Dengue Virus Activates Polyreactive, Natural IgG B Cells after Primary and Secondary Infection

BACKGROUND: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. METHODOLOGY/PRINCIPAL FINDINGS: We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection. CONCLUSIONS/SIGNIFICANCE: Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities" seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development

Up-scaling single cell-inoculated suspension culture of human embryonic stem cells

AbstractWe have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only ∼44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to ∼60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2ml cultures to serial passaging in 50ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines

Production of soluble factors associated with protection against dengue by monocyte subsets.

<p>Isolated monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Supernatants were harvested over the course of 6 days. (A) Levels of IFN-α were determined by a multi-subtype specific ELISA kit. (B and C) Levels of CXCL10 and TRAIL were determined using multiplex bead arrays. Results are mean ± SE for 6 different donors. There were no significant differences were found between infected CD16⁻ and CD16⁺ monocytes. (D) Supernatants from CD16⁻ and CD16⁺ monocytes exposed to dengue virus or medium without virus were harvested at day 6. These supernatants were passed through 100 kDa centrifuge filters to remove dengue virus. K562 cells were pretreated for 24 hours with either culture medium, supernatants of CD16⁻ or CD16⁺ monocytes with or without virus exposure. Pre-treated K562 cells were washed and infected with dengue virus at a MOI of 2. After 2 days, the extent of infection was determined by intracellular labeling of K562 cells with anti-NS1 antibody. The percentage of NS1⁺ K562 cells after 2 days is shown. Data are representative of 2 experiments using different donors. **, <i>p</i><0.005 between respective monocyte subset with and without virus.</p

IL-4 treatment enhances the susceptibility of the CD16⁺ monocyte subset to a greater extent.

<p>Isolated CD16⁻ or CD16⁺ monocyte subsets were pretreated with 25 ng/ml of IL-4 for two days. Cells were subsequently washed and harvested before exposure to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Percentages of CD16⁻ and CD16⁺ monocytes that are (A) NS1⁺ or (B) E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 5 different donors. (C) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed IL-4 treated CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 4 different donors. <b>*</b><i>p</i><0.05, between IL-4 treated CD16⁺ and IL-4 treated CD16⁻ monocytes with virus.</p

Susceptibility of monocyte subsets to dengue virus infection.

<p>(A) Flow cytometric profile of CD16⁻ and CD16⁺ monocytes after isolation. (B) Isolated CD16⁻ or CD16⁺ monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. After 2 days, monocytes were fixed, permeabilized and labeled with anti-E-protein and anti-NS1 specific antibodies. Quadrants for virus exposed monocytes (right panel) were set based on monocytes without virus exposure (left panel). Percentage positive cells in each quadrant are shown. Representative data for 6 different donors. (C and D) Percentages of CD16⁻ and CD16⁺ monocytes that are NS1⁺ or E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 6 different donors. (E) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 5 different donors.</p

Production of inflammatory cytokines by monocyte subsets.

<p>Monocyte subsets were exposed to dengue virus or medium without virus. Supernatants were harvested over the course of 6 days. Levels of (A) IL-1β (B) TNF-α (C) IL-6 (D) CCL2 (E) CCL3 and (F) CCL4 were measured using multiplex bead arrays. Results are mean ± SE of 5 different donors. <b>*</b><i>p</i><0.05, **, <i>p</i><0.005 between CD16⁺ and CD16⁻ monocytes with virus.</p

Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection

Broad specificity of dengue-induced antibodies.

<p>A) Total concentration of IgM, IgA and IgG in the plasma of dengue- and control OFI patients 1–3 days, day 4–7 and day 15–25 after onset of fever. Means±SD, n = 7–10 for dengue-negative and n = 9–16 for dengue-positive patients. A student's t test to compare dengue-positive with fever control samples was performed. B) Polio virus-specific antibodies in paired plasma samples from dengue (grey boxes) and fever control patients (white boxes) 1–3 days of fever, day 4–7 and day 15–25 after onset of fever. Data are combined from three individual experiments, n = 18 for dengue, n = 21 for OFI. A Two-Way Repeated Measures ANOVA test with Bonferroni Post-Hoc test showed a significant difference between dengue and OFI at day 15–25.</p

Rapid induction of cross-reactive IgG antibodies after dengue infection.

<p>Longitudinal plasma samples of dengue patients were tested by ELISA for dengue-specific IgM (A) and IgG antibodies (B). A and B) Four plasma dilutions from 1∶200 to 1∶25'000 were measured to exclude non-specific binding. For the combined illustration of all samples that were analysed the OD450 of one dilution (1∶5000 for IgG and 1∶1000 for IgM) was divided by the OD450 of a standard serum that was included on each plate. Time points: 1 = 1–3 days after fever, 2 = 4–7 days after fever, 3 = 15–25 days after fever. The same patient samples were analyzed for DENV1–4. Binding intensity correlated for all four serotypes, i.e. high binding to DENV1 would also apply to DENV2, 3 and 4. Primary versus secondary infection status was confirmed with the commercially available Panbio ELISA kit (Inverness Medical, Australia). The experiment was repeated for selected samples, confirming the original results. C) Concentrations of BAFF were measured in the plasma of dengue and flu patients at the indicated time points. D) moDCs were infected with DENV, heat-inactivated (HI) DENV, polyI:C or medium and BAFF was measured in the supernatant at different time points after infection. Data are presented as % of medium control and are the means±SEM of two independent experiments, done in triplicates. TSV01∶medium compared to polyIC∶medium is significantly different (p>0.05, Two-way ANOVA). The source of BAFF in DENV-infected patients is therefore unlikely to be DCs.</p