Ole Lagatie

Bulk B- and T-cell adaptive immune receptor (BCR/TCR) repertoire sequencing (AIRRseq) enables comprehensive analysis of adaptive immune diversity, but achieving balanced sequencing depth across heterogeneous clinical samples remains a major cost driver. Standard equimolar pooling prior to sequencing disproportionately allocates reads, leading to undersequencing of libraries with high sequence abundance and oversampling of those with few sequences. Here, a quantitative pooling strategy is described in which libraries are pooled based on estimated target-specific unique molecular identifier (UMI) counts rather than library molarity, ensuring a uniform number of supporting reads per target sequence UMI across samples. Libraries were prepared from human RNA using the NEBNext Immune Sequencing (IS) kit and quantified by qPCR during the second amplification (PCR2) step. Deeply sequenced samples from a dengue 1 human infection model (DHIM1) clinical study were used to model the log-linear relationship between PCR2 cycle threshold (Ct) values and detected sequence counts. This model was then applied to a second, independent batch of samples from a dengue 3 human infection model (DHIM3) clinical study to predict UMI abundance and guide equi-depth pooling prior to sequencing. Sequencing of these equi-depth pools demonstrated a near-uniform reads-per-sequence ratio across samples, confirming that this approach achieves balanced depth without oversampling and at reduced cost. Simulations across a range of between-sample UMI-count distributions reflecting real-world sample pools showed that equi-depth pooling is expected to reduce the total number of required sequencing reads by approximately 70% compared with equimolar pooling, with savings increasing as the spread of library sizes grows. Beyond cost efficiency, equi-depth pooling eliminates the complexity-dependent sampling bias inherent to equimolar pooling, ensuring uniform sequencing depth and comparable UMI recovery across small and large libraries. For new workflows or sample types, implementation requires either an existing calibration dataset or a pilot sequencing experiment to establish the relationship between PCR2 Ct values and relative UMI abundance. The equi-depth method thus provides a robust, scalable, and cost-efficient strategy for bulk AIRRseq studies where library sizes vary widely.

Background: Dengue virus (DENV) is the most widespread mosquito-borne viral pathogen, affecting nearly half of the global population. Current serological diagnostics fail to meet WHO ASSURED criteria, to define ideal point-of-care diagnostics, largely due to reliance on laboratory-based workflows or reduced specificity caused by cross-reactivity with related orthoflaviviruses.Methods: In this study, we applied orthoflavivirus proteome-wide peptide microarray screening (n = 10,150 peptides) on samples from dengue-, Zika- and yellow fever-convalescent and negative individuals (n = 80) to map dengue-specific antigenic regions with minimal cross-reactivity. Peptide biomarker candidates from the most reactive regions were selected and translated into multiplex peptide immunoassays, evaluated using well-characterized longitudinally collected DENV1-4 samples (n = 426) and non-dengue controls (n = 243), and further applied into a lateral flow rapid diagnostic test format.Findings: We identified both pan-dengue and serotype-specific markers across structural and non-structural viral proteins, with epitopes from C, NS1, and NS3 achieving ≥85% sensitivity at ≥95% specificity. Including peptide variants and designing multi-epitope antigens show further assay improvement. Finally, we demonstrated, for the first time, the translation of dengue peptide-based biomarker candidates into a lateral flow assay.Interpretation: These findings highlight linear epitope-based biomarkers as robust alternatives to conventional serological targets such as whole proteins or viral lysates, and support their further development for next-generation, point-of-care dengue diagnostics.

Dengue virus infection triggers complex innate and adaptive immune responses, yet the molecular mechanisms that shape early antiviral immunity remain incompletely defined. We performed longitudinal single-cell multi-omics profiling of peripheral blood mononuclear cells from four flavivirus-naïve adults experimentally infected with DENV-1, integrating 5′ scRNA-seq with paired surface proteomics. Across 95,841 high-quality cells collected at baseline and on days 8 and 10 post-infection, we observed a strong interferon-driven transcriptional response accompanied by marked immune-cell redistribution, including expansion of monocytes and transient reductions in dendritic cells and double-negative T cells. Cytotoxic and helper lymphocyte populations, particularly naïve, central memory, and effector memory CD8+ T cells, showed extensive crosstalk with monocytes and NK cells, reflecting coordinated cytokine production and cytotoxic activation. Early B cell activation was evident through increased immunoglobulin gene expression. Innate sensing pathways, including RIG-I and Toll-like signaling, were activated across NK, T, and B cell subsets, while also demonstrating enrichment of antigen processing and apoptosis programs. Pro-inflammatory and cytotoxic signatures peaked at day 8, supported by broad upregulation of interferon-stimulated, pro-apoptotic, and regulatory genes. Together, these findings define a robust IFN-driven antiviral state and coordinated activation of immune cell subsets, providing new insights into the immune dynamics of primary dengue infection.

Dengue virus (DENV) poses a major global health burden, with limited vaccine availability and concerns that immunization of dengue-naïve individuals may exacerbate disease severity due to antibody-dependent enhancement. This challenge highlights the need for deeper insight into primary immune responses. Using a controlled human challenge model, we conducted a longitudinal analysis of B-cell receptor repertoire dynamics during primary DENV1 infection. Our study reveals that the acute-phase response is dominated by memory-derived B-cell clones, indicating pre-existing cross-reactive immunity. Concurrently, we identified highly public, convergent B-cell clones arising from naïve B cells, characterized by shared sequence features across individuals. These clones exhibit atypical maturation kinetics: while they switch to the IgG isotype during the convalescent phase, they retain germline-like sequences with limited somatic hypermutation. This suggests that affinity maturation is delayed compared to canonical responses. Our findings provide mechanistic context for previous reports of natural antibody responses in dengue and refine current models of how neutralizing and potentially enhancing antibodies emerge following primary infection. By resolving the timing and origin of early B-cell responses, this work contributes to a more precise framework for understanding flavivirus immunity. These insights may guide vaccine design strategies that aim to elicit protective immunity without enhancing the risk of severe disease. One Sentence Summary Primary DENV1 infection elicits convergent clones that undergo atypical maturation.

Background: Dengue is a global health concern, with half of the world’s population at risk and no antiviral treatment available. This Phase 0 study investigated dengue infections among household contacts (HHCs) of dengue index cases (ICs) and assessed the feasibility of conducting a Phase 2 trial for a novel antiviral. Methods: Participants were enrolled in Nha Trang, Vietnam, from April 2022 to February 2023. Dengue ICs were identified within 72 h of fever onset, and their healthy adult HHCs enrolled within 48 h. Blood samples and questionnaires were collected bi-weekly for four weeks, with a follow-up visit on day 40. DENV RT-qPCR, NS1, and anti-DENV IgM/IgG ELISAs were performed. Results: Overall, 130 dengue ICs and 301 HHCs were enrolled, with 91.7% (276/301) completing all follow-up visits. Baseline anti-DENV IgG showed prior dengue infections in 262/301 HHCs (87.0%). Fifty HHCs were excluded from the HHC infection analysis based on evidence of a DENV infection (viral load [VL], NS1, IgM, and IgG results) at enrollment. During follow-up, 2.0% of HHCs (5/251) had DENV infections based on virological parameters (DENV RNA and/or NS1 positivity), and anti-DENV IgG/IgM seroconversion was detected in 7.2% (18/251). Conclusions: This study demonstrated the operational feasibility of a dengue IC-HHC design for a Phase 2 trial.

Current serologic tests for HIV screening and confirmation of infection present challenges to the adoption of HIV vaccines. The detection of vaccine-induced HIV-1 antibodies in the absence of HIV-1 infection, referred to as vaccine-induced seropositivity/seroreactivity, confounds the interpretation of test results, causing misclassification of HIV-1 status with potential affiliated stigmatization. For HIV vaccines to be widely adopted with high community confidence and uptake, tests are needed that are agnostic to the vaccination status of tested individuals (ie, positive only for true HIV-1 infection). Successful development and deployment of such tests will require HIV vaccine developers to work in concert with diagnostic developers. Such tests will need to match today's high-performance standards (accuracy, cost-effectiveness, simplicity) for use in vaccinated and unvaccinated populations, especially in low- and middle-income countries with high HIV burden. Herein, we discuss the challenges and strategies for developing modified serologic HIV tests for concurrent deployment with HIV vaccines.

While the detection of HIV infection is accomplished by several methods, the most common are serological tests that detect host antibodies produced in response to viral infection. However, the use of current serological tests may present a significant challenge to the adoption of an HIV vaccine in the future because the antibodies to HIV antigens detected in currently available tests also tend to be included as antigens in the HIV vaccines in development.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.