Carlos A. Sariol

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.

Transcriptional Activation of Interferon-Stimulated Genes but Not of Cytokine Genes after Primary Infection of Rhesus Macaques with Dengue Virus Type 1

ABSTRACT Macaques are the only animal model used to test dengue virus (DENV) vaccine candidates. Nevertheless, the pathogenesis of DENV in macaques is not well understood. In this work, by using Affymetrix oligonucleotide microarrays, we studied the broad transcriptional modifications and cytokine expression profile after infecting rhesus macaques with DENV serotype 1. Five days after infection, these animals produced a potent, innate antiviral immune response by inducing the transcription of signature genes from the interferon (IFN) pathway with demonstrated antiviral activity, such as myxoprotein, 2′,5′-oligoadenylate synthetase, phospholipid scramblase 1, and viperin. Also, IFN regulatory element 7, IFN-stimulated gene 15, and protein ligases linked to the ISGylation process were up-regulated. Unexpectedly, no up-regulation of IFN-α, -β, or -γ genes was detected. Transcription of the genes of interleukin-10 (IL-10), IL-8, IL-6, and tumor necrosis factor alpha was neither up-regulated nor down-regulated. Results were confirmed by real-time PCR and by multiplex cytokine detection in serum samples.

Dengue Virus Subverts the Interferon Induction Pathway via NS2B/3 Protease-IκB Kinase ε Interaction

ABSTRACT Dengue is the worlds most common mosquito-borne viral infection and a leading cause of morbidity throughout the tropics and subtropics. Viruses are known to evade the establishment of an antiviral state by regulating the activation of interferon regulatory factor 3 (IRF3), a critical transcription factor in the alpha/beta interferon induction pathway. Here, we show that dengue virus (DENV) circumvents the induction of the retinoic acid-inducible gene I-like receptor (RLR) pathway during infection by blocking serine 386 phosphorylation and nuclear translocation of IRF3. This effect is associated with the expression of nonstructural 2B/3 protein (NS2B/3) protease in human cells. Using interaction assays, we found that NS2B/3 interacts with the cellular IκB kinase ε (IKKε). Docking computational analysis revealed that in this interaction, NS2B/3 masks the kinase domain of IKKε and potentially affects its functionality. This observation is supported by the DENV-associated inhibition of the kinase activity of IKKε. Our data identify IKKε as a novel target of DENV NS2B/3 protease.

Safety and Immunogenicity of a Tetravalent Dengue Vaccine Candidate in Healthy Children and Adults in Dengue-Endemic Regions: A Randomized, Placebo-Controlled Phase 2 Study

BACKGROUND A safe, effective tetravalent dengue vaccine is a global health priority. The safety and immunogenicity of a live attenuated, recombinant tetravalent dengue vaccine candidate (TDV) were evaluated in healthy volunteers from dengue-endemic countries. METHODS This multicenter, double-blind, phase 2 study was conducted in Puerto Rico, Colombia, Singapore, and Thailand. During stage I, 148 volunteers aged 1.5-45 years were sequentially enrolled into 4 age-descending groups and randomized at a ratio of 2:1 to receive TDV or placebo. In stage II (group 5), 212 children aged 1.5-11 years were randomized at a ratio of 3:1 to receive TDV or placebo. Participants received a subcutaneous injection of TDV or placebo on days 0 and 90 and were followed for analysis of safety, seropositivity, and neutralizing antibodies to DENV-1-4. RESULTS Injection site pain, itching, and erythema (mostly mild) were the only solicited adverse events more frequently reported with TDV than with placebo in all age groups. After 2 TDV doses, seropositivity was >95% in all 5 groups for DENV-1-3 and 72.7%-100% for DENV-4; geometric mean titers ranged from 582 to 1187 for DENV-1, from 582 to 1187 for DENV-2, from 196 to 630 for DENV-3, and from 41 to 210 for DENV-4 among the 5 groups. CONCLUSIONS TDV was well tolerated and immunogenic in volunteers aged 1.5-45 years, irrespective of prevaccination dengue exposure.

Serial Cervicovaginal exposures with Replication-deficient SIVsm induce higher Dendritic Cell (pDC) and CD4+ T-Cell Infiltrates not associated with prevention but a More Severe SIVmac251 Infection of Rhesus Macaques

Objective:Intravaginal exposure to simian immunodeficiency virus (SIV) acutely recruits interferon-alpha (IFN-&agr;) producing plasmacytoid dendritic cells (pDC) and CD4+ T-lymphocyte targets to the endocervix of nonhuman primates. We tested the impact of repeated cervicovaginal exposures to noninfectious, defective SIV particles over 72 hours on a subsequent cervicovaginal challenge with replication competent SIV. Methods:Thirty-four female Indian Rhesus macaques were given a 3-day twice-daily vaginal exposures to either SIVsmB7, a replication-deficient derivative of SIVsmH3 produced by a T lymphoblast CEMx174 cell clone (n = 16), or to CEM supernatant controls (n = 18). On the fourth day, animals were either euthanized to assess cervicovaginal immune cell infiltration or intravaginally challenged with SIVmac251. Challenged animals were tracked for plasma viral load and CD4 counts and euthanized at 42 days after infection. Results:At the time of challenge, macaques exposed to SIVsmB7, had higher levels of cervical CD123 pDCs (P = 0.032) and CD4+ T cells (P = 0.036) than those exposed to CEM control. Vaginal tissues showed a significant increase in CD4+ T-cell infiltrates (P = 0.048) and a trend toward increased CD68+ cellular infiltrates. After challenge, 12 SIVsmB7-treated macaques showed 2.5-fold greater daily rate of CD4 decline (P = 0.0408), and viral load rise (P = 0.0036) as compared with 12 control animals. Conclusions:Repeated nonproductive exposure to viral particles within a short daily time frame did not protect against infection despite pDC recruitment, resulting instead in an accelerated CD4+ T-cell loss with an increased rate of viral replication.

Herpes B-virus seroreactivity in a colony of Macaca mulatta: data from the Sabana Seca Field Station, a new specific pathogen-free program.

Abstract:  The demand for B‐virus‐free animals for biomedical research is increasing, while at the same time the availability of such animals is decreasing. The establishment of Specific Pathogen‐Free (SPF) breeding macaque colonies is a priority of the National Institutes of Health. Nevertheless, it is well known that seroreactivity to B‐virus can be difficult to interpret, particularly as it can vary over time in a single animal. The aim of the present study was to implement a reliable algorithm to examine B‐virus reactivity among the rhesus monkey population of the Caribbean Primate Research Center. The sensitivity and specificity of our assay were determined using reports from two different laboratories as references. Whereas 95.4% of animals showed consistent serological status and 4.6% of animals recruited to this SPF program showed serovariability to B‐virus over the initial 2 years of examination. Implications for all SPF programs are discussed in this article.

Short communication: Lack of immune response in rapid progressor morphine-dependent and SIV/SHIV-infected rhesus macaques is correlated with downregulation of TH1 cytokines.

Our previous studies have shown two distinct disease patterns (rapid and normal onset of clinical symptoms) in morphine-dependent SHIV/SIV-inoculated rhesus macaques. We have also shown that control as well as 50% of morphine-dependent macaques (normal progressor) developed humoral and cellular immune responses whereas the other half of the morphine-dependent macaques (rapid progressor) did not develop antiviral immune responses after infection with SIV/SHIV. In the present study, we analyzed the association between cytokine production, immune response, and disease progression. To study the immunological effects of morphine at cytokine levels in the context of a lentiviral infection, we inoculated rhesus macaques with a mixture of SHIV(KU-18), SHIV(89.6)P, and SIV/17E-Fr. These animals were followed for a period of 56 weeks for cytokine level production in plasma. Drug-dependent rapid disease progressors exhibited an increase in IL-18 and IL-1Ra and a decrease in IL-12 levels in the plasma. Morphine-dependent normal progressors and control macaques exhibited an increase in both IL-18 and IL-12, whereas IL-Ra levels remained constant throughout the observation period. These results suggest that rapid disease progression in relation to morphine dependency may be the result of an altered cytokine profile.

Differential distribution of antibodies to different viruses in young animals in the free-ranging rhesus macaques of Cayo Santiago

Background  The breeding colony of free‐ranging rhesus macaques was established in 1938 in Cayo Santiago (CS) with animals collected in northern India. The seroprevalence to cercopithecine herpesvirus type 1 (B virus) and simian retroviruses has been studied previously.

Retraction: Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity (Proc Natl Acad Sci USA (2014) 111, (1939-1944) DOI: 10.1073/pnas.1317350111)

MICROBIOLOGY Retraction for “Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity,” by William B. Messer, Ruklanthi de Alwis, Boyd L. Yount, Scott R. Royal, Jeremy P. Huynh, Scott A. Smith, James E. Crowe, Jr., Benjamin J. Doranz, Kristen M. Kahle, Jennifer M. Pfaff, Laura J. White, Carlos A. Sariol, Aravinda M. de Silva, and Ralph S. Baric, which appeared in issue 5, February 4, 2014, of Proc Natl Acad Sci USA (111:1939–1944; first published January 2, 2014; 10.1073/ pnas.1317350111). The authors wish to note the following: “We have discovered a significant error that compromises our ability to interpret the in vitro and in vivo data reported in this paper. During recent follow-up studies with the rDENV-3/4 virus, we discovered that the original stocks used to generate the in vitro neutralization data and in vivo primate infection data reported in the manuscript contained contaminating wild-type DENV-4 virus. We have since determined this low-level contamination was sufficient to confound the chimeric DENV-3/4 results, rendering them uninterpretable. We emphasize that the escape mutant and recombinant protein data reported in the paper are in no way compromised by this contamination and stand on their own. Specifically, mapping of the critical 5J7 antibody epitope residues, shown in Fig. 1, did not utilize any recombinant chimeric DENV. The viral antibody escape mutants were derived from wild-type infectious clones and were sequence verified. The loss of antibody binding studies were conducted by Integral Molecular and used a recombinant protein expression system independent of the chimeric DENV. We deeply apologize for this inadvertent error. Accordingly, we have unanimously decided to retract the manuscript at this time. Given the large number of related constructs that we have subsequently generated in the laboratory, the strategy is sound; however, we cannot interpret the data and results reported in the manuscript.”