Ratio of dengue infections to notified cases by age in Colombo Municipal Council, 2009.

<p>Blue line: Model 1 (constant force of infection); Red line: Model 2 (different forces of infection above and below 6 years).</p

Observed prevalence by age (with 95% CIs) and model-predicted values.

<p>Blue line: Model 1 (constant force of infection); Red line: Model 2 (different forces of infection above and below 6 years).</p

Number and percentage of children positive for flavivirus antibodies, Colombo, Sri Lanka, 2008–9.

<p>Number and percentage of children positive for flavivirus antibodies, Colombo, Sri Lanka, 2008–9.</p

Dissecting the human serum antibody response to secondary dengue virus infections

<div><p>Dengue viruses (DENVs) are mosquito-borne flaviviruses and the causative agents of dengue fever and dengue hemorrhagic fever. As there are four serotypes of DENV (DENV1-4), people can be infected multiple times, each time with a new serotype. Primary infections stimulate antibodies that mainly neutralize the serotype of infection (type-specific), whereas secondary infections stimulate responses that cross-neutralize 2 or more serotypes. Previous studies have demonstrated that neutralizing antibodies induced by primary infections recognize tertiary and quaternary structure epitopes on the viral envelope (E) protein that are unique to each serotype. The goal of the current study was to determine the properties of neutralizing antibodies induced after secondary infection with a different (heterotypic) DENV serotypes. We evaluated whether polyclonal neutralizing antibody responses after secondary infections consist of distinct populations of type-specific antibodies to each serotype encountered or a new population of broadly cross-neutralizing antibodies. We observed two types of responses: in some individuals exposed to secondary infections, DENV neutralization was dominated by cross-reactive antibodies, whereas in other individuals both type-specific and cross-reactive antibodies contributed to neutralization. To better understand the origins of type-specific and cross-reactive neutralizing antibodies, we analyzed sera from individuals with well-documented sequential infections with two DENV serotypes only. These individuals had both type-specific and cross-reactive neutralizing antibodies to the 2 serotypes responsible for infection and only cross-reactive neutralizing antibodies to other serotypes. Collectively, the results demonstrate that the quality of neutralizing (and presumably protective) antibodies are different in individuals depending on the number of previous exposures to different DENV serotypes. We propose a model in which low affinity, cross-reactive antibody secreting B-cell clones induced by primary exposure evolve during each secondary infection to secrete higher affinity and more broadly neutralizing antibodies.</p></div

Force of dengue infection estimates and 95% CIs by JE vaccination status.

<p>Model 1: constant force of infection; Model 2: different forces of infection above and below 6 years. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002259#pntd-0002259-g002" target="_blank">Figure 2</a> footnote: * Estimates from a model adjusting for JE vaccination status.</p

Model of B cell maturation following sequential DENV infections.

<p>With each successive DENV infection, the ratio of serotype-specific (TS) and cross-reactive (CR) antibodies that contribute to DENV neutralization changes. During a primary infection (DENV1 in this example), dengue-specific naïve B cells are activated and these cells give rise to both memory B cells (MBCs) and antibody secreting long lived plasma cells (LLPCs). This primary response is dominated MBC and LLPCs clones producing low affinity, weakly neutralizing serotype CR antibodies. The primary response also contains rare MBC and LLPCs producing TS antibodies that strongly neutralize DENV1. Following a secondary infection with a new serotype (DENV3 in this example), the overall DENV-specific B-cell response will be dominated by the activation and expansion of DENV1 and 3 cross-reactive MBCs induced by the primary infection. MBCs producing CR antibodies that bind to the second infecting serotype with high affinity will be preferentially activated. These activated cells will re-enter germinal centers and undergo further rounds of somatic hyper mutation. CR B-cells with high affinity for the second serotype will be selectively expanded to give rise to cross-reactive MBC and LLPCs that strongly cross-neutralize multiple serotypes. In the figure this increase in affinity and neutralization is depicted by an increase in the color gradient (light yellow to bright orange) of CR B-cells. Following a tertiary infection (DENV2 in this example), this process is secondaryed again and results in a population of CR MBCs and LLPCs that dominate the neutralizing antibody response. While the B-cell clones producing TS strongly neutralizing antibodies are also likely to be maintained through each successive round of infection, the TS response will account for only a small fraction of the total neutralizing response.</p

Panel of naïve and late convalescent DENV-immune human sera.

<p>Panel of naïve and late convalescent DENV-immune human sera.</p

People exposed to secondary DENV infections develop neutralizing antibodies that target simple epitopes on recombinant E protein.

<p>Primary DENV2 or secondary DENV infection convalescent immune sera depleted of rE protein binding antibodies were assessed for neutralization of DENV2. In people exposed to secondary DENV infections, a greater proportion of DENV2 neutralizing antibodies bound to epitopes displayed on rE protein compared to people exposed to primary DENV2 infections (Mean of 75% vs. 26%; *P<0.018 unpaired Student t Test to compare means). The primary immune sera data are from 2 subjects analyzed in this study (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005554#pntd.0005554.s004" target="_blank">S4 Fig</a>) and two subjects analyzed in a previous study[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005554#pntd.0005554.ref008" target="_blank">8</a>]. All the secondary infection data are from samples analyzed for current study.</p

Neutralization properties of primary and secondary infection DENV-immune human sera depleted of DENV-specific antibodies.

<p>Polystyrene beads coated with either purified DENV2 or a mixture of purified DENV1, 3 and 4 were used to deplete DENV2 primary immune sera (DT001 and DT110) and DENV secondary infection immune sera (DT000, DT130, DT121, DT027 and DT025) of DENV-specific antibodies. After confirming depletion of relevant antibodies (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005554#pntd.0005554.s001" target="_blank">S1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005554#pntd.0005554.s002" target="_blank">S2</a> Figs), sera was tested for neutralization of DENV1-4. <b>A-B</b>. Levels of type-specific and cross-reactive neutralizing antibodies in 2 individuals exposed to primary DENV2 infections. <b>C-G</b>. Levels of type-specific and cross-reactive neutralizing antibodies in individuals exposed to secondary DENV infections.</p

Properties of DENV neutralizing antibodies after sequential infections with two different DENV serotypes.

<p>Subjects 985.8 <b>(A)</b> and 3428.8 <b>(C)</b> experienced sequential DENV2 → DENV3 infections. Subject 2934.7 <b>(B)</b> experienced sequential DENV1 → DENV3 infections. Polystyrene beads coated with DENV serotypes that infected each individual were used to deplete different populations of antibodies in the samples. After confirming depletion of relevant antibodies, the sera were tested for neutralization of DENV1-4 (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005554#pntd.0005554.s003" target="_blank">S3 Fig</a>). The graphs depict the levels of DENV serotype-specific and cross-reactive neutralizing antibodies in each subject after the second infection.</p