We analyzed the frequency and serotype-specificity of DENV-specific B cells following acute DENV infection by use of ELIspot assays [31
]. Our data show that B cells are readily activated in vitro to secrete antibodies at early and late time points in children with primary and secondary infection. Other recent studies have examined antibody responses to DENV infection [19
]. However, our study is novel in several respects. First, we used ELIspot assays to assess the frequency of DENV-specific memory B cells; other studies examined polyclonal antibody responses in serum samples or studied monoclonal antibodies, which may be a less accurate representation of the B-cell repertoire available to respond to a subsequent infection. Second, we used samples from children with primary and secondary DENV infection. Third, we included infections with all 4 serotypes of DENV. Fourth, we tested PBMCs obtained at early and late convalescence, thus allowing us to examine the repertoire of B cells available to respond over time.
Our analysis revealed significantly higher frequencies of DENV-1–specific B cells in PBMCs of subjects with primary DENV-1 infection during early convalescence. Although the comparison of subjects infected with different serotypes was planned, no differences have previously been reported in antibody responses in primary cases according to serotype. We utilized purified recombinant proteins produced under the same conditions in the ELIspot assay, which measures only the frequency of antigen-specific ASCs; it is therefore unlikely that this finding is an artifact of the assay conditions. Our finding is consistent with the results of a recent study by Huynh et al [32
], who reported that viral RNA levels in primary DENV-1 cases were significantly higher than those in primary DENV-2 or DENV-3 cases or in secondary DENV-1 cases. Another possible explanation for these results is a longer duration of viremia in primary infections compared with secondary infections, which is supported by our previous findings in this cohort [3
]. The main objective in this study was to compare the frequency of antigen-specific ASCs in primary versus secondary DENV infections. We selected a representative subset of subjects from our clinical study to include all serotypes and both dengue fever (DF) and DHF. Mean frequencies of E-specific B cells were not significantly different in patients with mild disease (DF) compared with those with severe disease (DHF; mean, 1,330 vs 867 APCs per 106
cells, respectively). However, the small number of subjects with the same DENV serotype, serologic response (primary vs secondary), and clinical outcome (DF vs DHF) limits the statistical power for important subgroup analyses.
We found striking serotype–cross-reactivity to the E protein (with B cells responding to heterologous serotypes as well as or better than to homologous serotypes) in children experiencing secondary DENV infections, in contrast to serotype-specific responses in primary infections. When detectable, DENV-specific B cells in many subjects had a different profile of serotype–cross-reactivity at the 6-month time point compared with the same subjects early during convalescence. Despite the higher frequency of DENV-1–specific B cells in early convalescence in children with primary DENV-1 infection, this higher frequency was not maintained 6 months later. Memory B cells that were present earlier may have trafficked to tissues such as bone marrow during late convalescence [33
]. Increased serotype–cross-reactivity at the later time point could indicate either preferential survival of the cross-reactive B cells or maturation of the B-cell response. Although we are unable to exclude the possibility that another exposure occurred between early and late convalescence, we believe that it is unlikely that some subjects may have been exposed to another DENV infection between the 2 time points. First, DENV infections tend to be seasonal in Thailand. Because we enrolled patients during the peak transmission season, the 6-month interval would tend to fall outside the peak season. In addition, prior studies have shown a period of resistance to re-infection lasting 3–6 months after an acute DENV infection. Our data suggest that DENV-specific memory B-cell responses in humans evolve over the first 6 months after infection.
Our finding that culture supernatants from PBMCs stimulated in vitro react with prM peptides indicates that some antibodies bind linear epitopes. Of particular interest were responses to the linear peptide prM 18 (WILRHPGFTIMAAILAY). A commonly used monoclonal antibody 2H2 recognized amino acids within the first membrane-spanning domain of the M protein (GFTVMAAILAYIIGTT) [9
]. To our knowledge, this is the first report of human antibodies directed against a linear prM epitope. Most known neutralizing epitopes are conformational, but continuous amino acid sequences corresponding to antigenic determinants of the proteins that elicit antibodies in DENV-infected patients have only been mapped to the E protein [34
]. Dejnirattisai et al [21
] found a large number of monoclonal antibodies directed against prM, which together with our findings indicates that both conformational and linear epitopes need to be further defined on the prM protein.
We used a B-cell ELIspot assay in these studies to measure the frequency and specificity of DENV-specific B cells in the peripheral blood. The development of B-cell ELIspot-based assays has only recently facilitated the study of human B-cell immune responses in the peripheral blood [31
]. The B-cell ELIspot assay detects B cells that secrete IgG capable of binding to DENV antigens, and it does not give further information about antibody function (eg, neutralization or enhancement). We found a trend toward significance in correlation between ASCs detected by ELIspot assay and HAI titers in patients with secondary DENV-1 infection and between ASCs detected by ELIspot assay and PRNT50
titers in patients with primary DENV-1 infection. Our data indicate that the relationship between DENV-specific antibody titers in the serum and the frequency of DENV-specific memory B cells in the blood is still not well defined and warrants further investigation. Halliley et al [27
] found that peak frequencies of ASCs following influenza virus immunization correlated with the increase in serum HAI titers.
We chose an antigen-independent method used by several groups to stimulate B cells in the peripheral blood, because frequencies of ASC precursors in PBMCs are extremely low [27
]. The predictive value of B-cell ELIspot assays for protection from infection remains to be established. The technical problems with neutralizing antibody assays for DENV, and the poor performance of these assays as a correlate of protective immunity, have been well described elsewhere [36
]. With the advent of clinical vaccine trials, there is considerable interest in additional measures of humoral immunity to DENV, and the B-cell ELIspot assay warrants further study as a potential biomarker of protective immunity.