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

Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.

Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics

Aedes aegypti Molecular Responses to Zika Virus: Modulation of Infection by the Toll and Jak/Stat Immune Pathways and Virus Host Factors

Zika (ZIKV) and dengue virus (DENV) are transmitted to humans by Aedes mosquitoes. However, the molecular interactions between the vector and ZIKV remain largely unexplored. In this work, we further investigated the tropism of ZIKV in two different Aedes aegypti strains and show that the virus infection kinetics, tissue migration, and susceptibility to infection differ between mosquito strains. We also compare the vector transcriptome changes upon ZIKV or DENV infection demonstrating that 40% of the mosquito’s midgut infection-responsive transcriptome is virus-specific at 7 days after virus ingestion. Regulated genes included key factors of the mosquito’s anti-viral immunity. Comparison of the ZIKV and DENV infection-responsive transcriptome data to those available for yellow fever virus and West Nile virus identified 26 genes likely to play key roles in virus infection of Aedes mosquitoes. Through reverse genetic analyses, we show that the Toll and the Jak/Stat innate immune pathways mediate increased resistance to ZIKV infection, and the conserved DENV host factors vATPase and inosine-5′-monophosphate dehydrogenase are also utilized for ZIKV infection

Proteins recovered from active anti-DENV fraction of <i>C</i>. <i>sp</i>. Panama.

<p>Proteins recovered from active anti-DENV fraction of <i>C</i>. <i>sp</i>. Panama.</p

Protein extracts of culture supernatants of <i>C</i>. <i>sp</i>. Panama inhibit DENV entry by mediating degradation of the E protein.

<p>(<b>A</b>) DENV titers following exposure to a protein extract of the <i>C</i>. <i>sp</i>. Panama culture supernatant before or after viral attachment to BHK-21 cells; significance determined using unpaired t-tests. Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>. (<b>B</b>) Representative negative stained transmission EM images of control DENV particles (arrow head) and those treated with <i>C</i>. <i>sp</i>. Panama culture supernatant protein extract (empty arrow head); low magnification: 93,000X, high magnification: 245,000X. (<b>C</b>) Size distribution of the viral particles as measured from the transmission EM images collected in B; mean and standard deviation indicated; significance determined using unpaired t-test. (<b>D</b>) Western blot (anti-E) of DENV proteins when exposed to the <i>C</i>. <i>sp</i>. Panama culture supernatant protein extract. (<b>E</b>) Fluorescence microscopy showing DENV attachment to BHK-21 cells in the presence of the protein extract of the <i>C</i>. <i>sp</i>. Panama. Cells were fixed with 4% PFA, stained for DNA (DAPI-blue), and F-actin (Phalloidin-green). Membrane-bound DENV (arrow head) were immunostained with 4G2 anti-E antibody (Red). ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001.</p

Aminopeptidase secreted by <i>C</i>. <i>sp</i>. Panama induces DENV E protein degradation, thereby inhibiting viral entry.

<p>(<b>A</b>) Protein sequence alignment of CSPP0261 and orthologs in <i>Pseudomonas aeruginosa</i> (Paer, UniProt: P14756) and <i>Vibrio proteolyticus</i> (Vpro, UniProt: Q00971). Conserved active and metal binding sites highlighted in blue (in both orthologs) and red (in <i>P</i>. <i>aeruginosa</i> ortholog). (<b>B</b>) Protein sequence alignment of CSPP0262 and ortholog in <i>Vibrio proteolyticus</i>. Conserved active and metal binding sites highlighted in blue (Vpro, UniProt: Q01693). (<b>C</b>) Western blot (anti-E-DENV) of DENV2 proteins when exposed to the culture supernatant of <i>C</i>. <i>sp</i>. Panama with or without supplementation with 10 μM bestatin or 100 μM phosphoramidon (P-ramidon). (<b>D</b>) DENV titers in BHK-21 cells following incubation of the virus with the <i>C</i>. <i>sp</i>. Panama culture supernatant with or without supplementation with 10 μM bestatin or 100 μM phosphoramidon (P-ramidon); significance determined using unpaired t-tests (ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001). Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>.</p

Anti-DENV activity of <i>Chromobacterium sp</i>. Panama is mediated by secreted proteinous factors.

<p>DENV virus titers: (<b>A</b>) in the presence or absence of culture supernatant of <i>C</i>. <i>cp</i>. Panama following infection in BHK-21 and C6/36 cells, as determined by plaque or focus forming assays, respectively; (<b>B</b>) in BHK-21 cells upon exposure to different solvent extracts of culture supernatants of <i>C</i>. <i>sp</i>. Panama; (<b>C</b>) in BHK-21 cells in the presence or absence of culture supernatant of <i>C</i>. <i>cp</i>. Panama incubated for 1 hour at the indicated temperatures; and (<b>D</b>) upon exposure to protein extract of the culture supernatant of <i>C</i>. <i>sp</i>. Panama prepared by 70% ammonium sulfate precipitation (Pellet), the desalted supernatant of that extraction (Sup.) or control buffer (0.1M Tris-HCl). Significance determined using unpaired t-tests; (ns, not significant; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001). Raw underlying data available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006443#pntd.0006443.s002" target="_blank">S2 Table</a>.</p

Engineered <i>Aedes aegypti</i> JAK/STAT Pathway-Mediated Immunity to Dengue Virus

<div><p>We have developed genetically modified <i>Ae</i>. <i>aegypti</i> mosquitoes that activate the conserved antiviral JAK/STAT pathway in the fat body tissue, by overexpressing either the receptor Dome or the Janus kinase Hop by the blood feeding-induced vitellogenin (Vg) promoter. Transgene expression inhibits infection with several dengue virus (DENV) serotypes in the midgut as well as systemically and in the salivary glands. The impact of the transgenes Dome and Hop on mosquito longevity was minimal, but it resulted in a compromised fecundity when compared to wild-type mosquitoes. Overexpression of Dome and Hop resulted in profound transcriptome regulation in the fat body tissue as well as the midgut tissue, pinpointing several expression signatures that reflect mechanisms of DENV restriction. Our transcriptome studies and reverse genetic analyses suggested that enrichment of DENV restriction factor and depletion of DENV host factor transcripts likely accounts for the DENV inhibition, and they allowed us to identify novel factors that modulate infection. Interestingly, the fat body-specific activation of the JAK/STAT pathway did not result in any enhanced resistance to Zika virus (ZIKV) or chikungunya virus (CHIKV) infection, thereby indicating a possible specialization of the pathway’s antiviral role.</p></div

Midgut transcriptomic profiles of transgenic mosquitoes compared to WT at 24 hpbm.

<p>Relative gene expression of (A) Fold change in Dome and Hop gene expression (fat body/midgut) (B) Fold change in DVRF1 gene expression in the midgut of the transgenic lines as compared to WT. (C) Number of differentially expressed transcripts in the midgut of the transgenic lines as compared to WT mosquitoes, classified according to functional groups as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref008" target="_blank">8</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005187#pntd.0005187.ref015" target="_blank">15</a>]. Abbreviations: CS, cytoskeletal and structural; CSR, chemosensory reception; DIV, diverse functions; DIG, blood and sugar food digestive; IMM, immunity; MET, metabolism; PROT, proteolysis; RSM, redox, stress, and mitochondrion; RTT, replication, transcription, and translation; TRP, transport; UKN, unknown functions. (D) Percentage of genes enriched or depleted in each functional group in the midguts of the VgDome or VgHop mosquitoes as compared to WT.</p

Generation of transgenic <i>Ae</i>. <i>aegypti</i> over-expressing Dome and Hop under the control of the Vg promoter.

<p>(A) Schematic of the piggyBac (pBac) transformation plasmids used to generate the VgDome and VgHop lines. pBacL, pBacR: pBac left and right arms, 3xP3: eye-specific promoter with either EGFP or DsRed as markers, Vg promoter: vitellogenin promoter, Dome: Dome coding sequence, Hop: Hop coding sequence, TrypT: trypsin terminator sequence. (B) Transcript abundance of transgenes and effector genes in the fat body of VgDome and VgHop lines from before blood feeding (0 hr) up to 48 hpbm. Each bar represents the relative fold change of Dome, Hop or DVRF1 (DENV restriction factor 1), compared between transgenic lines and WT <i>Ae</i>. <i>aegypti</i>. The S7 ribosomal gene was used to normalize cDNA templates. Error bars indicate standard error of the mean.</p