Hayden Siegfried

Walter Reed Army Institute of Research

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Richard G. Jarman

Walter Reed Army Institute of Research

Kaitlin Victor

LabCorp (United States)

Adam T. Waickman

SUNY Upstate Medical University

Jeffrey R. Currier

Walter Reed Army Institute of Research

Heather Friberg

Walter Reed Army Institute of Research

Anon Srikiatkhachorn

King Mongkut's Institute of Technology Ladkrabang

Damon W. Ellison

Walter Reed Army Institute of Research

Alan L. Rothman

Providence College

Gregory D. Gromowski

Walter Reed Army Institute of Research

Stefan Fernandez

Armed Forces Research Institute of Medical Science

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Walter Reed Army Institute of Research

Armed Forces Research Institute of Medical Science

SUNY Upstate Medical University

National Institutes of Health

University of Maryland, College Park

University of Maryland, Baltimore

Integral Molecular (United States)

International Vaccine Institute

National Institute of Allergy and Infectious Diseases

University of Massachusetts Chan Medical School

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

Scientific Reports (2020)

Mark A. Sanborn Tao Li Kaitlin Victor Hayden Siegfried Christian Fung

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.

Primer (cosmetics)

10.1038/s41598-020-65939-5

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Citations (16)

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections

PLoS Pathogens (2021)

Adam T. Waickman Heather Friberg Gregory D. Gromowski Wiriya Rutvisuttinunt Tao Li

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

Flavivirus

10.1371/journal.ppat.1009240

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Citations (29)

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques

npj Vaccines (2021)

Michael K. McCracken Caitlin Kuklis Chandrika B. Kannadka David A. Barvir Mark A. Sanborn

Abstract Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1–4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

Viremia

Dengue vaccine

Seroconversion

Heterologous

Priming (agriculture)

Antibody-dependent enhancement

10.1038/s41541-021-00339-y

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Citations (13)

Temporally integrated single cell RNA sequencing analysis of controlled and natural primary human DENV-1 infections

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Adam T. Waickman Heather Friberg Gregory D. Gromowski Wiriya Rutvisuttinunt Tao Li

Abstract Controlled dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To bridge this knowledge gap, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 controlled human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both controlled and natural DENV infection resulted in overlapping patterns of inflammatory gene upregulation, natural DENV infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by controlled and natural primary DENV infection are similar, but that natural DENV infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state

Flavivirus

10.1101/2020.07.16.206557

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Citations (0)

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

EBioMedicine (2020)

Adam T. Waickman Gregory D. Gromowski Wiriya Rutvisuttinunt Tao Li Hayden Siegfried

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.

Humoral immunity

Immunoglobulin M

10.1016/j.ebiom.2020.102733

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Citations (32)

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

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Mark A. Sanborn Tao Li Kaitlin Victor Hayden Siegfried Christian Fung

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. IMPORTANCE Dengue is one of the most widespread vector-borne viral diseases in the world. However, it is still unclear which cells harbor virus during DENV infection, and how viral reservoirs in serum and infected cells are related. These results demonstrate for the first time that intracellular DENV RNA can be identified and infected cells quantified by 5’ capture scRNAseq. This strategy provides a significantly greater throughput and higher sensitivity than previously published methods and has the potential to provide additional information on the genomic heterogeneity of intracellular DENV RNA.

Primer (cosmetics)

10.1101/2020.01.23.917757

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Citations (0)

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

The Journal of Immunology (2020)

Adam T. Waickman Gregory D. Gromowski Tao Li Hayden Siegfried Kaitlin Victor

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 molecular-level diversity, specificity, and functionality of the antibody response 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. Unexpectedly, we 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 and appeared to be derived from memory B cells, even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional proteomics and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive and DENV-neutralizing Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results identify a heretofore unappreciated role for DENV-reactive IgA in the humoral response to DENV infection, and may offer insight into the differential pathogenesis associated with primary and secondary DENV infections.

Humoral immunity

Memory B cell

10.4049/jimmunol.204.supp.247.5

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Citations (1)