Abstract Introduction Secondary dengue virus (DENV) infection can lead to severe disease through antibody-dependent enhancement (ADE). However, after recovery, individuals develop broad immunity to all four DENV serotypes, DENV1-4. Methods To investigate the features of natural DENV immunity that drive enhancement versus protection, we evaluated 45 adults who were flavivirus naïve or previously DENV exposed and challenged them with a live DENV3 vaccine (NCT05691530). We measured baseline antibodies using binding and function assays and DENV-specific T cells to evaluate their association with viremia. Results To identify the determinants of viremia, regardless of infection history, we grouped immune participants based on the area under the curve of their viremia using unbiased clustering: those with low viremia had no boost in neutralizing antibodies, consistent with sterilizing immunity (N = 8). Intermediate viremia was of similar magnitude to naïve participants (N = 12), and high viremia was significantly greater than in other groups, as observed during enhanced dengue (N = 11). We found that fold-enhancement of DENV3 infection at a 1:10 serum dilution on a monocytic U937 cell line expressing CD16 (FcγRIII) was the strongest predictor of viremia, while neutralizing antibodies to mature DENV3 and greater neutralization breadth had the strongest protective effects. Neutralization measured using partially immature virus was not directly protective and instead showed non-linear associations with viremia. Sera that competed with a broadly neutralizing antibody for binding to the quaternary envelope-dimer epitope on DENV3 also had lower viremia, while binding responses to other serotypes were not protective. IFN-γ—secreting CD8+ T cells correlated with reduced viremia. Conclusion While enhancing antibodies determine susceptibility to viremia, neutralizing antibodies and antiviral CD8+ T cell responses are protective. Our results may help inform evaluation of future dengue vaccines and therapeutics. Funding Source This research was supported by the Intramural Research Program of the National Institutes of Health (NIH) as well as NIH Bench-to-Bedside Program Funds - Award # 994875, the NIH Director’s Challenge Innovation Award, ReVAMPP grant 1 U19 AI181960-01, and P Topic Categories Immune Mechanisms of Human Disease (HUM)

Abstract Introduction Pre-existing dengue immunity shapes subsequent immune responses, which can either be protective or pathogenic, though the underlying mechanisms remain poorly defined. Using a live attenuated monovalent DENV3 vaccine, we investigated how baseline immunity shapes cytokine, plasmablast, and clinical biomarker responses. Methods Participants were classified as naïve, heterotypic to a single serotype, or polytypic based on screening neutralizing antibodies, then restratified using area under the curve of viremia by qRT-PCR. We profiled 30 cytokines by Luminex and assessed plasmablast responses by ELISpot alongside standard clinical biomarkers. Results Low-viremia participants, most of whom had polytypic immunity, had early increases in innate cytokines, such as IL-6, IL-7, GM-CSF, and IFN-γ, followed by downregulated TNF-alpha and IL-2R and elevated MCP-1, lymphocyte, and monocyte counts, consistent with efficient viral control. The intermediate-viremia group showed early elevations in IFN-α and Th1-associated cytokines, followed by expansion of DENV3-reactive IgG+ and IgA+ plasmablasts, and IP-10 and IL1-RA induction, indicating a coordinated antiviral response with moderate inflammation. High-viremia participants, who mostly had heterotypic immunity, had more delayed increases in DENV-specific IgG+ plasmablasts and broader and more sustained elevations in proinflammatory and tissue-activating cytokines, such as G-CSF, VEG-F, and MIP-1 chemokines. Conclusion Pre-existing dengue immunity coupled with post-vaccine viremia delineates individuals into distinct profiles. We observed transient innate activation and controlled inflammation in low viremia participants, a coordinated adaptive response in the intermediate viremia group, and low-level but broad and persistent inflammation in the high viremia group. Understanding these profiles can help refine dengue interventions to improve safety and protective efficacy. Funding Source This research was supported by the Intramural Research Program of the National Institutes of Health (NIH) as well as NIH Bench-to-Bedside Program Funds - Award # 994875, the NIH Director’s Challenge Innovation Award, ReVAMPP grant 1 U19 AI181960-01, and P Topic Categories Viral Immunology (VIR)

Background: Compared to the potent efficacy of autologous hematopoietic cell transplantation (AutoHCT) on multiple sclerosis (MS) lesion activity, benefit on disability progression is less clear. Paramagnetic rim lesions (PRLs) are imaging biomarkers associated with progression.Objectives: Evaluate effect of AutoHCT on PRLs using 7T magnetic resonance imaging (MRI).Methods: A single rater evaluated PRLs before/after AutoHCT on 7T MRI at an MS referral center.Results: Seven participants were included with mean age 38.9 years and median disease duration 9 years. Median PRL count pre-transplantation was 2. Post-AutoHCT (median 15 months in 5 participants) none of the PRLs resolved.Conclusion: These results provide low-level evidence that PRLs do not resolve following AutoHCT.

Hematopoietic cell transplantation (HCT) offers curative potential for inborn errors of immunity (IEI) patients, who often enter HCT with significant comorbidities and disease sequelae, active infections, and/or limited donor options. Strategies to enhance engraftment must be balanced against graft-versus-host disease (GVHD) risk and the need for prompt immune reconstitution.Recipients of T cell-replete HLA-matched (related or unrelated) or HLA-haploidentical grafts (n = 44) received serotherapy-free, radiation-free reduced-intensity conditioning (pentostatin/cyclophosphamide/2 days busulfan), with PTCy, sirolimus, plus/minus mycophenolate mofetil (MMF) as GVHD prophylaxis. Given promising outcomes with very low GVHD incidence in 20 patients who received MMF on post-HCT days 5–35 (MMF35) (1), MMF duration was reduced (MMF18) and/or omitted (MMF0) via a duration de-escalation schema for the subsequent 24 patients (supplemental table).Overall and graft-failure-free survival, along with engraftment kinetics, were largely unaffected by MMF duration, but patients who received MMF18 or MMF0 attained full donor T cell chimerism earlier (Figure 1) and required fewer unplanned donor cell infusions. Lymphocyte reconstitution differed in the T cell, but not natural killer (NK) cell, compartment at days 28 and 42, with higher T cell counts in the MMF18 and MMF0 groups even accounting for graft type, but the effect disappeared by day 60 (Figure 2). Viral complications, including BK-associated cystitis incidence and duration, were similar across cohorts (supplemental table, Figure 2).Figure 1.Engraftment and survival by mycophenolate mofetil (MMF) exposure. Overall survival (A), graft failure-free survival (B), neutrophil recovery (defined as >500 neutrophils/uL on 3 consecutive days post-HCT) (C), and platelet recovery (defined as >20,000 platelets/uL on 3 consecutive days post-HCT without transfusion in preceding 7 days) (D) do not vary with MMF exposure, except for neutrophil recovery, where the effect disappeared when only marrow grafts were analyzed. Donor CD3+ chimerism at days 28, 42, and 60 is significantly higher in patients with reduced MMF exposure, even accounting for graft type (E). Statistical significance depicted as: ns (p > 0.05), *, (p 0.05, *, p 0.0001.Importantly, there was only 1 infection-related death in the MMF18 and MMF0 cohorts (4%), as compared to 3 in the MMF35 cohort (15%). Among 7 haploidentical HCTs using MMF18, 1 patient experienced engraftment syndrome, later dying of chemotherapy-associated lung toxicity, while another died of GVHD complications and idiopathic pneumonia syndrome; both had received peripheral blood stem cell (PBSC) grafts.Overall, steroid-refractory acute GVHD occurred only in 2 recipients, both with haploidentical HCTs at MMF18, compared to none in the original MMF35 cohort. Of note, chronic GVHD, mostly mild, occurred only in PBSC recipients.Reduced MMF duration/omission in our platform is associated with excellent outcomes for matched HCTs. Mismatched HCT outcomes and the role of PBSC vs. marrow graft receipt require further study with larger patient numbers. Across patients, reducing MMF exposure is associated with earlier donor T cell reconstitution and no adverse effect on survival or engraftment.Tabular data are included as downloadable supplement files.

Antigen-specific responses to complex antigens encompass a range of cell states and reactivities to an array of epitopes, reflective of the heterogeneity in immune responses. Single-cell sequencing has created new opportunities when combined with flow cytometry for profiling of immune repertoire and cell phenotype. Rare B cells with fine specificities can be precisely isolated using flow cytometry, and with oligonucleotide-tagged antigen assemblies enable the isolation of a pool of cells reactive to different antigens in parallel from biological samples. Single-cell sequencing can then detect these tags to allow mapping of individual B cell reactivities to specific antigens, in addition to simultaneous profiling of cellular phenotypes. We establish workflows for these approaches with antigen reactivity frequencies typical of human peripheral blood, by conducting all staining prior to sorting to minimize loss of rare antigen-specific cells. We identify antigen:streptavidin ratio as a priority for optimization when using novel antigens to generate oligonucleotide-tagged assemblies for B cell staining, which is demonstrated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigens. We develop a novel approach of combining dual labelling using separately oligonucleotide- and fluor-tagged assemblies, to increase staining sensitivity sufficiently for exploratory detection of dengue-reactive cells in naturally exposed individuals. Together these findings support parallel profiling of cells with differing antigen-specificities, conferring new opportunities to advance understanding beyond characterization of individual clonotypes to their role in the broader humoral response. This can be important in pathogen, vaccine, malignancy, and autoreactivity responses, where both antigen-reactive cells at low frequencies and interactions between different epitope responses can shape clinical outcomes.

Few studies have assessed hypomorphic variants in ZAP70, leading to profound, late-onset combined immunodeficiency. A previously healthy 21-year-old man presented with primary EBV B cell lymphoproliferative disease (LPD). He developed hemophagocytic lymphohistiocytosis treated with rituximab, although EBV LPD rapidly relapsed. He subsequently underwent matched, unrelated donor hematopoietic cell transplantation complicated by fatal hepatic sinusoidal obstructive syndrome. The family history included profound immune dysregulation in his younger sister, characterized by juvenile systemic lupus erythematosus/juvenile idiopathic arthritis overlap diagnosed at 7 years of age, HPV+ warts, and EBV viremia. She failed therapy with methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, rituximab, tofacitinib, abatacept, tocilizumab, and adalimumab. After NIH evaluation, she was confirmed to be HLA:B27-positive and had a marked clinical response to ustekinumab without loss of viral control. Maternal history included epidermodysplasia verruciformis, recurrent infectious rectovaginal fistulas, and lymphocytic colitis. Whole-exome sequencing (WES) revealed novel homozygous, splice-site variants in ZAP70 (c.1623+5G>A) in all 3 affected family members. WES homology was consistent with founder effect without consanguinity, although the mother and father were fourth cousins. cDNA analysis of patient cells confirmed aberrant splicing between exons 12 and 13 (Figure 1). Figure 1. cDNA generated from RNA of the proband, his affected sister, and his affected mother confirmed aberrant splicing at Exon 12 and Exon 13 of ZAP70, consistent with the homozygous, splice-site variant found on whole-exome sequencing. All affected family members had CD4 lymphopenia, although CD8 central and effector memory T cells were relatively preserved. Naïve CD4+ and CD8+ T cells were nearly absent in the proband and his sister. Assays in cells from all affected individuals showed reduced ZAP70-dependent T cell receptor stimulation (Figure 2) and decreased T cell proliferation to mitogens. Maternal lymphocyte phenotyping showed increased NK cells with strong cytolytic function, which may have contributed to her relatively mild phenotype. Markedly increased phospho-S6 and PD-1 expression in CD8 cells as well as increased CD4+ and CD8+ TIGIT and PD-1 double-positive cells were consistent with immune dysregulation and T cell exhaustion. Figure 2. T cells from the proband, his affected sister, and his affected mother showed decreased total ZAP70 protein as well as decreased ZAP70 signaling (phospho-ZAP70, phospho-LAT, and phospho-ERK). Here we present a 3-member kindred with variable expressivity of a novel homozygous, hypomorphic, splice-site variant in ZAP70. While all family members suffered from severe viral infections, they also presented with multiple features not typically associated with ZAP70 deficiency, including late-onset CID, profound immune dysregulation, and relative preservation of total and memory CD8+ T cell populations.

Viral infections of the central nervous system (CNS) are a major cause of morbidity largely due to lack of prevention and inadequate treatments. While mortality from viral CNS infections is significant, nearly two thirds of the patients survive. Thus, it is important to understand how the human CNS can successfully control virus infection and recover. Since it is not possible to study the human CNS throughout the course of viral infection at the cellular level, here we analyzed a non-lethal viral infection in the CNS of nonhuman primates (NHPs). We inoculated NHPs intracerebrally with a high dose of La Crosse virus (LACV), a bunyavirus that can infect neurons and cause encephalitis primarily in children, but with a very low (≤ 1%) mortality rate. To profile the CNS response to LACV infection, we used an integrative approach that was based on comprehensive analyses of (i) spatiotemporal dynamics of virus replication, (ii) identification of types of infected neurons, (iii) spatiotemporal transcriptomics, and (iv) morphological and functional changes in CNS intrinsic and extrinsic cells. We identified the location, timing, and functional repertoire of optimal transcriptional and translational regulation of the primate CNS in response to virus infection of neurons. These CNS responses involved a well-coordinated spatiotemporal interplay between astrocytes, lymphocytes, microglia, and CNS-border macrophages. Our findings suggest a multifaceted program governing an optimal CNS response to virus infection with specific events coordinated in space and time. This allowed the CNS to successfully control the infection by rapidly clearing the virus from infected neurons, mitigate damage to neurophysiology, activate and terminate immune responses in a timely manner, resolve inflammation, restore homeostasis, and initiate tissue repair. An increased understanding of these processes may provide new therapeutic opportunities to improve outcomes of viral CNS diseases in humans.