Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis

Decreased Dengue Replication and an Increased Anti-viral Humoral Response with the use of Combined Toll-Like Receptor 3 and 7/8 Agonists in Macaques

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo

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

Effective control of early Zika virus replication by Dengue immunity is associated to the length of time between the 2 infections but not mediated by antibodies.

Little is known about the contribution of virus-specific and cross-reacting antibodies (Abs) or the cellular immune response generated by a primary dengue (DENV) infection on the course of a secondary zika (ZIKV) infection in vivo. Here we show that the length of time between DENV/ZIKV infections has a qualitative impact on controlling early ZIKV replication. Depletion of DENV2-specific Abs in sera confirmed that those type-specific Abs do not contribute to ZIKV control. We show that the magnitude and durability of the neutralizing antibodies (nAbs) induced by a secondary ZIKV infection is modest compared to the response induced after a secondary heterologous DENV infection. Our in vivo results are showing a complex interplay between the cellular and innate immune responses characterized by a high frequency of plasmacytoid dendritic cells (pDC) correlating with an increase in the frequency of DENV antigen specific T cells and a significant control of ZIKV replication which is time dependent. Taken together, our results suggest that early after ZIKV infection other mechanisms such as the innate and cellular immune responses may play a predominant role in controlling ZIKV replication. Regardless of the time elapsed between infections there was no evidence of in vivo antibody-dependent enhancement (ADE) of ZIKV by DENV immunity. These findings have pivotal implications while interpreting ZIKV pathogenesis in flavivirus-experimented populations, diagnostic results interpretation and vaccine designs and schedules among others

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

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

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