B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts

Abstract

Dengue virus (DENV) infection results in the production of both type-specific and cross-neutralizing antibodies. While immunity to the infecting serotype is long-lived, heterotypic immunity wanes a few months after infection. Epidemiological studies link secondary heterotypic infections with more severe symptoms, and cross-reactive, poorly neutralizing antibodies have been implicated in this increased disease severity. To understand the cellular and functional properties of the acute dengue virus B cell response and its role in protection and immunopathology, we characterized the plasmablast response in four secondary DENV type 2 (DENV2) patients. Dengue plasmablasts had high degrees of somatic hypermutation, with a clear preference for replacement mutations. Clonal expansions were also present in each donor, strongly supporting a memory origin for these acutely induced cells. We generated 53 monoclonal antibodies (MAbs) from sorted patient plasmablasts and found that DENV-reactive MAbs were largely envelope specific and cross neutralizing. Many more MAbs neutralized DENV than reacted to envelope protein, emphasizing the significance of virion-dependent B cell epitopes and the limitations of envelope protein-based antibody screening. A majority of DENV-reactive MAbs, irrespective of neutralization potency, enhanced infection by antibody-dependent enhancement (ADE). Interestingly, even though DENV2 was the infecting serotype in all four patients, several MAbs from two patients neutralized DENV1 more potently than DENV2. Further, half of all type-specific neutralizing MAbs were also DENV1 biased in binding. Taken together, these findings are reminiscent of original antigenic sin (OAS), given that the patients had prior dengue virus exposures. These data describe the ongoing B cell response in secondary patients and may further our understanding of the impact of antibodies in dengue virus pathogenesis.

Importance: In addition to their role in protection, antibody responses have been hypothesized to contribute to the pathology of dengue. Recent studies characterizing memory B cell (MBC)-derived MAbs have provided valuable insight into the targets and functions of B cell responses generated after DENV exposure. However, in the case of secondary infections, such MBC-based approaches fail to distinguish acutely induced cells from the preexisting MBC pool. Our characterization of plasmablasts and plasmablast-derived MAbs provides a focused analysis of B cell responses activated during ongoing infection. Additionally, our studies provide evidence of OAS in the acute-phase dengue virus immune response, providing a basis for future work examining the impact of OAS phenotype antibodies on protective immunity and disease severity in secondary infections.

FIG 1

Generation of MAbs from plasmablasts of patients with secondary DENV2 infection. (A) Percentages of plasmablasts (defined as CD19⁺ CD3⁻ CD27^hi CD38^hi lymphocytes) among total CD19⁺ B cells determined by flow cytometry. (B) Representative ELISPOT analysis showing total and dengue virus-specific IgG-secreting cells for Pt. 31 and Pt. 33. Each well shown contained 222 PBMCs. The numbers below the wells are spot counts. (C) Total and dengue virus-specific IgG-secreting cells per million PBMCs for all patients. Above each bar is the percentage of total IgG-secreting cells that were dengue specific. (D) Serum neutralization titers were determined by FFA, and FRNT₅₀ values are shown. FFAs were performed on serum samples collected the same day the plasmablasts were sorted for MAb synthesis. The values are from two or more independent experiments, with the mean FRNT₅₀ plotted. The dotted line represents the maximum dilution factor of serum tested (1:200). Each serum sample was tested against all four DENV serotypes in FFA. (E) The antibodies generated were tested for binding using the same antigen as in the ELISPOT assay. Antibodies from all the patients are shown.

FIG 2

Plasmablasts induced during secondary DENV infection are highly affinity matured and are clonally related. (A) V_H mutation frequency in the dengue patient cohort compared to historical data. Each circle represents the average number of V_H nucleotide mutations per donor. Somatic hypermutation frequencies in naive, memory, germinal center, and influenza virus-specific peripheral B cells were derived from previously published data (36). Statistical analyses were performed using an unpaired, two-tailed t test. *, P < 0.05; **, P < 0.005. (B) Ranges of V_H mutation frequencies in all four dengue patients. Each square represents the number of V_H mutations per MAb sequence. (C) R/S mutation ratios in the entire V_H gene, CDRs, and FR. The ratios are based on the average numbers of R and S mutations for all MAb sequences per patient. A ratio above 2.9 (dotted line) suggests antigenic selection. (D) Clonality in plasmablast-derived heavy-chain sequences for all the patients. The number at the center of each pie chart is the total number of heavy-chain sequences analyzed (MAb sequences plus unpaired V_H sequences that were not pursued for MAb synthesis). The asterisks indicate clonal groups for which a MAb exists in our panel. The percentage of clonal sequences is also shown.

FIG 3

Patient plasmablast-derived MAbs display highly heterogeneous binding and neutralization activities. (A) Each MAb was tested against all four dengue virus serotypes for binding and neutralization activity. To test binding, ELISAs were performed with rE proteins from DENV1 to -4. The values plotted represent the minimum concentrations required to obtain three times the background signal obtained with plain blocking buffer. Neutralization activity was determined by FFA, and FRNT₅₀ values are shown. FFAs were performed with duplicates in two or more independent experiments, and the mean value is plotted. The dotted lines represent the maximum concentrations of MAbs tested in ELISA (5 μg/ml) and FFA (20 μg/ml). (B) Summary of rE binding patterns of MAbs as determined by ELISA. Each bar represents one patient, and the sections within the bar indicate the extents of cross-reactivity, as shown in the legend on the far right. (C) Summary of DENV neutralization patterns of MAbs as determined by FFA. FRNT₅₀ values of ≤5 μg/ml were considered a positive result for neutralization. The bars are coded as for panel B.

FIG 4

Secondary DENV2 infection induced the activation of DENV1-specific plasmablasts in patients. (A) MAb FRNT₅₀ values against DENV1 to -4 as determined by FFA. Each circle represents one MAb, and the solid lines connect the same MAb across serotypes. The dotted lines represent the highest concentration of MAbs tested in the FFA (20 μg/ml). Antibodies below the dotted line failed to neutralize the virus by 50% at 20 μg/ml. (B) Comparison of binding versus neutralization activities of mononeutralizing MAbs. Binding was tested by rE ELISA and capture virus ELISA. 31.3H04, shown on the left of the dashed line, is provided as a contrasting example of a MAb with full cross-reactivity. Each MAb was tested against all four DENV serotypes in ELISA and FFA.