Unknown,Transcriptomics,Genomics,Proteomics

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An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause renavirus disease but does elicit protective immunity

ABSTRACT: Lassa fever (LF) is a rodent-borne viral disease that can be fatal for human beings. In this study, an attenuated Lassa vaccine candidate, ML29, was tested in SIV-infected rhesus macaques for its ability to elicit immune responses without instigating signs of virulent disease. ML29 is a reassortant between Lassa and Mopeia viruses that causes a transient infection in non-human primates and confers sterilizing protection from lethal Lassa viral challenge. However, since the LF endemic area of West Africa also has high HIV seroprevalence, it is important to determine whether vaccination could be safe in the context of AIDS. SIV-infected and uninfected rhesus macaques were vaccinated with the ML29 virus and monitored for classical and non-classical signs of arenavirus disease. Classical disease signs included viremia, rash, weight loss, high liver enzyme levels, and virus invasion of the central nervous system. Non-classical signs derived from profiling the blood transcriptome of virulent and non-virulent arenavirus infections included increased expression of interferon response genes and decreased expression of COX2, IL-1?, coagulation intermediates and nuclear receptors needed for stress signaling. Here it is demonstrated that SIV-infected and uninfected rhesus macaques responded similarly to ML29 vaccination, and that none developed signs of arenavirus disease or persistence. Furthermore, 5 of 5 animals given a heterologous challenge with a lethal dose of LCMV-WE survived without developing disease signs. 30 RNA samples from Monkey PBMC: 4 uninf. Monkey PBMC, 8 SIV-infected Monkey PBMC(From 8 Monkeys), 5 SIV+ML29-sc infected week1(Monkey PBMC), 5 SIV+ML29-sc infected week2(Monkey PBMC), 1 SIV+ML29-ig infected week1(Monkey PBMC), 1 SIV+ML29-ig infected week2(Monkey PBMC), 2 SIV+Arm-sc infected week1(Monkey PBMC), 2 SIV+Arm-sc infected week2(Monkey PBMC), 1 only ML29-iv infected week1(Monkey PBMC), 1 only ML29-iv infected week2(Monkey PBMC)

ORGANISM(S): Macaca mulatta

SUBMITTER: Maria Salvato

PROVIDER: E-GEOD-33687 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Expression profiling by NanoString nCounter gene expression system Inflammatory response genes play an important role during acute HIV and SIV infection. Using an SIV macaque model for AIDS and CNS disease, we measured mRNA expression of 92 genes before and at multiple time points during acute SIV infection in three tissues: Peripheral blood mononuclear cells, mesenteric lymph nodes (MLN) and peripheral blood mononuclear cells (PBMC). Studying the overall changes of mRNA expressions over time or analyzing the correlation between the gene measurements and SIV RNA in plasma can result in limited interpretations. This is due to several reasons including but not limited to: 1) lack of prior information on how cells react to changes in gene expressions; and 2) biological responses typically involve many genes working together. To approach this problem, we combine multiple preprocessing methods with two multivariate analysis methods, namely principal component analysis and partial least square regression, to create a multiplexed set of 12 “judges”. Each of the judges simultaneously observes all the variables and emphasizes a unique type of gene expression response that could be significant, for example, depending on whether the cell responds to the absolute or relative size of gene expression changes. By incorporating multiple potential biological models of response, it is possible to identify genes that are consistently ranked as high “contributing” genes in different scenarios, i.e., genes that have a higher weight when we classify the data based on different classification schemes. We then use statistical analysis to verify that the consistently high-ranking genes are also statistically significant. We also investigate whether genes are tissue-specific, identify clusters of genes that co-vary together and study their correlation with regard to other gene clusters. The multiplex component analysis method introduced in this paper is a powerful tool to analyze complex gene datasets, identify significant genes, and generate testable hypotheses. In this study we inoculated 20 pig-tailed macaques with two SIV strains (SIV/17E-Fr and the swarm SIV/DeltaB670), and mock-inoculated 4 animals for control. Infected animals were sacrificed at 4 time points, as following: 6 animals sacrificed at 4 days, 6 at 7 days, 6 at 14 days and 2 at 21 days post-infection. Animals were perfused before necropsy to decrease blood contamination in the tissues. Organs were then culled, and RNA was extraction for NanosString analysis using a set of 92 probes specifically generated for macaque genes. Data were then analyzed by multiple preprocessing methods with two multivariate analysis methods. The following files contains raw data for the listed samples, respectively; GAMA Nanostring RAW MLN.xlsx - all MLN samples GAMA Nanostring RAW PBMC.xlsx - all PBMC samples GAMA Nanostring RAW Spleen 1.xls - PT01-04SP, PT71-74SP, PT141-144SP GAMA Nanostring RAW Spleen 2.xls - PT41-44SP, PT75-76SP, PT145-146SP, PT211-212SP GAMA Nanostring RAW Spleen 3.xls - PT45-46SP Please note that, in the associated publication, we describe a multiplexed component analysis to identify genes that highly contribute to inflammation during SIV acute infection. This analysis was done by compiling results generated by different types of data transformations and data normalizations (more than 12 for each tissue) and the relevant algorithms/mathematical processes will be discussed in detail. Therefore, only raw data is presented in this record (i.e. each sample data table contains raw data).

Project description:In SIV/HIV infection, the gastrointestinal tissue dominates as an important site due to the impact of massive mucosal CD4 depletion and immune activation-induced tissue pathology. Unlike AIDS-susceptible rhesus macaques, natural hosts do not progress to AIDS and resolve immune activation earlier. Here, we examine the role of dendritic cells in mediating immune activation and disease progression. We demonstrate that plasmacytoid dendritic cells (pDC) in the blood upregulate ?7-integrin and are rapidly recruited to the colorectum following a pathogenic SIV infection in rhesus macaques. These pDC were capable of producing proinflammatory cytokines and primed a Tc1 response in vitro. Consistent with the upregulation of ?7-integrin on pDC, in vivo blockade of ?4?7-integrin dampened pDC recruitment to the colorectum and resulted in reduced immune activation. The upregulation of ?7-integrin expression on pDC in the blood was also observed in HIV-infected humans but not in chronically SIV-infected sooty mangabeys that show low levels of immune activation. Our results uncover a new mechanism by which pDC influence immune activation in colorectal tissue following pathogenic immunodeficiency virus infections. SIV negative controls (n=4) and week 12 post SIV infected (n=4) groups of Rhesus macaques and SIV negative controls (n=4) and week 55 post SIV infected (n=4) groups of Sooty mangabeys colorectal tissue biopsies were collected in to RNA later reagent (Qiagen) and were homogenized with syringe and needle method. RNA was extracted with Rneasy mini kit (Qiagen) and was used for microarray experiments. Rhesus GeneChip assays were performed in the Yerkes Microarray Core Facility (www.microarray.emory.edu) , one of the Affymetrix Microarray Core Labs.The 0.5µg of total RNA sample was analyzed on Rhesus Macaque Genome GeneChip that consists of over 52,000 probe sets (Affymetrix, Santa Clara, CA). Target RNA labeling, hybridization and post-hybridization processing were performed following the Affymetrix GeneChip Expression Analysis standard protocols. In brief, The 5 ?g of RNA sample was first reverse-transcribed using T7-Oligo(dT) Promoter Primer and SuperScript II in the first-strand cDNAs synthesis reaction. Following RNase H-mediated second-stranded cDNA synthesis, the double-stranded cDNAs were purified by use of a GeneChip sample clean-up module and served as templates in the generation of biotinylated complementary RNAs (cRNAs) in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix by in vitro transcription (IVT) reaction. The biotinylated cRNAs were cleaned up, fragmented, and hybridized to the rhesus macaque expression arrays at 45°C for 16 h with constant rotation at 60 rpm. The gene chips were then washed and stained with Affymetrix fluidics stations 450 and scanned on Affymetrix scanner 3000. The images are processed to collect raw data with GeneChip Operating Software (GCOS) 1.4. Tissue: Colorectal tissue Time after SIV infection: 12 weeks for SIV infected Rhesus macaques, 55 weeks for Sooty mangabeys Infection: SIVmac251 infection for Rhesus macaques, SIVsm infection for Sooty mangabeys

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An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause renavirus disease but does elicit protective immunity

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