Resistance to infection, early and persistent suppression of simian immunodeficiency virus SIVmac251 viremia, and significant reduction of tissue viral burden after mucosal vaccination in female rhesus macaques.

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4ß7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

Tat engagement of p38 MAP kinase and IRF7 pathways leads to activation of interferon-stimulated genes in antigen-presenting cells.

As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.

HIV-1 Tat second exon limits the extent of Tat-mediated modulation of interferon-stimulated genes in antigen presenting cells.

BACKGROUND: We have shown that HIV-1 Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters is key to IFN-stimulated genes (ISG) activation in immature dendritic cells and macrophages. RESULTS: We evaluated how Tat alleles and mutants differ in cellular gene modulation of immature dendritic cells and monocyte-derived macrophages and what similarities this modulation has with that induced by interferons. The tested alleles and mutants modulated to different degrees ISG, without concomitant induction of interferons. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all modulated genes to a significantly greater extent than full-length wild type, two-exon Tat, indicating that Tat second exon is critical in reducing the innate response triggered by HIV-1 in these cells. Mutants with reduced LTR transactivation had a substantially reduced effect on host gene expression modulation than wild type TatSF2. However, the more potent LTR transactivator TatSF2A58T modulated ISG expression to a lower degree compared to TatSF2. A cellular gene modulation similar to that induced by Tat and Tat mutants in immature dendritic cells could be observed in monocyte-derived macrophages, with the most significant pathways affected by Tat being the same in both cell types. Tat expression in cells deleted of the type I IFN locus or receptor resulted in a gene modulation pattern similar to that induced in primary immature dendritic cells and monocyte-derived macrophages, excluding the involvement of type I IFNs in Tat-mediated gene modulation. ISG activation depends on Tat interaction with MAP2K3, MAP2K6, and IRF7 promoters and a single exon Tat protein more strongly modulated the luciferase activity mediated by MAP2K3, MAP2K6, and IRF7 promoter sequences located 5' of the RNA start site than the wild type two-exon Tat, while a cysteine and lysine Tat mutants, reduced in LTR transactivation, had negligible effects on these promoters. Chemical inhibition of CDK9 or Sp1 decreased Tat activation of MAP2K3-, MAP2K6-, and IRF7-mediated luciferase transcription. CONCLUSIONS: Taken together, these data indicate that the second exon of Tat is critical to the containment of the innate response stimulated by Tat in antigen presenting cells and support a role for Tat in stimulating cellular transcription via its interaction with transcription factors present at promoters.

Evaluation of ELISA-Based Multiplex Peptides for the Detection of Human Serum Antibodies Induced by Zika Virus Infection across Various Countries.

Zika virus (ZIKV) is a mosquito-borne Flavivirus with a positive-sense RNA genome, which are generally transmitted through the bite of an infected Aedes mosquito. ZIKV infections could be associated with neurological sequelae that, and otherwise produces similar clinical symptoms as other co-circulating pathogens. Past infection with one member of the Flavivirus genus often induces cross-reactive antibodies against other flaviruses. These attributes complicate the ability to differentially diagnose ZIKV infection from other endemic mosquito-borne viruses, making it both a public health issue as well as a diagnostic challenge. We report the results from serological analyses using arbovirus-specific peptides on 339 samples that were previously collected from 6 countries. Overall, we found that our multiplexed peptide-based ELISA was highly efficient for identifying ZIKV antibodies as early as 2 weeks post infection, and that it correlates with microneutralization, plaque reduction neutralization tests (PRNTs) and commercial tests for ZIKV in previously characterized samples. We observed that seropositivity varied by patient cohort, reflecting the sampling period in relation to the 2015-2016 ZIKV outbreak. This work evaluates the accuracy, specificity, and sensitivity of our peptide-based ELISA method for detecting ZIKV antibodies from geographically diverse regions. These findings can contribute to ongoing serological methods development and can be adapted for use in future studies.

Peptide arrays of three collections of human sera from patients infected with mosquito-borne viruses.

Background: Global outbreaks caused by emerging or re-emerging arthropod-borne viruses (arboviruses) are becoming increasingly more common. These pathogens include the mosquito-borne viruses belonging to the Flavivirus and Alphavirus genera. These viruses often cause non-specific or asymptomatic infection, which can confound viral prevalence studies. In addition, many acute phase diagnostic tests rely on the detection of viral components such as RNA or antigen. Standard serological tests are often not reliable for diagnosis after seroconversion and convalescence due to cross-reactivity among flaviviruses. Methods: In order to contribute to development efforts for mosquito-borne serodiagnostics, we incubated 137 human sera on individual custom peptide arrays that consisted of over 866 unique peptides in quadruplicate. Our bioinformatics workflow to analyze these data incorporated machine learning, statistics, and B-cell epitope prediction. Results: Here we report the results of our peptide array data analysis, which revealed sets of peptides that have diagnostic potential for detecting past exposure to a subset of the tested human pathogens including Zika virus. These peptides were then confirmed using the well-established ELISA method. Conclusions: These array data, and the resulting peptides can be useful in diverse efforts including the development of new pan-flavivirus antibodies, more accurate epitope mapping, and vaccine development against these viral pathogens.

Characterizing the Different Effects of Zika Virus Infection in Placenta and Microglia Cells.

Zika virus (ZIKV) is a neuropathic virus that causes serious neurological abnormalities such as Guillain-Barre syndrome in adults and congenital Zika syndrome (CZS) in fetuses, which makes it an important concern for global human health. A catalogue of cells that support ZIKV replication, pathogenesis, and/or the persistence of the virus still remains unknown. Here, we studied the behavior of the virus in human placenta (JEG-3) and human microglia (HMC3) cell lines in order to better understand how different host tissues respond during infection. We quantified the host transcriptional response to ZIKV infection in both types of cells at 24 and 72 h post-infection. A panel of 84 genes that are involved in the innate or adaptive immune responses was used to quantify differential expression in both cell lines. HMC3 cells showed a unique set of significant differentially expressed genes (DEGs) compared with JEG-3 cells at both time points. Subsequent analysis of these data using modern pathway analysis methods revealed that the TLR7/8 pathway was strongly inhibited in HMC3 cells, while it was activated in JEG-3 cells during virus infection. The disruption of these pathways was subsequently confirmed with specific small interfering RNA (siRNA) experiments that characterize their role in the viral life cycle, and may partially explain why ZIKV infection in placental tissue contributes to extreme neurological problems in a developing fetus.