Plasmablast Expansion Following the Tetravalent, Live-Attenuated Dengue Vaccine Butantan-DV in DENV-Naïve and DENV-Exposed Individuals in a Brazilian Cohort

Abstract

An effective vaccine against the dengue virus (DENV) should induce a balanced, long-lasting antibody (Ab) response against all four viral serotypes. The burst of plasmablasts in the peripheral blood after vaccination may reflect enriched vaccine-specific Ab secreting cells. Here we characterize the acute plasmablast responses from naïve and DENV-exposed individuals following immunization with the live attenuated tetravalent (LAT) Butantan DENV vaccine (Butantan-DV). The frequency of circulating plasmablasts was determined by flow cytometric analysis of fresh whole blood specimens collected from 40 participants enrolled in the Phase II Butantan-DV clinical trial (NCT01696422) before and after (days 6, 12, 15 and 22) vaccination. We observed a peak in the number of circulating plasmablast at day 15 after vaccination in both the DENV naïve and the DENV-exposed vaccinees. DENV-exposed vaccinees experienced a significantly higher plasmablast expansion. In the DENV-naïve vaccinees, plasmablasts persisted for approximately three weeks longer than among DENV-exposed volunteers. Our findings indicate that the Butantan-DV can induce plasmablast responses in both DENV-naïve and DENV-exposed individuals and demonstrate the influence of pre-existing DENV immunity on Butantan DV-induced B-cell responses.

Keywords: dengue; dengue infection; dengue vaccine; humoral response; plasmablast.

Figure 1

Study design. Naïve and DENV-pre-exposed volunteers received a single subcutaneous dose of the lyophilized vaccine consisting of 1,000 PFU of each attenuated virus or placebo (qualified Leibovitz L-15 medium only). Blood samples were collected pre-vaccination (baseline, day 0) and at days 3, 6, 9, 11-12, 15, 21-22, 28, 56 and 91 post-vaccination, and used for the assessment of vaccine induce-viremia, DENV neutralizing antibodies (nAbs) titers using plaque reduction test (PRNT), and plasmablast responses using flow cytometry.

Figure 2

Logarithm-transformed DENV neutralizing antibody (nAb) titers from naïve (white) (A) and DENV pre-exposed (gray) (B) volunteers at baseline and post-vaccination. Data was generated by 50% plaque-reduction neutralization titer assay (PRNT50). Circles and triangles represent median log titers for each DENV serotype in placebo and vaccinees volunteers, respectively. Horizontal lines span the 25^th-75^th percentiles (interquartile range). The values on the right of each time point column represents the p-values obtained after the analysis of placebo and vaccinees nAb titers using non-parametric Wilcoxon rank-sum test.

Figure 3

Representative flow cytometric analysis of circulating plasmablast in naïve (DENV (–)) and DENV-pre-exposed (DENV(+)) volunteers enrolled in the Phase II clinical trial evaluated before (baseline) and after vaccination (days 6, 11-12, 15, 21-22). All plots represent the frequency of CD27⁺ CD38⁺ cells gated on live CD3^- CD14^- CD19⁺ cells.

Figure 4

Plasmablast expansion (as the percentage of CD20^- CD27⁺ CD38⁺ cells among the CD19⁺ population) after Butantan-DV vaccination. Results are presented as pooled total plasmablast cells analyzed in a Boolean analysis. (A, B) Frequency of plasmablast in fresh blood from DENV (–) (A) and DENV(+) (B) volunteers at baseline and days 6, 11-12, 15 and 21-22 post-immunization. D, days post-vaccination. P, placebo. V, vaccinee. Horizontal bars represent median and interquartile range. Statistical analysis was performed using a non-paired t test. (C) Difference in the frequency of plasmablast comparing the values obtained at baseline and at the peak of plasmablast expansion (D15 after Butantan-DV vaccination) in naïve [DENV (–)] and DENV pre-exposed [DENV(+)) vaccinees. DENV(+) volunteers were divided into individuals pre-exposed to 1 or 2 or more serotypes, according to PRNT50 results to all four DENV serotypes at baseline. Data were analyzed with the Mann-Whitney U test.