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

Abstract

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.

Fig 1. Subject characterization and experimental overview.

A) Kinetics of RNAemia and DENV IgM/IgG seroconversion in experimental primary DENV-1 infection subjects. Serum IgM/IgG titers calculated as highest serum dilution providing a 2X signal over background. B) Kinetics of IgM/IgG seroconversion and duration of fever in natural primary DENV-1 infection subjects analyzed in this study. DENV-reactive serum IgM/IgG levels shown as EIA units.

Fig 2. Annotation and quantification of major leukocyte populations following experimental or natural primary DENV-1 infection.

A) Integrated UMAP projection of scRNAseq data from all annotated leukocyte populations derived from all experimental primary DENV-1 infection subjects (n = 3, 8 time points per subject) and natural primary DENV-1 infection subjects (n = 2, 3 time points per subject). B) Expression of key linage defining gene products across all annotated leukocyte populations captured in this analysis. C) Integrated UMAP projection of all annotated leukocyte populations split by subject, including all time points. D) Relative population abundance in all subjects split by time point.

Fig 3. Temporal and transcriptional characterization of experimental primary DENV-1 infection and identification of conserved gene signatures.

A) Quantification of differential gene expression across all major leukocyte populations following experimental primary DENV-1 infection. Subject- and population-specific differentially expressed genes (DEGs) were defined by a Wilcoxon rank-sum test with a Bonferroni correction relative to study day 0 for each subject. B) Population-restricted frequency and temporal dynamics of cDEGs across all experimental primary DENV-1 infection subjects. Conserved DEGs defined as DEGs observed in all three subjects at the same time point relative to baseline. C) Expression of select upregulated cDEGs from study day 10 within the indicated cell populations across all study time points. D) Expression of select suppressed cDEGs from study day 10 within the indicated cell populations across all study time points.

Fig 4. Temporal and transcriptional characterization of natural primary DENV-1 infection and identification of conserved gene signatures.

A) Quantification of differential gene expression across all major leukocyte populations following natural primary DENV-1 infection. Subject- and population-specific differentially expressed genes (DEGs) were defined by a Wilcoxon rank-sum test with a Bonferroni correction relative to the 6-month control sample for each subject. B) Population-restricted frequency of cDEGs from both natural primary DENV-1 infection subjects. C) Expression of select induced cDEGs within the indicated cell populations across all study time points. D) Expression of select induced cDEGs within the indicated cell populations across all study time points.

Fig 5

Transcriptional profile overlap of experimental and natural primary DENV-1 infection A) Frequency and cell-population distribution of infection-induced cDEGs identified following experimental or natural primary DENV-1 infection. Experimental primary DENV-1 infection cDEGs restricted to study day 10. B) Frequency and overlap of infection-induced cDEGs identified following experimental or natural primary DENV-1 infection C) Frequency and cell-population distribution of infection-suppressed cDEGs identified following natural primary or experimental DENV-1 infection. Experimental primary DENV-1 infection cDEGs restricted to study day 10. D) Frequency and overlap of infection-suppressed cDEGs identified following natural or experimental primary DENV-1 infection. F) Schematic representation of the differential gene expression responses observed following either natural or experimental primary DENV infection.