DHF Project

Category: Publications

  • 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.

  • Peripheral follicular helper T cells in acute viral diseases: a perspective on dengue

    in

    Sánchez-Vargas LA, Mathew A

    Future Virol 2019 Mar;14(3):161-169

    PMID: 31073324

    Abstract

    Follicular helper T cells (T) are a predominant subset of CD4 T cells specialized in providing help to B cells in germinal centers and necessary to generate T cell-dependent antibody responses. Peripheral T (pT) are the counterpart of T found in the circulation, which resemble T in many aspects of their phenotype and function. The CD4 pT subset has received a lot of interest recently because they are easy to access and have the potential to serve as a biomarker for long-lasting humoral immunity. This review will discuss recent findings of pT in human acute viral diseases with a focus on dengue infection.

  • Using Multiple Scale Space-Time Patterns in Variance-Based Global Sensitivity Analysis for Spatially Explicit Agent-Based Models

    in

    Kang JY, Aldstadt J

    Comput Environ Urban Syst 2019 May;75:170-183

    PMID: 31728075

    Abstract

    Sensitivity analysis (SA) in spatially explicit agent-based models (ABMs) has emerged to address some of the challenges associated with model specification and parameterization. For spatially explicit ABMs, the comparison of spatial or spatio-temporal patterns has been advocated to evaluate models. Nevertheless, less attention has been paid to understanding the extent to which parameter values in ABMs are responsible for mismatch between model outcomes and observations. In this paper, we propose the use of multiple scale space-time patterns in variance-based global sensitivity analysis (GSA). A vector-borne disease transmission model was used as the case study. Input factors used in GSA include one related to the environment (introduction rates), two related to interactions between agents and environment (level of herd immunity, mosquito population density), and one that defines agent state transition (mosquito extrinsic incubation period). The results show parameters related to interactions between agents and the environment have great impact on the ability of a model to reproduce observed patterns, although the magnitudes of such impacts vary by space-time scales. Additionally, the results highlight the time-dependent sensitivity to parameter values in spatially explicit ABMs. The GSA performed in this study helps in identifying the input factors that need to be carefully parameterized in the model to implement ABMs that well reproduce observed patterns at multiple space-time scales.