Upregulation of plasma cell modules at viremia positive time points during reinfection and their absence post-vaccine boosts.

(A) Enrichment plots depicting differential enrichment of LM22 (PLASMA_CELLS and T_CELLS_FOLLICULAR_HELPER) and BTM (antiviral IFN signature (M75)) modules for the Early acute time point of the reinfection ([R]) relative to primary infection ([P]) using the following contrast: (REARLY-ACUTE−RPRE-REINFECTION)–(PEARLY-ACUTE−PPRE-INFECTION). Primary infection data were obtained from (Rosenberg et al, 2018). Each gene forming the indicated module is represented by vertical black lines along the x-axis. The green line connects enrichment scores (ES) indicated by the y-axis and genes. Red-dashed lines indicate the maximum and minimum ES reached. False discovery rate (FDR) and normalized ES (NES) are presented with each enrichment plot. (B-D) Immune cell subsets (LM22 and BTM datasets indicated on sidebar) transcriptomic signature enrichment among the list of ranked differentially expressed genes at primary infection (Resolvers; n = 6), prime vaccine timepoints (n = 6), secondary infection (Resolvers; n = 7) and boost vaccine timepoints (n = 6). Pre-Infection times were used as a baseline for primary and secondary infections timepoints. d0 and d56 were used as a baseline for prime vaccine and boost vaccine time points, respectively. The x-axis indicates timepoints. The y-axis indicates immune subset enriched in at least one timepoint with a Benjamini−Hochberg-corrected gene set enrichment analysis (GSEA) p value (B) innate immune cells, (C) B cells and (D) T cells. The presence (Grey) or absence (White) of viral RNA is indicated below corresponding primary and secondary infection samples.</p

HCV-specific antibodies in the plasma of spontaneous resolvers increase and neutralize H77 HCVpp.

(A) Longitudinal anti-E2 (Left and Middle panels) and NS3 (Right) IgG responses in plasma measured by ELISA and represented as OD450–570 for each subject (see Fig 1A for time point definitions). HCV antigens are indicated on top of each graph. Each symbol represents one subject (n = 8). The genotype of each infection is indicated next to each subject symbol as (primary infection | reinfection). (B) Longitudinal plasma neutralizing activity from all subjects against H77 HCVpp, J6/JFH1 HCVpp, and S52 HCVpp at 1:100 dilution, presented as percentage of neutralization relative to HCV-naive donor controls (n = 4). The dotted line delineates the 50% neutralization threshold. Results are presented as the mean of 3 independent experiments. For (A) and (B) repeated measure ANOVA with Tukey’s post hoc test was used. *P P C) Spearman correlations of all pairwise combinations of the designated parameters at the early acute time point. The intensity of the color and size of the circles reflect the values of corresponding correlations coefficients. Positive correlations are displayed in orange while negative correlations are displayed in purple. (TIFF)</p

Concerted increase of HCV-specific T and B cell responses during acute reinfection.

Results are presented per subjects with from top to bottom: (HCV RNA) Viremia status presented as viremia positive (+) and negative (-). (Longitudinal IFN-γ ELISpot responses and MHC class I tetramer+ CD8+ T cells). PBMCs from the indicated time points were tested in an IFN-γ ELISpot assay against overlapping peptide pools representing the HCV H77 (SR2, SR3, SR5, SR6, SR7 and SR8) or J4 polyprotein (SR1 and SR4). The frequencies of IFN-γ spot forming cell (SFC) per million PBMCs from all subjects are presented in each top graph. When available, longitudinal MHC class I tetramer+ CD8+ T cells frequencies were assessed. The following tetramers were utilized: A1-1436 (SR2), A2-1073 (SR5), B7-41 (SR7 and SR8) and B27-2841 (SR4 and SR6). Subjects SR4 and SR6 ELISpot results were previously published in [6]. Pre-reinfection time-point different from the ones mentioned in Table 1 were used for subjects SR2 (-133) and SR3 (-815). (ELISA) Anti-E2 (gt1a and gt2a) and NS3 (Right) IgG responses in plasma measured by ELISA and represented as OD450–570. (H77 HCVpp) Plasma neutralizing activity against H77 HCVpp presented as percentage of neutralization relative to naïve donors. The dotted line delineates the 50% neutralization threshold. (J6-E2 Tetramer) Longitudinal J6-E2 Tetramer+ MBCs frequencies. No PBMC samples were available at pre-reinfection for subject SR8 to evaluate the frequency of J6-E2 Tetramer+ MBCs.</p

LM22 and BTMs modules differentially enriched during HCV infections in PWID and post HCV vaccine prime and boost in healthy subjects.

(XLSX)</p

Activation and expansion of E2-specific MBCs following HCV reinfection.

(A) Representative gating strategy for detection of class-switched, E2-specific MBCs (CD3–CD14–CD16–CD56–CD19+CD27+IgM–E2tet+) in total PBMCs. (B) Longitudinal frequencies of E2-specific (J6-E2 Tet+) MBCs at the indicated time points (see Fig 1A). Threshold of detection of E2-specific MBCs (dotted line) was 0.277% (mean detection + 2SD from 5 naïf PWID). (C) Global t-SNE projection of CD3–CD14–CD16–CD56–CD19+ B cells from three subjects (SR3, SR7 and SR8) at the Early Acute time point, concatenated and overlaid. Each t-SNE projection reflects the expression of the indicated protein marker. (D) t-SNE projection of B cell clusters identified by FlowSOM clustering. (E) Heatmap shows mean fluorescence intensities (MFI) of each protein marker (columns), for each detected FlowSOM cluster (rows). (E) Representative gating strategy of resting MBCs (CD71–), activated MBCs (CD71+CD20hiCD38int-lo), antibody-secreting MBCs (CD71+CD20loCD38hi) shown for the total MBCs population (grey contour plots), and HCV E2-specific MBCs (purple contour plots). (F) Phenotypes of E2-specific MBCs in resting (blue), activated (orange), or antibody-secreting (green) states (cells that did not meet these categories are designated as Other and shown in grey) at Early Acute and Late Acute time points (n = 3; SR3, SR7 and SR8). Significance was determined by Two-way repeated measure ANOVA with Tukey’s post hoc test (ns: not significant).</p

Subjects’ Demographics and Clinical information for samples analyzed by RNA-seq.

Subjects’ Demographics and Clinical information for samples analyzed by RNA-seq.</p

Representative gating strategy for identifying HCV-specific CD8+ T cells using MHC class I tetramers.

Representative staining from the Late acute time point for all subjects studied are presented. Numbers in the upper right quadrant represent % Tetramer+CD8+CD3+ T cells. (TIFF)</p

Early acute reinfection transcriptional profile is enriched in B and T cells signatures.

(A-C) Heatmaps displaying the sample level enrichment analysis (SLEA) z-score of each of the LM22 and BTMs modules using leading edge genes of the Early acute versus pre-reinfection contrast among all reinfected subjects. Rows represent individual modules from either LM22 (light blue) or BTMs (grey). Each module category is indicated on the colored sidebar. Modules are further divided into: (A) innate immune cells, (B) B cells and (C) T cells. Colors indicate the SLEA z-score and show whether the immune subset is upregulated (SLEA z-score > 0; red) or downregulated (SLEA z-score (TIFF)</p

Study design and differential gene expression analysis.

(A) Reinfection study design. (B) Principal component analysis (PCA) of the top 500 genes expressed in the reinfection group. As described in Materials and Methods, patient-specific gene expression variation was removed using the function removeBatchEffect. Time points and groups are indicated by different colors and shapes, respectively. The ellipses denote the core area with a confidence interval of 68% for each group. (C) Number of differentially expressed genes (DEGs) across all reinfection time points. Pre-reinfection time-points were used as a baseline. Genes differentially expressed are selected using a Benjamin-Hochberg adjusted P value (D) Vaccine study design. Vaccine data were obtained from Hartnell et al. [18]. (E) PCA of the top 500 genes expressed in the vaccine group. (F) Number of differentially expressed genes (DEGs) across all vaccine samples. d0 and d56 were used as a baseline for prime and boost vaccine time points, respectively. Genes differentially expressed are selected using a Benjamin-Hochberg adjusted P value < 0.05.</p

Changes in gene expression in DC-induced latently infected CD4⁺ T cells.

<p>(<b>A</b>) Fold change scatterplot comparing gene expression between HIV T (+DC) (CD4⁺ T cells cultured with DC and HIV), and Mock T (+DC) (CD4⁺ T cells cultured with only DC) relative to their controls, HIV T (CD4⁺ T cells cultured with HIV) and Mock T (CD4⁺ T cells cultured in media alone) respectively. The solid line indicates absolute 2-fold change. (<b>B</b> and <b>C</b>) Top two gene interaction networks as ranked by Ingenuity Pathway Analysis. The networks were built from the list of differentially expressed genes induced by HIV T (+DC), relative to Mock T (+DC) after subtracting HIV T and Mock T from each group respectively. Genes highlighted in red were up-regulated and those in blue were down-regulated. The different node shapes indicate genes in different functional categories according to the legend. The interactions between the different nodes are shown as solid (direct interaction) or dashed (indirect interaction) lines (edges). (<b>D</b>) Fold change in gene expression values for selected genes from the Illumina BeadArrays plotted against Real-Time PCR (qPCR) deltaCt values for each target gene. PCR targets were mapped to BeadArray probes by matching the official gene symbols.</p