DENV2 E85-VRP-immunization reduced viral load during antibody-mediated severe dengue disease.

<p>AG129 mice were immunized with 1×10⁶ IU DENV2 E85-VRP i.p. 14 and 5 days prior to challenge with 5×10⁸ GE DENV. The baseline and ADE groups were included as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003723#ppat-1003723-g001" target="_blank">figure 1</a>. Viral RNA levels were measured in the liver 3 days after challenge. (A) One group of immunized mice was challenged in the presence of exogenous anti-DENV antibody 2H2 (black diamonds), and another group of immunized mice was challenged in the presence of a non-specific isotype control C1.18 (black triangles). (B) One group of mice was immunized with 1×10⁶ IU VRP expressing GFP (VRP-GFP, white triangles) instead of the DENV-E ectodomain (DENV2 E85-VRP, black triangles). Each symbol depicts one mouse, P-values from two-tailed unpaired t-test with Welch's correction, confidence interval 95%, * P≤0.05, ** P≤0.01, *** P≤0.001. Dotted line and symbols in gray as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003723#ppat-1003723-g001" target="_blank">figure 1</a>.</p

Role of Humoral versus Cellular Responses Induced by a Protective Dengue Vaccine Candidate

<div><p>With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.</p></div

Contribution of T cells to protection from DENV challenge after DENV2 E85-VRP-immunization.

<p>(A and B) AG129 mice were immunized with 1×10⁶ DENV2 E85-VRP i.p. 14 and 5 days prior to challenge with 5×10⁸ GE DENV. The “baseline” and “ADE” groups were included. Viral RNA was quantified in the liver 3 days post infection. (A) Prior to challenge, CD4⁺ T cells (black circles) or CD8⁺ T cells (black diamonds) were depleted. This experiment was repeated twice with a total of 7–8 mice per group with similar results, one experiment is shown. (B) Mice were challenged with 5×10⁸ GE DENV either in the absence (open symbols) or in the presence (black symbols) of exogenous anti-DENV antibody. Half of the mice were depleted of their CD8⁺ T cell population prior to challenge (diamonds). (C and D) IL-6 and IL-10 were measured in the serum 3 days after challenge with virus in mice either not immunized or immunized as described in A. In addition, CD4⁺ or CD8⁺ T cells were depleted as indicated. Each symbol depicts one mouse, P-values from two-tailed unpaired t-test with Welch's correction, confidence interval 95%, * P≤0.05, ** P≤0.01, *** P≤0.001. Dotted line and symbols in gray as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003723#ppat-1003723-g001" target="_blank">figure 1</a>.</p

Passive transfer of DENV2 E85-VRP-immune serum or adoptive transfer of DENV2 E85-VRP-immune B cells can increase the viral RNA levels in the liver upon infection with DENV.

<p>(A) 50, 200 or 500 µl DENV2 E85-VRP-immune serum (from AG129 mice immunized i.p. 14 and 5 days prior to serum collection) were transferred i.v. into naïve AG129 recipient mice one day prior to challenge with 5×10⁸ GE DENV. One group received 500 µl of VRP immune serum i.v. 3, 2 and 1 day before challenge (total 1.5 ml). One control group received 1.5 ml naïve serum (500 µl 3, 2 and 1 day prior to challenge), another control group received no serum and was infected in the presence of anti-DENV Ab (ADE) and the last control group received no serum and was infected in the presence of an isotype control Ab of irrelevant specificity (baseline). Viral RNA was quantified in the liver by qRT-PCR 3 days after challenge. (B) 2×10⁷ DENV2 E85-VRP-primed splenic B cells (from AG129 mice immunized i.p. 14 and 5 days prior to B-cell isolation) were transferred i.v. into naïve AG129 recipient mice one day prior to challenge with 5×10⁸ GE DENV. The “baseline” and “ADE” control groups were included as in A. Each symbol depicts one mouse, P-values from two-tailed unpaired t-test with Welch's correction, confidence interval 95%, * P≤0.05, ** P≤0.01, *** P≤0.001.</p

DENV2 E85-VRP-immunization protects from DENV challenge.

<p>AG129 mice were immunized with 1×10⁶ IU DENV2 E85-VRP either i.f. (black circles) or i.p. (black triangles) 14 and 5 days prior to challenge with 5×10⁸ GE DENV. Two groups of mice were not immunized prior to infection with DENV, one of which was infected in the presence of anti-DENV antibody 2H2, resulting in antibody-mediated enhancement of disease (black squares, ADE group) and the other one was infected in the presence of antibody C1.18 which is an isotype control of irrelevant specificity (white squares, baseline group). Viral RNA levels were measured in the liver 3 days after challenge (A) and survival was monitored (B). One day prior to challenge, serum DENV-specific IgG were measured by ELISA on DENV-coated plates (C) and the neutralization capacity of the serum was determined by PRNT₅₀ (D). Each symbol depicts one mouse except in B where n = 4–5, and in C where each symbol represents the mean of 4 animals, P-values from two-tailed unpaired t-test with Welch's correction, confidence interval 95% (A, C, D) or Gehan-Breslow-Wilcoxon test (B), *P≤0.05, **P≤0.01, ***P≤0.001. The dotted line represents the limit of detection of the assay. Samples with undetectable levels of DENV2 RNA are represented in gray under the detection limit. As they have no numerical value, they were not taken into account to calculate the mean.</p

DENV2 E85-VRP provides long-term protection; and the interval between the two immunizations modulates the degree to which the vaccine-induced protection relies on CD8⁺ T cells.

<p>(A) AG129 mice were immunized with 1×10⁶ IU DENV2 E85-VRP i.p. 42 and 33 days before challenge with 5×10⁸ GE DENV. Half of the mice were depleted of their CD8⁺ T cell population (black circles), and one group was left untreated before challenge (baseline). Viral RNA was quantified in the liver 3 days after challenge. (B) AG129 mice were immunized twice with 1×10⁶ IU DENV2 E85-VRP i.p. either 28 or 9 days apart. 28 days after the second immunization, mice were challenged with 5×10⁸ GE DENV. Half of the mice were depleted of their CD8⁺ T cell population. Viral RNA was quantified in the liver 4 days after challenge. (C) AG129 mice were immunized as in B (28 or 9 days apart). 33 days after the second immunization, mice were challenged with 5×10⁸ GE DENV. Before challenge, half of the mice were depleted of their CD4⁺ T cell population. Viral RNA was quantified in the liver 3 days after challenge. (D, E, F) AG129 mice were immunized with 1×10⁶ IU DENV2 E85-VRP i.p. 28 days apart (-61/-33) or 9 days apart (-42/-33), and on day 0, serum levels of DENV-specific IgG were measured by ELISA (D), neutralizing titers were determined by PRNT₅₀ (E), and DENV-specific IgG1, IgG2a, IgG2b and IgG3 were measured by ELISA (F). Each symbol depicts one mouse, except for D where n = 4 for the groups receiving B cells and 2 for the groups receiving no B cells (base and ADE) and F where n = 5 for experimental groups, naïve serum was included as a negative control, P-values from two-tailed unpaired t-test with Welch's correction, confidence interval 95%, * P≤0.05, ** P≤0.01, *** P≤0.001. Dotted line and symbols in gray as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003723#ppat-1003723-g001" target="_blank">figure 1</a>.</p

Toll-like receptor agonists abrogate viremia.

<p>A: Left panel, a peak of viremia was detected at day 4 after DENV infection in two of three animals (44Z and AM28). However, this peak was completely absent in the four animals of the DENV/TLR group at 48 hours after the TLR3 and 7/8 agonists [poly (I:C) and CL097M-012, respectively] were administered (right panel). Results are expressed in milliequivalent genomes per milliliter. B: Plasma levels of DENV protein NS1 were not significantly different between the DENV group and the treated group (DENV/TLR). However, NS1 levels were significantly higher in the DENV group compared to the Control group (p<0.018), whereas they were not significantly different between the DENV/LTR (despite the Dengue infection) and the Control groups. This confirmatory assay corroborates the role of TLRs in controlling DENV replication. The results reflect the ratio of the sample O.D./Control O.D. according to the manufacturer's instructions.</p

Dengue virus inhibits TLR-induced activation of mDC.

<p>A: Left panel, 48 hours after the first TLR stimulation, mDC were activated in the groups receiving TLRs but not in the group receiving only the virus. Coincident with the peak of viremia at day 4, an inhibitory effect of DENV on the mDC subset was evidenced by the frequency of activated cells in the DENV group, which was significantly lower compared to the DENV/TLR and TLR groups. Right panel, 10 days after the infection (72 hours after second TLR stimulation) the mDC subset was still significantly activated in both groups that received TLRs when compared to the Control group. However, the inhibitory effect of DENV was absent as there were no significant differences between the DENV and DENV/TLR groups, supporting the role of DENV replication in the inhibitory effect on mDC activation on day 4. B: Ten days after infection and 72 hours after the secondary TLR stimulation the frequency of activated pDC was significantly higher in the DENV/TLR and TLR groups compared to the DENV and Control groups. However, an inhibitory effect of DENV on this subset of DC was not observed. Asterisks denote significant differences, (*) p <0.05, (**) p <0.01, and (***) p<0.00.</p

TLR3-7/8 stimulations in synergism with DENV induce quantitative and qualitative modifications of the humoral response.

<p>A: Ten days after the infection, the frequency of activated B cells was significantly higher in DENV/TLR group when compared to all other groups, showing a synergistic effect of DENV with the TLR agonists in this activation. DENV alone was able to induce activation of B cells when compared to the TLR and Control groups. However, in our model, TLR agonists alone were not sufficient to induce B-cell activation. B: The activation profile of B cells correlates with the levels of anti-DENV antibodies. Antibody levels were significantly higher in the DENV/TLR group compared to the DENV group. C: As early as 10 days after infection and coincident with activation of B cells, the switch to IgG1 was significantly diminished in the DENV/TLR group. This difference had disappeared by 15 days after the infection, but it was re-established on day 30. D: Although antibody O.D. values were limited, the difference in the IgG1 and IgG2 levels translated into a significant increase of the IgG2/IgG1 ratio (>11 and 6 times on days 10 and 30 after the infection, respectively). Asterisks denote significant differences, (*) p<0.05, (**) p<0.01, and (***) p<0.001.</p

Serum levels of CXCL-10 and IL-1Ra are increased significantly after TLR stimulation and are inhibited by DENV.

<p>A: Coincident with the higher level of mDC activation, serum levels of CXCL-10 increased significantly only in the TLR group when compared to the Control group. Higher levels, albeit not significant, were also found in the DENV/TLR group. The lowest levels among the experimental groups were found in animals of the DENV group. This trend in the serum levels of CXCL-10 among different groups confirms a stimulatory effect of the TLR agonist but at the same time suggests an inhibitory role of Dengue virus in the secretion of this cytokine. B: Serum levels of IL-1Ra peaked 48 hours after the first TLR stimulation. An inhibitory effect of DENV at this time point (also coincident with the viremia peak) is supported by significantly higher values of this cytokine in the TLR group when compared to the DENV and DENV/TLR groups. Both groups, DENV/TLR and TLR, showed significantly higher serum levels of this cytokine when compared to the DENV and Control groups. Ten days after the infection (72 hours after second TLR stimulation), IL1-Ra values were still increased in both groups receiving TLR agonists. Significantly higher levels were present up to 15 days after the infection (8 days after the last TLR stimulation). However, at the later time point, there was no significant difference between the DENV/TLR and TLR groups. Asterisks denote significant differences, (*) p <0.05, (**) p<0.01, and (***) p<0.001.</p