Antibody and memory B cell responses to the dengue virus NS1 antigen in individuals with varying severity of past infection

Abstract

To further understand the role of NS1-specific antibodies (Abs) in disease pathogenesis, we compared neutralizing antibody levels (Nabs), NS1-Ab levels, IgG antibody subclass profiles and NS1-specific memory B-cell responses (Bmems) in individuals, with varying severity of past dengue. Nabs (Neut50 titres) were assessed using the Foci Reduction Neutralization Test (FRNT) and in-house ELISAs were used to assess NS1-Abs and NS1-Ab subclasses for all four DENV serotypes in individuals with past DF (n = 22), those with past DHF (n = 14) and seronegative (SN) individuals (n = 7). B-cell ELISpot assays were used to assess NS1-specific Bmem responses. 15/22 (68.18%) individuals with past DF and 9/14 (64.29%) individuals with past DHF had heterotypic infections. Neut50 titres were found to be significantly higher for DENV1 than DENV2 (p = 0.0006) and DENV4 (p = 0.0127), in those with past DHF, whereas there was no significant difference seen in titres for different DENV serotypes in those with past DF. Overall NS1-Ab to all serotypes and NS1-specific IgG1 responses for DENV1, 2 and 4 serotypes were significantly higher in those with past DHF than individuals with past DF. Those with past DHF also had higher IgG1 than IgG3 for DENV1 and DENV3, whereas no differences were seen in those with past DF. Over 50% of those with past DF or DHF had NS1-specific Bmem responses to >2 DENV serotypes. There was no difference in the frequency of Bmem responses to any of the DENV serotypes between individuals with past DF and DHF. Although the frequency of Bmem responses to DENV1 correlated with DENV1-specific NS1-Abs levels (Spearman r = 0.35, p = 0.02), there was no correlation with other DENV serotypes. We found that those with past DF had broadly cross-reactive Nabs, while those with past DHF had higher NS1-Ab responses possibly with a different functionality profile than those with past DF. Therefore, it would be important to further evaluate the functionality of NS1-specific antibody and Bmem responses to find out the type of antibody repertoire that is associated with protection against severe disease.

Keywords: B-cell ELISpots; NS1 antigen; antibodies; dengue; dengue fever; disease severity; neutralizing antibodies.

FIGURE 1

Neutralizing antibody levels (Neut50 titres) to DENV1, DENV2, DENV3 and DENV4 in those with varying severity of past dengue infections. Neutralizing antibody (Nab) levels were measured by FRNTs in those who had past DF (n = 22) and past DHF (n = 11). The differences in the Nab (Neut50 titres) between different DENV serotypes were analysed in those with past DF (a) and past DHF (b) using the Friedman test. All tests were two sided. The lines represent the median and the interquartile range.

FIGURE 2

NS1‐specific antibody responses to the four DENV serotypes in those with varying severity of past dengue infections. NS1‐specific antibodies (Abs) to the DENV serotypes were assessed by an in‐house ELISA in those with past DF (n = 22), those with past DHF (n = 14) and seronegative individuals (n = 7). Comparison of NS1‐Ab responses between the serotypes in those with past DF (a) and past DHF (b) were analysed using the Friedman test. The differences between NS1‐specific antibody responses in individuals with past DF, past DHF and seronegative (SN) individuals were analysed using the Mann–Whitney U test (c). Comparison of DENV2 NS1‐Ab responses in those with past DF, those with past DHF and seronegative (SN) individuals with the DENV2 NS1‐specific Ab responses of convalescent samples of individuals with primary DENV2 infections (n = 17) were analysed using the Mann–Whitney U test (d). All tests were two‐tailed. The horizontal dotted line represents the positive cut‐off (mean ± 2 SD of the background responses). The lines represent the median and the interquartile range.

FIGURE 3

NS1‐specific IgG1 and IgG3 responses to the four DENV serotypes in those with varying severity of past dengue infections. NS1‐specific IgG1 and IgG3 antibody levels were measured for the four DENV serotypes in individuals with past DF (n = 22), those with past DHF (n = 14) and seronegative (SN) individuals (n = 7) using an in‐house ELISA. Comparison in IgG1 responses (a) and IgG3 responses (b) between those with past DF, DHF and seronegative individuals were analysed using the Mann–Whitney U test. The horizontal dotted line represents the positive cut‐off (mean ± 2 SD of the background responses). Comparison between IgG1 and IgG3 responses in those with past DF, past DHF and seronegative individuals to DENV1 (c), DENV2 (d), DENV3 (e) and DENV4 (f) were analysed using the Wilcoxon signed rank test. All tests were two‐tailed. The lines indicate median and the interquartile range.

FIGURE 4

NS1‐specific memory B‐cell responses (Bmem) to all four DENV serotypes in an individual with past DHF. The Bmem responses were measured by B‐cell ELISpot assays to DENV NS1 for DENV1 (1A), DENV2 (2A), DENV3 (3A) and DENV4 (4A), and responses to culture media, which was used as negative controls (1B, 2B, 3B and 4B). The positive control wells show the total IgG‐secreting cells (5) for the same individual. Each test was been carried out in duplicate.

FIGURE 5

The frequency of NS1‐specific memory B‐cell (Bmem) responses to the four DENV serotypes in individuals with varying severity of past dengue infections. The Bmem responses were measured by B‐cell ELISpot assays in those who had past DF (n = 22), past DHF (n = 11) and seronegative individuals for the four DENV serotypes (SN) (n = 7). The differences in the frequency of Bmem responses for four DENV serotypes were analysed in those with past DF (a) and past DHF (b) using the Friedman test. Comparisons in NS1‐specific antibody responses between individuals with past DF, past DHF and seronegative (SN) individuals were analysed using the Mann–Whitney U test (c). All tests were two‐tailed. The lines indicate median and the interquartile range.