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

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

[1]  V. L. La Russa,et al.  Dengue-2 virus infection of human bone marrow: characterization of dengue-2 antigen-positive stromal cells. , 1996, The American journal of tropical medicine and hygiene.

[2]  R. O. Spertzel,et al.  Protective and toxic effects of a nuclease-resistant derivative of polyriboinosinic-polyribocytidylic acid on Venezuelan equine encephalomyelitis virus in rhesus monkeys. , 1979, The Journal of infectious diseases.

[3]  R. Koup,et al.  HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Douglas T. Golenbock,et al.  Flavivirus Activation of Plasmacytoid Dendritic Cells Delineates Key Elements of TLR7 Signaling beyond Endosomal Recognition1 , 2006, The Journal of Immunology.

[5]  F. Ennis,et al.  Human Dendritic Cells Are Activated by Dengue Virus Infection: Enhancement by Gamma Interferon and Implications for Disease Pathogenesis , 2001, Journal of Virology.

[6]  S. Yoksan,et al.  In vivo and in vitro studies on the morphological change in the monkey epidermal Langerhans cells following exposure to dengue 2 (16681) virus. , 1996, The Southeast Asian journal of tropical medicine and public health.

[7]  A. Nisalak,et al.  Immunopathologic mechanisms of dengue hemorrhagic fever and dengue shock syndrome. , 1994, Archives of virology. Supplementum.

[8]  S. Green,et al.  Immunopathological mechanisms in dengue and dengue hemorrhagic fever , 2006, Current opinion in infectious diseases.

[9]  K. Abel,et al.  The relationship between simian immunodeficiency virus RNA levels and the mRNA levels of alpha/beta interferons (IFN-alpha/beta) and IFN-alpha/beta-inducible Mx in lymphoid tissues of rhesus macaques during acute and chronic infection. , 2002, Journal of virology.

[10]  K. Abel,et al.  Transcriptional Activation of Interferon-Stimulated Genes but Not of Cytokine Genes after Primary Infection of Rhesus Macaques with Dengue Virus Type 1 , 2007, Clinical and Vaccine Immunology.

[11]  T. Burgess,et al.  Differential Effects of Dengue Virus on Infected and Bystander Dendritic Cells , 2005, Journal of Virology.

[12]  A. Nisalak,et al.  A Blunted Blood Plasmacytoid Dendritic Cell Response to an Acute Systemic Viral Infection Is Associated with Increased Disease Severity 1 , 2003, The Journal of Immunology.

[13]  J. V. D. van der Meer,et al.  Cytokine patterns during dengue shock syndrome. , 2003, European cytokine network.

[14]  Pei-Yong Shi,et al.  NS5 of Dengue Virus Mediates STAT2 Binding and Degradation , 2009, Journal of Virology.

[15]  J. Hiscott,et al.  Manipulation of the nuclear factor-κB pathway and the innate immune response by viruses , 2006, Oncogene.

[16]  Bali Pulendran,et al.  Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity , 2006, The Journal of experimental medicine.

[17]  J. Durdik,et al.  Presence of activated antigen-binding B cells during immunization enhances relative levels of IFN-gamma in T cell responses. , 1998, Journal of immunology.

[18]  A. Fernández-Sesma,et al.  Dengue Virus Inhibits the Production of Type I Interferon in Primary Human Dendritic Cells , 2010, Journal of Virology.

[19]  M. Eller,et al.  CD40 Ligand Enhances Dengue Viral Infection of Dendritic Cells: A Possible Mechanism for T Cell-Mediated Immunopathology12 , 2006, The Journal of Immunology.

[20]  S. Vasudevan,et al.  Role of T cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever , 2006, Reviews in medical virology.

[21]  G. Foster,et al.  Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression , 2005, Journal of Virology.

[22]  Ross M. Kedl,et al.  Immunization with HIV-1 Gag Protein Conjugated to a TLR7/8 Agonist Results in the Generation of HIV-1 Gag-Specific Th1 and CD8+ T Cell Responses , 2005, The Journal of Immunology.

[23]  Alan L Rothman,et al.  Dengue: defining protective versus pathologic immunity. , 2004, The Journal of clinical investigation.

[24]  A. Gettie,et al.  Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses. , 2002, Journal of immunological methods.

[25]  S. A. Oliveira,et al.  Increased pro-inflammatory cytokines (TNF-alpha and IL-6) and anti-inflammatory compounds (sTNFRp55 and sTNFRp75) in Brazilian patients during exanthematic dengue fever. , 1999, Memorias do Instituto Oswaldo Cruz.

[26]  Michael G. Katze,et al.  Distinct RIG-I and MDA5 Signaling by RNA Viruses in Innate Immunity , 2007, Journal of Virology.

[27]  R. Sauerwein,et al.  Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections. , 1995, The Journal of infectious diseases.

[28]  Yi-Ling Lin,et al.  Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3K-dependent NF-kappaB activation. , 2006, Microbes and infection.

[29]  S. Akira,et al.  Toll-like receptors and innate immunity , 2006, Journal of Molecular Medicine.

[30]  Chun-Nan Lee,et al.  Human TLR3 recognizes dengue virus and modulates viral replication in vitro , 2009, Cellular microbiology.

[31]  R. Purcell,et al.  Failure to Demonstrate Circulating Interferon During Incubation Period and Acute Stage of Transfusion-Associated Hepatitis , 1971, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[32]  R. Lanciotti,et al.  Serotype-Specific Detection of Dengue Viruses in a Fourplex Real-Time Reverse Transcriptase PCR Assay , 2005, Journal of Clinical Microbiology.

[33]  A. Plebani,et al.  Viral Double-Stranded RNA Triggers Ig Class Switching by Activating Upper Respiratory Mucosa B Cells through an Innate TLR3 Pathway Involving BAFF1 , 2008, The Journal of Immunology.

[34]  A. Nisalak,et al.  Human immune responses to dengue viruses. , 1990, The Southeast Asian journal of tropical medicine and public health.

[35]  A. Rothman,et al.  Viral replication and paracrine effects result in distinct, functional responses of dendritic cells following infection with dengue 2 virus , 2008, Journal of leukocyte biology.

[36]  A. Fernández-Sesma,et al.  Inhibition of the Type I Interferon Response in Human Dendritic Cells by Dengue Virus Infection Requires a Catalytically Active NS2B3 Complex , 2010, Journal of Virology.

[37]  S. Akira,et al.  CpG directly induces T-bet expression and inhibits IgG1 and IgE switching in B cells , 2003, Nature Immunology.

[38]  R. Johnston,et al.  Flow Cytometry-Based Assay for Titrating Dengue Virus , 2005, Journal of Clinical Microbiology.

[39]  S. Morrison,et al.  Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies. , 1988, The EMBO journal.

[40]  Mario Roederer,et al.  Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates , 2006, The Journal of experimental medicine.

[41]  Pieter H. Reitsma,et al.  Differential Gene Expression Changes in Children with Severe Dengue Virus Infections , 2008, PLoS neglected tropical diseases.

[42]  Arthur M. Krieg,et al.  The Toll-Like Receptor 7 (TLR7) Agonist, Imiquimod, and the TLR9 Agonist, CpG ODN, Induce Antiviral Cytokines and Chemokines but Do Not Prevent Vaginal Transmission of Simian Immunodeficiency Virus When Applied Intravaginally to Rhesus Macaques , 2005, Journal of Virology.

[43]  Kevin R Porter,et al.  Functional characterization of ex vivo blood myeloid and plasmacytoid dendritic cells after infection with dengue virus. , 2009, Virology.

[44]  A. Lanzavecchia,et al.  Toll‐like receptor stimulation as a third signal required for activation of human naive B cells , 2006, European journal of immunology.

[45]  G. Screaton,et al.  A Complex Interplay among Virus, Dendritic Cells, T Cells, and Cytokines in Dengue Virus Infections1 , 2008, The Journal of Immunology.

[46]  P. Desprès,et al.  Innate immune responses to dengue virus. , 2005, Archives of medical research.

[47]  A. Osterhaus,et al.  Kinetics of Dengue Virus-Specific Serum Immunoglobulin Classes and Subclasses Correlate with Clinical Outcome of Infection , 2001, Journal of Clinical Microbiology.

[48]  K. Abel,et al.  Rapid Virus Dissemination in Infant Macaques after Oral Simian Immunodeficiency Virus Exposure in the Presence of Local Innate Immune Responses , 2006, Journal of Virology.

[49]  M. Guzmán,et al.  Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis , 2010, PLoS neglected tropical diseases.

[50]  R. Steinman,et al.  Synthetic Double-Stranded RNAs Are Adjuvants for the Induction of T Helper 1 and Humoral Immune Responses to Human Papillomavirus in Rhesus Macaques , 2009, PLoS pathogens.

[51]  D. Chang,et al.  Infection of Human Dendritic Cells by Dengue Virus Causes Cell Maturation and Cytokine Production1 , 2001, The Journal of Immunology.

[52]  Adolfo García-Sastre,et al.  Inhibition of interferon signaling by dengue virus , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[53]  A. Rothman,et al.  Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo , 2009, Journal of medical virology.

[54]  M. Neuberger,et al.  Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies , 1987, The Journal of experimental medicine.

[55]  A. Luster,et al.  Both CXCR3 and CXCL10/IFN-Inducible Protein 10 Are Required for Resistance to Primary Infection by Dengue Virus1 , 2006, The Journal of Immunology.

[56]  K. Abel,et al.  The Relationship between Simian Immunodeficiency Virus RNA Levels and the mRNA Levels of Alpha/Beta Interferons (IFN-α/β) and IFN-α/β-Inducible Mx in Lymphoid Tissues of Rhesus Macaques during Acute and Chronic Infection , 2002, Journal of Virology.

[57]  M. Blettner,et al.  Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. , 2006, The Journal of infectious diseases.

[58]  S. Baron,et al.  Effect of a nuclease-resistant derivative of polyriboinosinic-polyribocytidylic acid complex on yellow fever in rhesus monkeys (Macaca mulatta). , 1977, The Journal of infectious diseases.

[59]  D. Gubler.  Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. , 2002, Trends in microbiology.

[60]  D. Chang,et al.  Infection of Human Dendritic Cells by Dengue Virus Activates and Primes T Cells Towards Th0‐Like Phenotype Producing Both Th1 and Th2 Cytokines , 2004, Immunological investigations.