Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients

Abstract

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Figure 1. Increased turnover of B and T cells in acute DENV infection

(A) Representative flow cytometry contour plots illustrating the gating strategy to define B cell and T cell subsets (left), and expression of Ki-67 and active caspase-3 (Cas-3) on B cells from a healthy naïve individual and individuals with DF and DFC (right). Boxes are defined based on fluorescence with isotype control Ab. (B) Percentage (left) and absolute number (right) of T cells and B cells within the PBMC of each group. Symbols represent individual subjects and horizontal lines represent means. (C) Percentage of T cells and B cells expressing Ki-67 and active caspase-3 in the different groups. (D) Correlation between the percent of T cells and B cells expressing Ki-67 and active caspase-3. HN=healthy naïve, OFI=other febrile illness, DF=dengue fever, DFC=complicated dengue fever, HR=healthy recovered, P=primary infection, S=secondary infection. *P<.05, ** P< .01 and *** P<.001 vs. OFI; # P<.05 vs. secondary DFC (dengue groups only).

Figure 2. Increased activation of B cells in acute DENV infection

(A) Representative flow cytometry contour plots illustrating the gating strategy to determine expression of CD69 and CD95 on B cells in a healthy naïve individual and individuals with DF and DFC. Boxes are defined based on fluorescence with relevant isotype control Ab. (B) Percentage of B cells expressing CD69 and CD95 in the different groups. Symbols represent individual subjects and horizontal lines represent means. (C) Correlation between the percent of B cells expressing CD95 and active caspase-3. HN=healthy naïve, OFI=other febrile illness, DF=dengue fever, DFC=complicated dengue fever, HR=healthy recovered, P=primary infection, S=secondary infection. * P<.05 vs. OFI.

Figure 3. Marked expansion of plasmablasts in severe secondary DENV infection

(A) Representative flow cytometry contour plots illustrating the gating strategy to define naïve B cells (black), resting memory B cells (blue), atypical memory B cells (orange), activated memory B cells (green) and plasmablasts (red) in a healthy individual and individuals with OFI, DF and DFC. (B) Percentage of B cells that are plasmablasts in 45 patient samples with either primary (closed diamonds) or secondary (open squares) acute DENV infection analyzed at different intervals after the onset of symptoms. The interval between the dotted lines indicates the peak response. (C) Percentage of naïve B cells, resting memory B cells, atypical memory B cells, activated memory B cells and plasmablasts at 4 to 7 days of symptoms in the different groups. Symbols represent individual subjects and horizontal lines represent means. (D) The relative proportion of B cell subsets in each group shown as stacked bars. HN=healthy naïve, OFI=other febrile illness, DF=dengue fever, DFC=complicated dengue fever, P=primary infection, S=secondary infection. * P<.05, ** P< .01 vs. OFI; # P<.05, ## P<.01, ### P<.001 vs. secondary DFC.

Figure 4. Plasmablasts in severe secondary DENV infection are DENV-specific and serotype cross-reactive but preferentially react with the infecting serotype

(A) Representative Elispot assay depicting plasmablasts secreting Ab reactive with BSA, influenza virus or DENV-3 compared with the total number of IgG-secreting cells from a healthy naïve individual (HN) and a patient with secondary DFC (DFC-S). Numerals represent number of spots. 10,000 cells were used for each condition except for total IgG and DENV-3 in the DFC-S individual where 1,000 cells were used. (B, C) Graphical representation of the number of Ab-secreting cells per 1 million PBMC (B) and the percent of all Ab-secreting cells (C) that are reactive with BSA, influenza virus and DENV-3 in healthy naïve individuals compared with individuals with secondary DFC. **P<.01, ***P<.001 vs. BSA; ### P<.001 vs. influenza virus. **P<.01, ***P<.001 vs. BSA control; ### P<.001 vs. influenza virus. (D) Representative Elispot assay depicting plasmablasts secreting Ab reactive with BSA, DENV-1, DENV-2 or DENV-3 in a patient with secondary DFC. Numerals represent number of spots. 2,500 cells were used per well. (E) Graphical representation of the number of Ab-secreting cells per 1 million PBMC that are reactive with BSA or each of the 3 DENV serotypes in individuals with secondary DFC. ***P<.001 vs. BSA; ## P<.01 vs. DENV-3.

Figure 5. Lack of correlation between plasmablast response and serum neutralizing Ab titers

(A) Reciprocal 50% neutralizing Ab (PRNT₅₀) titers to each of the four DENV serotypes in sera of patients with primary and secondary DENV infections and either DF or DFC, measured at 4 to 7 days after onset of symptoms (A) and at convalescence (C), as determined by plaque reduction neutralization test. Convalescent samples were taken 15 to 54 days after onset of symptoms except for two secondary DFC patients (closed triangle and closed square) taken 2 and 3 days after the acute sample, respectively. Each symbol represents the response of an individual patient across each of the four DENV serotypes. Individuals with secondary DFC correspond to those with the same symbols in Figure 5 where applicable. (B) Correlation between percent plasmablasts at day 4 to 7 after onset of symptoms and the corresponding PRNT₅₀ titer of serum Ab against DENV-1 and DEN-3 in patients with secondary DF (closed circles) and DFC (open squares).