Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge.

Liver macrophage-associated inflammation correlates with SIV burden and is substantially reduced following cART.

Liver disease is a leading contributor to morbidity and mortality during HIV infection, despite the use of combination antiretroviral therapy (cART). The precise mechanisms of liver disease during HIV infection are poorly understood partially due to the difficulty in obtaining human liver samples as well as the presence of confounding factors (e.g. hepatitis co-infection, alcohol use). Utilizing the simian immunodeficiency virus (SIV) macaque model, a controlled study was conducted to evaluate the factors associated with liver inflammation and the impact of cART. We observed an increase in hepatic macrophages during untreated SIV infection that was associated with a number of inflammatory and fibrosis mediators (TNFα, CCL3, TGFß). Moreover, an upregulation in the macrophage chemoattractant factor CCL2 was detected in the livers of SIV-infected macaques that coincided with an increase in the number of activated CD16+ monocyte/macrophages and T cells expressing the cognate receptor CCR2. Expression of Mac387 on monocyte/macrophages further indicated that these cells recently migrated to the liver. The hepatic macrophage and T cell levels strongly correlated with liver SIV DNA levels, and were not associated with the levels of 16S bacterial DNA. Utilizing in situ hybridization, SIV-infected cells were found primarily within portal triads, and were identified as T cells. Microarray analysis identified a strong antiviral transcriptomic signature in the liver during SIV infection. In contrast, macaques treated with cART exhibited lower levels of liver macrophages and had a substantial, but not complete, reduction in their inflammatory profile. In addition, residual SIV DNA and bacteria 16S DNA were detected in the livers during cART, implicating the liver as a site on-going immune activation during antiretroviral therapy. These findings provide mechanistic insights regarding how SIV infection promotes liver inflammation through macrophage recruitment, with implications for in HIV-infected individuals.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Deep sequencing of HIV-infected cells: insights into nascent transcription and host-directed therapy.

UNLABELLED: Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4(+) T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expressed genes at 12 h postinfection by Total RNAseq than by mRNAseq. These expression changes were also evident by concurrent changes in introns and were recapitulated by later mRNA changes, revealing an unexpectedly significant delay between transcriptional initiation and mature mRNA production early after HIV-1 infection. We computationally derived and validated the underlying regulatory programs, and we predicted drugs capable of reversing these HIV-1-induced expression changes followed by experimental confirmation. Our results show that combined total and mRNA transcriptome analysis is essential for fully capturing the early host response to virus infection and provide a framework for identifying candidate drugs for host-directed therapy against HIV/AIDS. IMPORTANCE: In this study, we used mass sequencing to identify genes differentially expressed in CD4(+) T cells during HIV-1 infection. To our surprise, we found many differentially expressed genes early after infection by analyzing both newly transcribed unprocessed pre-mRNAs and fully processed mRNAs, but not by analyzing mRNAs alone, indicating a significant delay between transcription initiation and mRNA production early after HIV-1 infection. These results also show that important findings could be missed by the standard practice of analyzing mRNAs alone. We then derived the regulatory mechanisms driving the observed expression changes using integrative computational analyses. Further, we predicted drugs that could reverse the observed expression changes induced by HIV-1 infection and showed that one of the predicted drugs indeed potently inhibited HIV-1 infection. This shows that it is possible to identify candidate drugs for host-directed therapy against HIV/AIDS using our genomics-based approach.

Deep transcriptional sequencing of mucosal challenge compartment from rhesus macaques acutely infected with simian immunodeficiency virus implicates loss of cell adhesion preceding immune activation.

Pathology resulting from human immunodeficiency virus (HIV) infection is driven by protracted inflammation; the primary loss of CD4(+) T cells is caused by activation-driven apoptosis. Recent studies of nonhuman primates (NHPs) have suggested that during the acute phase of infection, antiviral mucosal immunity restricts viral replication in the primary infection compartment. These studies imply that HIV achieves systemic infection as a consequence of a failure in host antiviral immunity. Here, we used high-dose intrarectal inoculation of rhesus macaques with simian immunodeficiency virus (SIV) SIVmac251 to examine how the mucosal immune system is overcome by SIV during acute infection. The host response in rectal mucosa was characterized by deep mRNA sequencing (mRNA-seq) at 3 and 12 days postinoculation (dpi) in 4 animals for each time point. While we observed a strong host transcriptional response at 3 dpi, functions relating to antiviral immunity were absent. Instead, we observed a significant number of differentially expressed genes relating to cell adhesion and reorganization of the cytoskeleton. We also observed downregulation of genes encoding members of the claudin family of cell adhesion molecules, which are coexpressed with genes associated with pathology in the colorectal mucosa, and a large number of noncoding transcripts. In contrast, at 12 dpi the differentially expressed genes were enriched in those involved with immune system functions, in particular, functions relating to T cells, B cells, and NK cells. Our findings indicate that host responses that negatively affect mucosal integrity occur before inflammation. Consequently, when inflammation is activated at peak viremia, mucosal integrity is already compromised, potentially enabling rapid tissue damage, driving further inflammation. Importance: The HIV pandemic is one of the major threats to human health, causing over a million deaths per year. Recent studies have suggested that mucosal antiviral immune responses play an important role in preventing systemic infection after exposure to the virus. Yet, despite their potential role in decreasing transmission rates between individuals, these antiviral mechanisms are poorly understood. Here, we carried out the first deep mRNA sequencing analysis of mucosal host responses in the primary infection compartment during acute SIV infection. We found that during acute infection, a significant host response was mounted in the mucosa before inflammation was triggered. Our analysis indicated that the response has a detrimental effect on tissue integrity, causing increased permeability, tissue damage, and recruitment of SIV target cells. These results emphasize the importance of mucosal host responses preceding immune activation in preventing systemic SIV infection.

Assessment and improvement of Indian-origin rhesus macaque and Mauritian-origin cynomolgus macaque genome annotations using deep transcriptome sequencing data.

BACKGROUND: The genome annotations of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques, two of the most common non-human primate animal models, are limited. METHODS: We analyzed large-scale macaque RNA-based next-generation sequencing (RNAseq) data to identify un-annotated macaque transcripts. RESULTS: For both macaque species, we uncovered thousands of novel isoforms for annotated genes and thousands of un-annotated intergenic transcripts enriched with non-coding RNAs. We also identified thousands of transcript sequences which are partially or completely 'missing' from current macaque genome assemblies. We showed that many newly identified transcripts were differentially expressed during SIV infection of rhesus macaques or during Ebola virus infection of cynomolgus macaques. CONCLUSIONS: For two important macaque species, we uncovered thousands of novel isoforms and un-annotated intergenic transcripts including coding and non-coding RNAs, polyadenylated and non-polyadenylated transcripts. This resource will greatly improve future macaque studies, as demonstrated by their applications in infectious disease studies.

Annotation of long non-coding RNAs expressed in collaborative cross founder mice in response to respiratory virus infection reveals a new class of interferon-stimulated transcripts.

The outcome of respiratory virus infection is determined by a complex interplay of viral and host factors. Some potentially important host factors for the antiviral response, whose functions remain largely unexplored, are long non-coding RNAs (lncRNAs). Here we systematically inferred the regulatory functions of host lncRNAs in response to influenza A virus and severe acute respiratory syndrome coronavirus (SARS-CoV) based on their similarity in expression with genes of known function. We performed total RNA-Seq on viral-infected lungs from eight mouse strains, yielding a large data set of transcriptional responses. Overall 5,329 lncRNAs were differentially expressed after infection. Most of the lncRNAs were co-expressed with coding genes in modules enriched in genes associated with lung homeostasis pathways or immune response processes. Each lncRNA was further individually annotated using a rank-based method, enabling us to associate 5,295 lncRNAs to at least one gene set and to predict their potential cis effects. We validated the lncRNAs predicted to be interferon-stimulated by profiling mouse responses after interferon-α treatment. Altogether, these results provide a broad categorization of potential lncRNA functions and identify subsets of lncRNAs with likely key roles in respiratory virus pathogenesis. These data are fully accessible through the MOuse NOn-Code Lung interactive database (MONOCLdb).

Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq.

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Macrophage-associated wound healing contributes to African green monkey SIV pathogenesis control.

Natural hosts of simian immunodeficiency virus (SIV) avoid AIDS despite lifelong infection. Here, we examined how this outcome is achieved by comparing a natural SIV host, African green monkey (AGM) to an AIDS susceptible species, rhesus macaque (RM). To asses gene expression profiles from acutely SIV infected AGMs and RMs, we developed a systems biology approach termed Conserved Gene Signature Analysis (CGSA), which compared RNA sequencing data from rectal AGM and RM tissues to various other species. We found that AGMs rapidly activate, and then maintain, evolutionarily conserved regenerative wound healing mechanisms in mucosal tissue. The wound healing protein fibronectin shows distinct tissue distribution and abundance kinetics in AGMs. Furthermore, AGM monocytes exhibit an embryonic development and repair/regeneration signature featuring TGF-ß and concomitant reduced expression of inflammatory genes compared to RMs. This regenerative wound healing process likely preserves mucosal integrity and prevents inflammatory insults that underlie immune exhaustion in RMs.

Author Correction: Macrophage-associated wound healing contributes to African green monkey SIV pathogenesis control.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regional astrocyte IFN signaling restricts pathogenesis during neurotropic viral infection.

Type I IFNs promote cellular responses to viruses, and IFN receptor (IFNAR) signaling regulates the responses of endothelial cells of the blood-brain barrier (BBB) during neurotropic viral infection. However, the role of astrocytes in innate immune responses of the BBB during viral infection of the CNS remains to be fully elucidated. Here, we have demonstrated that type I IFNAR signaling in astrocytes regulates BBB permeability and protects the cerebellum from infection and immunopathology. Mice with astrocyte-specific loss of IFNAR signaling showed decreased survival after West Nile virus infection. Accelerated mortality was not due to expanded viral tropism or increased replication. Rather, viral entry increased specifically in the hindbrain of IFNAR-deficient mice, suggesting that IFNAR signaling critically regulates BBB permeability in this brain region. Pattern recognition receptors and IFN-stimulated genes had higher basal and IFN-induced expression in human and mouse cerebellar astrocytes than did cerebral cortical astrocytes, suggesting that IFNAR signaling has brain region-specific roles in CNS immune responses. Taken together, our data identify cerebellar astrocytes as key responders to viral infection and highlight the existence of distinct innate immune programs in astrocytes from evolutionarily disparate regions of the CNS.

Integrated Omics Analysis of Pathogenic Host Responses during Pandemic H1N1 Influenza Virus Infection: The Crucial Role of Lipid Metabolism.

Pandemic influenza viruses modulate proinflammatory responses that can lead to immunopathogenesis. We present an extensive and systematic profiling of lipids, metabolites, and proteins in respiratory compartments of ferrets infected with either 1918 or 2009 human pandemic H1N1 influenza viruses. Integrative analysis of high-throughput omics data with virologic and histopathologic data uncovered relationships between host responses and phenotypic outcomes of viral infection. Proinflammatory lipid precursors in the trachea following 1918 infection correlated with severe tracheal lesions. Using an algorithm to infer cell quantity changes from gene expression data, we found enrichment of distinct T cell subpopulations in the trachea. There was also a predicted increase in inflammatory monocytes in the lung of 1918 virus-infected animals that was sustained throughout infection. This study presents a unique resource to the influenza research community and demonstrates the utility of an integrative systems approach for characterization of lipid metabolism alterations underlying respiratory responses to viruses.

Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate.

We describe the development of fetal brain lesions after Zika virus (ZIKV) inoculation in a pregnant pigtail macaque. Periventricular lesions developed within 10 d and evolved asymmetrically in the occipital-parietal lobes. Fetal autopsy revealed ZIKV in the brain and significant cerebral white matter hypoplasia, periventricular white matter gliosis, and axonal and ependymal injury. Our observation of ZIKV-associated fetal brain lesions in a nonhuman primate provides a model for therapeutic evaluation.

Next-generation sequencing reveals a controlled immune response to Zaire Ebola virus challenge in cynomolgus macaques immunized with vesicular stomatitis virus expressing Zaire Ebola virus glycoprotein (VSVΔG/EBOVgp).

Vesicular stomatitis virus expressing Zaire Ebola virus (EBOV) glycoprotein (VSVΔG/EBOVgp) could be used as a vaccine to meet the 2014 Ebola virus outbreak. To characterize the host response to this vaccine, we used mRNA sequencing to analyze peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques after VSVΔG/EBOVgp immunization and subsequent EBOV challenge. We found a controlled transcriptional response that transitioned to immune regulation as the EBOV was cleared. This observation supports the safety of the vaccine.

A comprehensive collection of systems biology data characterizing the host response to viral infection.

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.

Next-generation sequencing of small RNAs from HIV-infected cells identifies phased microrna expression patterns and candidate novel microRNAs differentially expressed upon infection.

HIV infection of CD4(+) T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection. IMPORTANCE New sequencing technologies allow unprecedented views into changes occurring in virus-infected cells, including comprehensive and largely unbiased measurements of different types of RNA. In this study, we used next-generation sequencing to profile dynamic changes in cellular microRNAs occurring in HIV-infected cells. The sensitivity afforded by sequencing allowed us to detect changes in microRNA expression early in infection, before the onset of viral replication. A phased pattern of expression was evident among these microRNAs, and many that were initially suppressed were later overexpressed at the height of infection, providing unique signatures of infection. By integrating additional mRNA data with the microRNA data, we identified a role for microRNAs in transcriptional regulation during infection and specifically a network of microRNAs involved in the expression of a known HIV cofactor. Finally, as a distinct benefit of sequencing, we identified candidate nonannotated microRNAs, including one whose downregulation may allow HIV-1 replication to proceed fully.

Blockade of the Fas/FasL system improves pneumococcal clearance from the lungs without preventing dissemination of bacteria to the spleen.

BACKGROUND: The Fas/FasL system is both proapoptotic and proinflammatory. FasL is inhibited by decoy receptor-3 (DcR3), a naturally occurring decoy receptor. We determined the effects of systemic blockade of the Fas/FasL system by a DcR3 analog (DcR3-a) in mice with pneumococcal pneumonia. METHODS: Streptococcus pneumoniae (7.2 x 105 or 1.9 x 107 cfu/mL) was instilled intratracheally into untreated C57Bl/6 mice, C57Bl/6 mice treated with DcR3-a, or Fas-deficient lpr mice, and the mice were studied 48 h later. RESULTS: After instillation of the lower bacterial dose, disruption of the Fas/FasL system by either DcR3-a or the lpr mutation resulted in improved clearance of bacteria in the lungs (mean +/- SE, 4.6+/-2.1 x 10(6) and 3.5 +/- 1.6 x 10(6) cfu/lung, respectively, vs. 21.9+/-9.3 x 10(6) cfu/lung in untreated C57Bl/6 mice; P<.05) and decreased percentage of polymorphonuclear neutrophils in bronchoalveolar lavage fluid (mean +/- SE, 19.3%+/-9.5% and 20.2%+/-7.8%, respectively, vs. 55.0%+/-12.2% in untreated C57Bl/6 mice; P<.05). These changes were associated with decreased lung concentrations of the proinflammatory cytokines tumor necrosis factor- alpha and macrophage inflammatory protein-2 and with a decrease in apoptotic cells in the alveolar walls. CONCLUSION: Blockade of the Fas/FasL system by DcR3-a in the lungs improves clearance of bacteria in mice with pneumococcal pneumonia.