Twenty-five subjects were determined for T cell assays: 13 who had a symptomatic secondary DENV infection (six with DENV1, six with DENV2, and one without a serotype determined), six who had a subclinical infection, and six with no DENV infection. dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural Jionoside B1 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 Jionoside B1 of IFN- IL-2 IFN-/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 Rabbit Polyclonal to NDUFA4 demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses. Dual Color IL-2 and IFN- Enzymatic ELISPOT Assay The ELISPOT assay was performed according to the manufacturer’s instructions (CTL, Cleveland, OH, USA). Cryopreserved PBMC were thawed and plated at a density of 1C2 105 cells/well. Peptide pools were added at a final concentration of 2 g/ml/peptide. As a positive control, PBMC were Jionoside B1 incubated with anti-CD3 and anti-CD28 antibodies at final concentrations of 1 1 and 0.1 g/ml, respectively. As a negative control, PBMC were incubated with medium. PBMC were stimulated for 45 h at 37C with 5% CO2. As a positive control, every plate had PBMC from a well-characterized DENV-immune subject tested with the same conditions. The number of spots per well was determined using an automated ELISPOT reader (S5UV analyzer, CTL, Cleveland, OH, USA) with the double color software. Determinations from duplicate wells were averaged. Data were analyzed by subtracting the mean number of spots in the wells with cells and medium-only from the mean counts of spots in wells with cells and antigen and expressed as spot-forming cells (SFC) per 106 PBMC. If the response to anti-CD3/CD28 antibodies was below 500 IFN- SFC per million PBMC, the sample was excluded. Statistical Analysis Statistical analysis was performed using GraphPad Prism software V 8.00 (GraphPad Software Inc., La Jolla, CA). The non-parametric Mann-Whitney or Wilcoxon signed rank test was used to compare two groups as appropriate. Statistical significance was set at 0.05. Results Characteristics of the Study Population PBMC from 27 subjects were selected for study based on available data from 5-years of follow-up (Table S2). Sixteen subjects were selected for B cell assays: six who had symptomatic DENV infection, four who had a subclinical infection and six subjects with no DENV infection. Twenty-five subjects were selected for T cell assays: 13 who had a symptomatic secondary DENV infection (six with DENV1, six with DENV2, and one without a serotype determined), six who had a subclinical infection, and six with no DENV infection. For the symptomatic and subclinical infection groups, we selected subjects who experienced an infection in the first or second year of the study to allow us to test PBMC collected 1C2 years before and 3C4 years after secondary DENV infection. HAI antibody titers to the four DENV serotypes at baseline (start of the study) were higher in the subclinical and no DENV infection groups compared to the symptomatic DENV1 and DENV2 groups ( 0.05) (Figure S1). Memory B Cell Responses to DENV To study.