DHF Project

Category: Publications

  • An Innovative, Prospective, Hybrid Cohort-Cluster Study Design to Characterize Dengue Virus Transmission in Multigenerational Households in Kamphaeng Phet, Thailand

    in

    Anderson KB, Buddhari D, Srikiatkhachorn A, Gromowski GD, Iamsirithaworn S, Weg AL, Ellison DW, Macareo L, Cummings DAT, Yoon IK, Nisalak A, Ponlawat A, Thomas SJ, Fernandez S, Jarman RG, Rothman AL, Endy TP

    Am. J. Epidemiol. 2020 Jul;189(7):648-659

    PMID: 31971570

    Abstract

    Difficulties inherent in the identification of immune correlates of protection or severe disease have challenged the development and evaluation of dengue vaccines. There persist substantial gaps in knowledge about the complex effects of age and sequential dengue virus (DENV) exposures on these correlations. To address these gaps, we were conducting a novel family-based cohort-cluster study for DENV transmission in Kamphaeng Phet, Thailand. The study began in 2015 and is funded until at least 2023. As of May 2019, 2,870 individuals in 485 families were actively enrolled. The families comprise at least 1 child born into the study as a newborn, 1 other child, a parent, and a grandparent. The median age of enrolled participants is 21 years (range 0-93 years). Active surveillance is performed to detect acute dengue illnesses, and annual blood testing identifies subclinical seroconversions. Extended follow-up of this cohort will detect sequential infections and correlate antibody kinetics and sequence of infections with disease outcomes. The central goal of this prospective study is to characterize how different DENV exposure histories within multigenerational family units, from DENV-naive infants to grandparents with multiple prior DENV exposures, affect transmission, disease, and protection at the level of the individual, household, and community.

  • Next-generation sequencing of 11 HLA loci in a large dengue vaccine cohort from the Philippines

    in

    Geretz A, Cofer L, Ehrenberg PK, Currier JR, Yoon IK, Alera MTP, Jarman R, Rothman AL, Thomas R

    Hum. Immunol. 2020 Aug;81(8):437-444

    PMID: 32654962

    Abstract

    HLA genotyping by next-generation sequencing (NGS) has evolved with significant advancements in the last decade. Here we describe full-length HLA genotyping of 11 loci in 612 individuals comprising a dengue vaccine cohort from Cebu province in the Philippines. The multi-locus individual tagging NGS (MIT-NGS) method that we developed initially for genotyping 4-6 loci in one MiSeq run was expanded to 11 loci including HLA-A, B, C, DPA1, DPB1, DQA1, DQB1, DRB1, and DRB3/4/5. This change did not affect the overall coverage or depth of the sequencing reads. HLA alleles with frequencies greater than 10% were A*11:01:01, A*24:02:01, A*24:07:01, A*34:01:01, B*38:02:01, B*15:35, B*35:05:01, C*07:02:01, C*04:01:01, DPA1*02:02:02, DPB1*05:01:01, DPB1*01:01:01, DQA1*01:02:01, DQA1*06:01:01, DQB1*05:02:01, DQB1*03:01:01, DRB1*15:02:01, DRB1*12:02:01, DRB3*03:01:03, DRB4*01:03:01, and DRB5*01:01:01. Improvements in sequencing library preparation provide uniform and even coverage across all exons and introns. This has led to a marked reduction in allele imbalance and dropout. Furthermore, including more loci, such as DRB3/4/5, decreases cross-mapping and incorrect allele assignment at the DRB1 locus. The increased number of loci sequenced for each sample does not reduce the number of samples that can be multiplexed on a single MiSeq run and is therefore more cost-efficient. We believe that such improvements will help HLA genotyping by NGS to gain momentum over other conventional methods by increasing confidence in the calls.

  • Analysis of cell-associated DENV RNA by oligo(dT) primed 5′ capture scRNAseq

    in

    Sanborn MA, Li T, Victor K, Siegfried H, Fung C, Rothman AL, Srikiatkhachorn A, Fernandez S, Ellison D, Jarman RG, Friberg H, Maljkovic Berry I, Currier JR, Waickman AT

    Sci Rep 2020 Jun;10(1):9047

    PMID: 32493997

    Abstract

    Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5′ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5′ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5′ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5′ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5′ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

  • Transcriptional and clonal characterization of B cell plasmablast diversity following primary and secondary natural DENV infection

    in

    Waickman AT, Gromowski GD, Rutvisuttinunt W, Li T, Siegfried H, Victor K, Kuklis C, Gomootsukavadee M, McCracken MK, Gabriel B, Mathew A, Grinyo I Escuer A, Fouch ME, Liang J, Fernandez S, Davidson E, Doranz BJ, Srikiatkhachorn A, Endy T, Thomas SJ, Ellison D, Rothman AL, Jarman RG, Currier JR, Friberg H

    EBioMedicine 2020 Apr;54:102733

    PMID: 32315970

    Abstract

    Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.

  • Longitudinal Analysis of Memory B and T Cell Responses to Dengue Virus in a 5-Year Prospective Cohort Study in Thailand

    in

    Sánchez-Vargas LA, Kounlavouth S, Smith ML, Anderson KB, Srikiatkhachorn A, Ellison DW, Currier JR, Endy TP, Mathew A, Rothman AL

    Front Immunol 2019;10:1359

    PMID: 31263466

    Abstract

    Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ > IL-2 > IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses.