in the departments of clinical chemistry and immuno/autoimmune diagnostics (p < 0.001). These were predominantly quantitative discrepancies (false low/false negative or false high/false positive). Root cause analysis showed a clear dominance of analytical causes (p < 0.001), while preanalytical and systemic causes were identified less frequently. In most cases, corrective measures, such as re-analyses, recalibrations, process adjustments, or staff training, were implemented promptly. Hard structural measures, such as changing methods or discontinuing tests, were rarely necessary. Conclusion: In a clinical laboratory, EQA is an important tool for structured error analysis and continuous quality improvement. Consistent processing of deviating EQA results goes hand in hand with stable analytical performance and a low error rate.

BackgroundTherapeutic agents targeting the PD1-PDL1 interaction are of great clinical value, however accurately predicting which patients are most likely to benefit is challenging. Improved predictive biomarkers for anti-PD1 therapy are clearly needed. Quantifying PD1 saturation by PDL1 in tumor tissue has the potential to serve as such a biomarker. Here we report a novel bioassay called the PD1 Ligand Receptor Complex Aptamer (LIRECAP) assay and demonstrate it can be used to quantify the saturation of PD1 by PDL1 in formalin-fixed paraffin-embedded tumor biospecimens. ResultsThe PD1 LIRECAP assay was developed by identifying a pair of RNA aptamers. One aptamer preferentially binds to unoccupied PD1 (P aptamer) and the other to the PD1-PDL1 complex (C aptamer). P and C aptamers were added together to a formalin-fixed sample, and bound aptamer extracted. A 2-color qRT-PCR assay using a single set of primers was used to determine the ratio of the sample-bound C to P aptamers (C:P ratio) which reflected PD1 saturation by PDL1 in the sample. Quantification of PD1 saturation by PDL1 as determined by the PD1 LIRECAP assay correlated closely with PD1-mediated signaling and PD1-PDL1 proximity. Analysis of sarcoma FFPE biospecimens confirmed the assay is technically reproducible on clinical biospecimens. There were significant differences in PD1 saturation by PDL1 between patients as well as considerable intratumoral heterogeneity. ConclusionsThe PD1 LIRECAP assay is novel assay that can be used to quantify PD1 saturation by PDL1 in clinical biospecimens. The assay is technically feasible, reproducible, and has the potential to serve as a superior predictive biomarker for PD1/PDL1-based therapy. Similar assays based on this platform could be used in other systems and settings to quantify interaction between two molecules.

Dengue disease remains a significant global health threat, with current vaccines exhibiting variable efficacy and safety concerns. Virus-like particles (VLPs) offer a promising alternative by mimicking native virus structures without infectious genomes. We engineered a mammalian expression plasmid encoding Dengue-1 prM and E proteins, optimized for secretion using Japanese Encephalitis virus signal sequences, and transiently expressed it in HeLa cells. Purified VLPs exhibited spherical morphology ([~]39 nm diameter) consistent with native virions, as confirmed by transmission electron microscopy. Immunization of mice with these VLPs elicited robust Dengue-1 specific IgG antibody responses. Our study demonstrates production of immunogenic Dengue-1 VLPs in HeLa cells, highlighting their potential as a vaccine candidate and a tool for serodiagnosis. Further characterization of VLP epitopes and protective efficacy is warranted to advance vaccine development. ImportanceDengue remains a significant global health challenge, with serotype 1 being one of the dominant strains causing recurrent outbreaks in Nepal. Existing vaccines demonstrate limited efficacy and pose significant safety concerns, particularly in seronegative populations. To address these limitations, this study explores virus-like particles (VLPs) as a safer alternative vaccine platform. VLPs elicit robust immunogenicity by mimicking the structure of native virus while completely lacking genetic components. This study combines DENV1 structural proteins with optimized expression systems to enhance immunogenicity. This work is particularly significant as the first dengue vaccine research conducted in Nepal, directly addressing antigenic mismatches between existing commercial vaccines and locally circulating viral strains. Furthermore, the study provides scalable platform for developing region-specific dengue vaccines for other serotypes and flaviviruses.

Measurement of antibody responses to viral infection is essential for surveillance, diagnostics, epidemiological research, and natural history of infection studies. However, current methods to detect virus-specific antibodies are often resource-intensive and impractical for deployment in outbreak settings or in field-based studies. This manuscript presents two economical, high-throughput immunoassays--the cytoblot immunoassay (CBA) and strip immunoblot assay (SIA)--for detecting and quantifying anti-lymphocytic choriomeningitis mammarenavirus (LCMV) antibodies in mouse serum. To validate, we tested serum from acutely or persistently experimentally infected mice. Both assays detected LCMV-specific IgG and IgM antibodies with high sensitivity and specificity across multiple timepoints. By facilitating the study of immune responses in rodent reservoirs, these tools can enhance our understanding of zoonotic viral transmission, provide scalable platforms for outbreak preparedness, and serve as adaptable models for the development of rapid serological assays for other viral pathogens.

IntroductionMalaria is a leading health problem with high disease burden and mortality rates worldwide. Currently approved vaccines target the sporozoite form of Plasmodium falciparum that initially infects the liver, but only provide modest protection against malaria in young children. There is an urgent need to develop next-generation malaria vaccines that target multiple parasite developmental stages for greater efficacy. Antibodies to merozoites, which are involved in blood-stage replication, and are associated with clinical illness, have multiple functional activities and can protect against malaria. A promising merozoite vaccine candidate is Merozoite Surface Protein 2 (PfMSP2). Antibodies to PfMSP2 can promote multiple antibody Fc-mediated functional activities to clear merozoites. MethodsWe developed and evaluated monovalent and bivalent (3D7 and FC27 variants) PfMSP2-based mRNA vaccines. We designed and codon-optimised mRNA, which was validated for in vitro expression in mammalian cells, and subsequently formulated as lipid nanoparticles for vaccination of mice in a 3-dose regimen. Vaccination with recombinant PfMSP2 protein with adjuvant was performed for comparison. We evaluated the induction of antibodies and functional activities relevant to protective immunity. ResultsmRNA vaccines induced prominent IgG responses using monovalent (3D7 allele) and bivalent (3D7 and FC27 alleles) vaccines encoding near full-length PfMSP2, and antibodies recognised the surface of whole merozoites. Vaccine responses were equivalent to, or superior than, a recombinant protein-based PfMSP2 vaccine. The bivalent vaccine induced equivalent antibodies to the two PfMSP2 alleles. Vaccination induced cytophilic IgG subclasses with multiple functional activities, including complement fixation, binding of human Fc{gamma}-receptors I and IIa, and opsonic phagocytosis. ConclusionsPfMSP2 is highly immunogenic using the mRNA vaccine platform and induces antibodies with multiple functional activities associated with protective immunity in humans. Combining PfMSP2 with other merozoite and sporozoite antigens is a promising strategy to develop highly efficacious vaccines to achieve malaria control and elimination goals.

Here we evaluated the performance of a previously published tiling PCR primer scheme by Ringlander et al. (2022) for whole-genome amplification of Hepatitis B virus (HBV) in combination with Oxford Nanopore sequencing. The primer set originally developed for Ion Torrent sequencing was adapted by removing platform-specific adapters and tested using clinical serum or plasma samples submitted for routine HBV genotyping and resistance testing. Two multiplexing strategies were compared: a single PCR pool containing all primers and a two-pool strategy with non-overlapping amplicons. Sequencing reads were processed using a Nanopore analysis pipeline, and genome coverage and amplicon performance were compared across samples spanning a wide Ct range and representing HBV genotypes A-E. Across all samples, the median genome coverage was approximately 50%, although recovery varied widely, ranging from complete failure to nearly full genomes. Combining all primers into a single PCR reaction, or separating overlapping amplicons into different reactions, had little overall impact on genome recovery, and no consistent differences between the two pooling strategies were observed. In contrast, amplification efficiency differed markedly between individual amplicons. Amplicons 1-5 generally produced higher sequencing depth, whereas amplicons 6-10 frequently showed low coverage and contributed to incomplete genome recovery. Genome coverage was strongly associated with Ct values, with higher coverage observed in samples with lower Ct values, while coverage was broadly similar across genotypes. These results demonstrate that the Ringlander et al. primer scheme can be adapted for multiplex PCR and Nanopore sequencing of HBV, but uneven amplicon performance limits consistent full-genome recovery and highlights the need for further optimization of HBV tiling PCR designs.

The vast diversity of B and T cell receptors generated through the recombination of Variable (V), Diversity (D), and Joining (J) gene segments plays a critical role in adaptive immunity. Profiling immune repertoires at the DNA level provides a robust and stable approach to capture the clonal composition of these receptors. immunoPETE is an assay designed to target recombined human T-cell Receptor Beta (TRB), T-cell Receptor Delta (TRD), and Immunoglobulin Heavy (IGH) chain genes directly from genomic DNA. Simultaneous profiling of B and T cell receptor chains in a single reaction provides internally normalized clone counts and facilitates the study of B-T cell interactions. Full-length amplicon consensus sequences representative of original template DNA molecules are accurately reconstructed using Unique Molecular Identifiers (UMIs). An in-house pipeline compiles VDJ rearrangements from the Complementarity-Determining Region 3 (CDR3) of TRB, TRD and IGH chains into comprehensive readouts at cell-level resolution. In this study, we describe the immunoPETE end-to-end workflow, followed by a comprehensive benchmarking of its performance in adaptive immune profiling. Where applicable, we used both natural and contrived samples and characterized the assays accuracy, linearity, and reproducibility across several metrics: retrieving CDR3 sequences, determining B and T cell ratios, total cell count, yield, fraction of functional rearrangements, clonal diversity, composition of dominant clones, pairwise similarity, and V/J gene usage frequencies. Furthermore, we assessed its quantitative limits concerning the total number of lymphocytes and the detection of rare clones. As an example of its applications, we show that adding immune biomarkers extracted from immunoPETE data to clinical factors improves prediction of progression-free survival in a cohort of non-muscle invasive bladder cancer (NMIBC) patients. Finally, we discuss the broad applications of immunoPETE in the study of aging, cancers, infections, and autoimmune disorders with reference to select published studies.

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway. Author summaryIn response to antigenic stimulation, cognate B cells become activated and form germinal centers in lymphoid tissues. Germinal center B cells modify their antigen receptors through somatic hypermutation (SHM) of immunoglobulin variable region gene exons, with antigen selecting for high affinity B cells by providing survival advantage. This mechanism accounts for antibody affinity maturaton over the gradual course of an immune response. Affinity maturation is critical for generating potent, neutralizing antibodies against diverse strains of the human immunodeficiency virus-1 (HIV-1). These broadly neutralizing antibodies (bnAbs) are heavily mutated, reflecting lengthy affinity maturation over years of chronic infection. Recapitulating the affinity maturation process is a major challenge for bnAb induction by vaccination. In immunization experiments, bnAb development often stalls at rate limiting steps that involve infrequent, but functionally important, mutational events. Overcoming such obstacles requires boost immunogens that can stimulate the stalled B cells to acquire the requisite mutations. To this end, we recapitulated the maturation arrest of a bnAb lineage by expressing a stalled antibody in mouse germinal center B cells. Using this mouse model, we developed boost immunization conditions that advanced the antibody maturation beyond a roadblock to attain neutralizing activities against heterogenous viruses.

BackgroundIn the course of testing mAb-based therapies to eradicate the persistent reservoir of HIV infection, we investigated the efficacy and mode of killing of HIV-infected cells by two categories of cytotoxic immunoconjugates (CICs) targeted by the same mAb, an immunotoxin (IT) and antibody-drug conjugate (ADC). MethodsWe performed metabolic and transcriptional analyses of treatment effects on the persistently-infected cell line H9/NL4-3. Cells were treated with CICs consisting of the anti-gp41 mAb 7B2 conjugated to either deglycosylated ricin A chain (dgA) or to the highly cytotoxic anthracycline derivative PNU-159682. At intervals up to 24 hr, intracellular metabolites were quantified by 1H nuclear magnetic resonance spectroscopy, and the transcriptome analyzed by RNA-Seq. ResultsSix hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. 7B2-dgA treated cells exhibited elevated intracellular levels of many amino acids, and activation of gene pathways for apoptosis, intracellular signaling, and immune activation. By 24 hr, both 7B2-dgA and 7B2-PNU treated cells differed markedly from untreated. Many of the changes observed following 7B2-PNU treatment at 24 hr were similar to those observed at 6hr following 7B2-dgA, likely indicating processes involved in cell death, but a number of alterations were unique to either IT or ADC treated cells. ConclusionsAn IT and ADC showed both similarities and differences in their cytotoxic effects. These results raise the question of whether the mode of cell killing could be a determinant of clinical efficacy. Although these studies were aimed at targeting the persistent reservoir of HIV infection, they have relevance for the design of CICs to treat cancer and other conditions. SUMMARYThe use of cytotoxic immunoconjugates, wherein an antibody is attached to a cellular poison, is effective in the treatment of cancer and other conditions. We seek to extend these results to treating HIV and other chronic viral infections. We analyzed the molecular mechanisms of cell killing when the same antibody was attached to different toxic structures. We report that each immunoconjugate induced both common and distinct patterns of killing. Such differences may have clinical relevance.

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral V{gamma}9V{delta}2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized V{gamma}9V{delta}2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human V{gamma}9V{delta}2 T cells for ACT. We characterized peripheral blood V{gamma}9V{delta}2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic V{gamma}9V{delta}2 T-cell products as a scalable platform for next-generation cancer immunotherapies.

BackgroundHumans and mice display elevated levels of IL-27, an immunosuppressive cytokine shown to increase during neonatal bacterial sepsis and compromise survival. This study explores two hypotheses for regulation of IL-27 expression: 1) decreased DNA methylation in newborns that contributes to increased expression of IL-27 genes; 2) neonatal hormones regulate IL-27 expression through upstream hormone response elements (HREs). MethodsWhole genome methyl-seq analysis of neonatal and adult blood-derived macrophages identified differentially methylated regions (DMRs) at steady-state. Quantitative PCR (qPCR) measured expression of IL-27 genes (IL27p28 and EBI3) in human and murine neonatal macrophages stimulated in vitro with synthetic glucocorticoid or progesterone. Confocal microscopy and chromatin immunoprecipitation (ChIP) of glucocorticoid receptor (GR) assessed translocation into the nucleus and binding to the EBI3 promoter. ResultsThe IL-27p28 promoter contained DMRs that were increased in the neonatal cohort. The analysis did not identify DMRs within the EBI3 promoter. Dexamethasone stimulation increased EBI3 gene expression in human and murine neonatal macrophages. GR localized to the nucleus in response to dexamethasone and was enriched at the EBI3 upstream regulatory region. ConclusionThese data suggest glucocorticoid (GC) signaling increases EBI3 expression. This has importance in the context of antenatal GC administration that may increase IL-27 levels. Impact Statement{blacksquare} Elevated expression of IL-27 in early life impairs the host response to invasive bacterial infection in neonates. {blacksquare}Understanding the regulatory mechanisms contributing to increased IL-27 during the neonatal period is necessary to reduce susceptibility to infection in this vulnerable population. {blacksquare}The methylation status of the IL-27 genes in macrophages from neonatal and adult blood donors does not suggest regulation of differential expression with age. {blacksquare}Glucocorticoids are a signal that can induce EBI3 gene expression in a GR-dependent manner. {blacksquare}Glucocorticoid therapy for premature infants may increase IL-27 expression and promote enhanced susceptibility to infection.

Current clinical management of multiple myeloma (MM) relies on bone marrow (BM) biopsies for minimal residual disease (MRD) assessment. While BM biopsies are the gold standard, their invasive nature and potential to miss extramedullary or patchy disease necessitate sensitive, non-invasive liquid biopsy platforms. In this study, we evaluated the analytical performance of the CellSearch CMMC assay to determine its utility for deep-MRD monitoring. Using a standard 4 mL whole blood input, the assay achieves a WBC-normalized sensitivity of 2.45 x 10-7, supported by a limit of quantitation of 5 cells per run. Given this high analytical sensitivity, the assay provides a robust negative predictive value, rendering false-negative findings highly unlikely in populations with detectable peripheral disease. These findings characterize the CellSearch CMMC assay as a highly sensitive, analytically validated platform for non-invasive deep-MRD level longitudinal surveillance monitoring. When integrated into a clinical workflow that accounts for its specificity profile, the platform offers a patient-friendly complement to serial BM biopsies, with the potential to reduce their frequency in appropriate clinical contexts.

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.