Center for Biologics Evaluation and Research

Immune imprinting, in which prior antigenic exposures biases recall toward dominant epitopes, constrains the breadth and durability of influenza vaccine protection. Here we show that targeted variation across multiple hemagglutinin (HA) head sites (A, B, and D) between sequential A(H3N2) vaccines reprograms epitope hierarchy-redirecting recall toward conserved, subdominant head and stem epitopes. In a controlled ferret model mimicking imprinting-like recall in humans, antigenically distant priming accelerates neutralizing antibody induction, broadens reactivity, enhances cross-protection, and reduces viral shedding after drifted virus challenge. Epitope mapping and structural analysis confirms redirection toward conserved epitopes; single-cell transcriptomics and ELISpot assays reveal amplified germinal center B cell and Th1 responses. This "epitope hierarchy reshaping" links targeted antigenic variation to enhanced B cell competition, amplified T cell help, and improved viral control. This principle is likely applicable to vaccines against other rapidly evolving viruses where strong imprinting effects similarly limit immune breadth.

Alignment-free signals in coding sequences provide a scalable route to taxonomic inference, quality control of large phylogenies, and rapid screening of genomic data. We present a model-agnostic framework that represents genomes or coding-sequence collections using codon usage (64-D) and codon-pair usage (4096-D) profiles, and we introduce Taxonomic Consistency (TC), a simple, rank-aware external index, for evaluating supervised predictions or unsupervised clustering against the hierarchical taxonomy. Across multiple taxonomic ranks (e.g., Domain, Order, etc.), compact supervised models and standard clustering methods are assessed with both internal (Silhouette) and external (TC) validation. In large-scale experiments, handling class imbalance and applying principled normalization had a greater impact on performance than sequence-level preprocessing, and codon usage profiles yielded the highest TC and coherent unsupervised structure, while codon-pair features provided complementary resolution within specific clades. We release open, versioned code and reproducible workflows to facilitate adoption in ecological and evolutionary pipelines. Taken together, alignment-free codon and codon-pair representations, paired with TC for transparent external validation, offer a practical complement to tree-based methods-especially for rapid screening, sanity checks, and exploratory analyses at higher ranks.

Despite advances in blood safety, emerging infectious agents continue to pose risks to the blood supply. Traditional nucleic acid testing assays primarily target known pathogens, limiting the detection of novel microbes. Nanopore metagenomic sequencing enables agnostic identification of diverse pathogens, potentially enhancing transfusion safety surveillance. We assessed the analytical performance and limit of detection (LoD) of a nanopore MinION metagenomic sequencing workflow for pathogen detection in clinical plasma samples. DNA/RNA was extracted from 14 archived samples from individuals with confirmed infections (HIV, HBV, HCV, WNV), followed by cDNA synthesis, barcoded library preparation, and nanopore sequencing. Quantitative reference panels were tested, and data were analyzed using a comprehensive bioinformatics pipeline. Metagenomic sequencing generated over 2 million reads, with 2.3% of reads mapping to microbial sequences. Key transfusion-transmissible viruses, including West Nile virus (WNV), HIV, and hepatitis C virus (HCV), were reliably detected, while protocol modifications enabled HBV identification. Strain-level characterization identified HIV-1 subtype B, HCV genotypes 1a, 2b, 4a, HBV genotype C, and WNV lineage 1A. High viral loads produced extensive genome coverage, while lower loads yielded limited recovery. Co-infections, including human pegivirus (HPgV-2) and torque teno virus (TTV), were identified. The workflow detected viral targets at 103 genome copy equivalents (GCEs)/mL, with > 50% genome coverage achieved at 104 GCE/mL. Nanopore metagenomic sequencing enables comprehensive detection of blood-borne pathogens in plasma samples. This approach offers a promising complementary strategy for enhancing transfusion safety surveillance, and the demonstrated strain-level characterization supports its potential utility for blood safety applications.

Abacavir (ABC)-induced hypersensitivity is associated with HLA-B*57:01, with tolerance maintained by regulatory T cells (Treg). This study hypothesized that the balance between Treg and CD8+ T-cells is influenced by the level of human leucocyte antigen (HLA) expression to determine hypersensitivity versus tolerance to abacavir. HLA-B*57:01 transgenic (Tg) mice and Tg mice lacking H2-KbDb (Tg/KO) were treated with ABC with or without IFN-α or IFN-γblockade. Immune cell phenotype and functions were assessed via flow cytometry, and splenic transcriptomic profiles were analyzed to evaluate gene expression changes associated with treatment. ABC treatment of Tg/KO mice, which only express HLA-B*57:01 as an MHC-I allele, led to the expansion of drug-specific CD8+ T-cells, enhanced antigen presenting cell (APC) activity, and reduction in Tregs. These changes were accompanied by increased IFN-α levels and plasmacytoid dendritic cell (pDC) maturation. Depletion of pDCs or blocking of IFN-αR, in contrast to IFN-γ blockade, restored Treg populations and diminished CD8+ T-cell responses. These findings demonstrate that IFN-α, primarily produced by pDCs, disrupts Treg expansion, whereas IFN-γ predominantly enhances APC maturation and activation. Transcriptomic analysis revealed key genes regulated exclusively by ABC treatment in the presence of both IFN-α and IFN-γ signaling and demonstrated that IFN-α signaling predominantly modulates innate and adaptive immune genes while supporting the downregulation of Treg-related genes. In vivo upregulation of HLA expression modulates the balance between drug-specific and regulatory T cells, thereby promoting optimal pDC expansion and enhancing IFN-α production, which counteracts Treg-mediated ABC tolerance. IFN-γ secretion by drug-specific CD8+ T-cells promoted APC and effector T-cell function that led to the development of ABC-associated hypersensitivity reactions in immunocompetent HLA Tg mice.

Background: Physiologically based pharmacokinetic (PBPK) modeling is applied to address clinical pharmacology issues including dose selection and exposure assessments for special populations (e.g., pediatrics, and renally or hepatically impaired patients). The objective of this study was to evaluate the predictive performance of a PBPK model for dosing assessment of intravenous immunoglobulin (IVIG) and anti-D immunoglobulin (anti-D Ig) products in pregnant women. Methods: A minimal PBPK (mPBPK) model that incorporates pregnancy-specific physiological parameters and allometric scaling approaches was developed and evaluated for predicting the exposure of IVIG and anti-D Ig in pregnant women. The concentration versus time data were obtained from the published literature. Results: The IVIG (n = 22) and anti-D Ig (n = 29) concentrations were predicted using the mPBPK model with an average fold error of 1.17 and 1.22, respectively. A total of 100% and 95% of IVIG concentrations were predicted within the 0.5–2-fold and 0.5–1.5-fold prediction error ranges, respectively. For anti-D Ig, predictions fell within the 0.5–2-fold and 0.5–1.5-fold ranges for 93% and 76% concentrations, respectively. A mPBPK model-based simulation following administration of 0.5 g/kg IVIG in 100 virtual nonpregnant and pregnant subjects revealed that the maximum plasma concentration (Cmax) was 15% lower and trough concentration (Ctrough) was 8% lower during the third trimester of pregnancy compared to nonpregnant subjects. In contrast, with flat dosing, Cmax and Ctrough were 32% and 26% lower in pregnant subjects, respectively. Overall, the model demonstrated reasonable predictive performance, and bodyweight-based dosing regimen is an acceptable approach that results in minimal change in exposure of IVIG in pregnant women.

The nucleus and uropod are the largest and most mechanically distinct structures in migrating amoeboid lymphocytes, including NK, B, and T cells. The biophysical properties of these structures may shape the ability of immune cells to navigate dense tissue microenvironments during immune surveillance. Using bead-spring and agent-based cell models, we explore the biomechanical contributions of the nucleus, uropod, septin-templated cortical rings, actomyosin cytoskeleton, and extracellular matrix obstacles to lymphocyte migration. Our results support a migration model in which, following cell-matrix collisions, septins mediate the formation of cortical rings that hydraulically seal cytoplasmic compartments on each side of the passing nucleus, generating a pressure difference that propels the nucleus forward. This hydraulically driven nuclear piston actively enhances migration through confined spaces. Concurrently, the uropod emerging from the peristaltic collapse of rear compartments stabilizes directional persistence and prevents T cell repolarization. We show that such polarity stabilization boosts immune surveillance efficiency. Together, these models redefine the nucleus as an active component of the migratory engine and the uropod as a locomotion stabilizer. Furthermore, the models offer a predictive framework towards engineering of immune cell motility in complex tissue microenvironments with broad implications for cancer immunotherapy, aging, and regenerative medicine.

Chimeric antigen receptor-expressing NK (CAR-NK) cells represent an advancing frontier in cancer immunotherapy, building upon decades of natural killer cell research and recent breakthroughs in CAR technology. While early CAR-NK manufacturing protocols have demonstrated feasibility, existing manufacturing methods, whether utilizing cord blood or peripheral blood sources, often require extended culture periods and intensive labor, creating bottlenecks for widespread therapeutic application. To address these manufacturing hurdles, we have developed an optimized protocol for ex vivo CAR-NK cell production from human peripheral blood that incorporates lessons learned from previous methodologies while introducing novel efficiency improvements. This protocol offers a practical solution for scalable CAR-NK cell manufacturing that can be readily adapted across different production facilities, potentially accelerating the clinical development of CAR-NK therapies.

CD47 is an antiphagocytic ("don't eat me") signal expressed on all cells that inhibits programmed cell removal of self, and loss of this molecule by aging erythrocytes is associated with increased susceptibility to clearance by macrophages. Previously, we have demonstrated that absence of CD47 confers resistance to infection with nonlethal murine malaria Plasmodium yoelii 17XNL. Furthermore, CD47 blockade with an anti-CD47 monoclonal antibody promotes survival and reduces pathologic features of experimental cerebral malaria (ECM) during infection with lethal murine malaria P. berghei ANKA (Pb-A). To further define the immunological basis of CD47 regulation of ECM, we compared Pb-A infection in wild-type (WT) versus CD47 genetic knockout (CD47-/-) mice on the C57BL/6 background. Absence of CD47 resulted in partial but significant (log-rank P < 0.001) resistance to ECM and was associated with diminished sequestration of natural killer (NK) cells, CD4+ T cells, and CD8+ T cells (immune cell subsets essential for ECM pathogenesis) within the brain and lower levels of circulating interleukin-10 and MIP1β cytokines. Most importantly, our data show a critical role for CD47 in the maturation, activation, and recruitment of brain-sequestered NK cells. In mice experiencing ECM, CD47 associates with the exclusive expression of Ly49h (an activation receptor that elicits IFN-γ and cytolytic activity upon stimulation) by a subset of brain-sequestered CD11b+CD49b+ NK cells. Our studies provide insights on a previously undefined role of CD47 in NK cell-mediated pathogenesis of ECM and an increased understanding of the role of CD47 in NK cell biology.