WNPRC IMMUNOLOGY & VIROLOGY UNIT

WNPRC 免疫学

• 批准号: 8173096
• 负责人: David I Watkins
• 金额: $ 10.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173096
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alleles; Antibodies; Antigens; Ascaridil; Australia; Back; Binding; Biological Assay; Blood specimen; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Separation; Cells; Cercopithecus tantalus; Characteristics; Charge; Coculture Techniques; Collaborations; Communicable Diseases; Computer Retrieval of Information on Scientific Projects Database; Consult; Consultations; Custom; Data; Data Analyses; Detection; Development; Diagnostic tests; Epitopes; Extramural Activities; Flow Cytometry; Funding; Gagging; Generations; Genetic; Grant; HIV Antibodies; HIV-1; HLA-B 2705 antigen; Histocompatibility; Histocompatibility Antigens Class I; Hour; Human; Immune response; Immunogenetics; Immunologic Deficiency Syndromes; Immunology; In Situ; Income; Infection; Institution; Laboratories; Leukocytes; Lymphoid Tissue; MHC Class I Genes; MHC Class II Genes; Macaca; Macaca mulatta; Manuscripts; Membrane; Messenger RNA; Methods; Mission; Molecular Biology; Mutagenesis; Mutation; National Cancer Institute; New England; Pathology; Peptides; Pluripotent Stem Cells; Population; Price; Primates; Printing; Process; Production; Protocols documentation; RNA Interference; Recombinants; Reporter; Research; Research Personnel; Resource Allocation; Resources; Reverse Transcriptase Polymerase Chain Reaction; SIV; Sampling; Schools; Science; Serum; Services; Shipping; Ships; Shivering; Silent Mutation; Site-Directed Mutagenesis; Sorting - Cell Movement; Source; Speed; Spices; Staining method; Stains; System; T cell response; T-Lymphocyte; T-Lymphocyte Epitopes; Techniques; Testing; Training; Transgenes; Translations; United States National Institutes of Health; Vaccinated; Vaccination; Vaccines; Variant; Vial device; Viral; Viral load measurement; Virus; Virus Replication; Work; Yellow Fever; Yellow Fever Vaccine; bean; cytokine; data acquisition; design; fast protein liquid chromatography; fitness; induced pluripotent stem cell; instrument; interest; macrophage; member; monocyte; mutant; neutralizing monoclonal antibodies; nonhuman primate; novel; peripheral blood; progenitor; reagent testing; research study; response; simian human immunodeficiency virus; vector; virology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To provide expert support to AIDS research conducted at the Primate Center. 1. PROGRESS AND CONCERNS: The Virology Services Unit (VS) performed over 1,700 SIV virus load determinations in FY2009. This is fewer than in FY2008, as our core users pared back on the scope of their studies. We anticipate increased demand in FY2010 as established users are funded to perform more and larger experiments. In anticipation of this increased demand, we tested a new workflow using the high-throughput Roche LightCycler 480 quantitative PCR platform in February 2010. Transition to this workflow will facilitate an expansion of our services and will simplify batch processing of samples as we now charge all users, both intra- and extramural, for virus load determinations. Our chargeback system for extramural investigators had 2 users and generated revenues of $18,255. VS also produced over 4,000 vials of high-titer SIVmac239 stock and 3 custom SIV mutants for WNPRC investigators. In response to investigators interested in evaluating the fitness of multiple mutant strains of SIV, we deployed a "barcode virus" reporter system. We used site-directed mutagenesis to insert a genetic "barcode" of silent mutations into the region of SIVmac239 gag targeted by our standard QRT-PCR assay. These mutations do not affect virus fitness, but they abrogate detection using the primers and probes of our standard assay. This "barcode" virus thus provides a standard against which any viral variant can be directly compared in competing coculture assays. The Tetramer Core of Immunology Services Unit (IS) produced approximately 99,000 tetramer tests in 2009. 25,508 tests were shipped to ten different investigators at nine different institutions, bringing in $76,524 in chargeback income. We also distributed 7,155 tests to laboratories on campus, including the David H. O'Connor laboratory in Pathology, the Thomas Friedrich laboratory at the Vet School and the Watkins Laboratory in Pathology. All three of these labs are also part of the WNPRC. Our FPLC instruments were also utilized by non-primate center investigators, assisting them in preliminary studies, or when their FPLC was not working. We are currently pursuing MHC class II tetramers, and expect to produce our first tetramers in early 2010. We have expanded the alleles that are available for tetramer production in 2009, to include several cynomolgus alleles. We will continue expanding our allele availability in 2010, as more and more cyno and rhesus alleles are found to be important in SIV studies. In addition, we are developing Elisas for class II tetramers, as well as a diagnostic test for peptide binding. Our group makes all of the p27 antibody used by our lab, and also shipped 200 tests of this reagent to Stephen Kent's lab in Australia. For extramural investigators more than 1200 blood samples were processed and shipped out, and 1680 IFN-¿ Elispot tests and 327 intracellular cytokine staining tests were performed. IS also provides expert support to Primate Center, UW-Madison and extramural investigators wishing to use our flow cytometry facilities. More than 2,500 hours were used for flow data acquisition during this year. These activities together resulted in an additional ~$55,000 in chargebacks. In 2009 we purchased a new custom-made BD-LSR II machine to expand our services. We have established or supported the development of several multicolor staining protocols. These included staining panels to test different functional capabilities of antigen specific T cell populations, and panels that identify innate immune responses after yellow fever vaccination. To promote better data analysis in flow cytometry we have introduced the use of Pestle and Spice data analysis softwares. To expand our services with BL-3 level sorting capabilities Dr Rakasz will be trained to operate a FACSAria high speed cell sorter in February. She will have access to a BL-3 level sorting facility on campus and will perform cell sort for investigators who has this request. 2. ALLOCATION OF RESOURCE ACCESS: The central mission of IVS is to provide expert support to AIDS and infectious disease related research conducted at the Primate Center by WNPRC or outside investigators. In fiscal 2009 IS served 9 on-campus and 15 off-campus laboratories, which were funded by both federal and non-federal grants. VS supported 4 on-campus and 2 off-campus users in FY2009. 3. DISSEMINATION: Drs. Friedrich, Wilson and Rakasz, the PIs of VS and IS units, consult closely with users of the service, helping to design experiments and interpret results. We request that projects utilizing Virology and Immunology Services acknowledge the service in manuscripts and presentations. 4. TRAINING: Dr. Friedrich consults regularly with recognized leaders in SIV virology and molecular biology to develop and refine our techniques. For example, custom SIV mutagenesis methods were developed in collaboration with Dr. Ronald Desrosiers at the New England Primate Center. Quantitative RT-PCR techniques were developed in consultation with Dr. Jeffrey Lifson at the National Cancer Institute. IS staff have been trained in flow cytometry techniques at Beckton Dickinson. Dr Rakasz and Ms Kim Weisgrau, members of the flow cytometry facility regularly train new investigators to acquire their data independently on the flow machines at their disposal. PUBLICATIONS: Bonaldo MC, Martins MA, Rudersdorf R, Mudd PA, Sacha JB, Piaskowski SM, Costa Neves PC, Veloso de Santana MG, Vojnov L, Capuano S 3rd, Rakasz EG, Wilson NA, Fulkerson J, Sadoff JC, Watkins DI, Galler R. Recombinant Yellow Fever Vaccine Virus 17D Expressing SIVmac239 Gag Induces SIV-Specific CD8+ T Cell Responses in Rhesus Macaques. J Virol. 2010 Jan 20. [Epub ahead of print] PMID: 20089645 Maness NJ, Wilson NA, Reed JS, Piaskowski SM, Sacha JB, Walsh AD, Thoryk E, Heidecker GJ, Citron MP, Liang X, Bett AJ, Casimiro DR, Watkins DI. Robust, vaccine-induced CD8(+) T lymphocyte response against an out-of-frame epitope. J Immunol. 2010 Jan 1;184(1):67-72. PMID: 19949108 Hessell AJ, Rakasz EG, Tehrani DM, Huber M, Weisgrau KL, Landucci G, Forthal DN, Koff WC, Poignard P, Watkins DI, Burton DR. Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. J Virol. 2010 Feb;84(3):1302-13..PMID: 19906907 Vojnov L, Reed JS, Weisgrau KL, Rakasz EG, Loffredo JT, Piaskowski SM, Sacha JB, Kolar HL, Wilson NA, Johnson RP, Watkins DI. Effective simian immunodeficiency virus-specifici CD8+ T cells lack an easily detectable, shared characteristic. J Virol. 2010 Jan;84(2):753-64. PMID: 19889785 Valentine LE, Loffredo JT, Bean AT, Le¿n EJ, MacNair CE, Beal DR, Piaskowski SM, Klimentidis YC, Lank SM, Wiseman RW, Weinfurter JT, May GE, Rakasz EG, Wilson NA, Friedrich TC, O'Connor DH, Allison DB, Watkins DI. Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques. J Virol. 2009 Nov;83(22):11514-27. PMID: 19726517 Maness NJ, Sacha JB, Piaskowski SM, Weisgrau KL, Rakasz EG, May GE, Buechler MB, Walsh AD, Wilson NA, Watkins DI. Novel translation products from simian immunodeficiency virus SIVmac239 Env-encoding mRNA contain both Rev and cryptic T-cell epitopes. J Virol. 2009 Oct;83(19):10280-5. PMID: 19605480 Loffredo JT, Sidney J, Bean AT, Beal DR, Bardet W, Wahl A, Hawkins OE, Piaskowski S, Wilson NA, Hildebrand WH, Watkins DI, Sette A. Two MHC class I molecules associated with elite control of immunodeficiency virus replication, Mamu-B*08 and HLA-B*2705, bind peptides with sequence similarity. J Immunol. 2009 Jun 15;182(12):7763-75.PMID: 19494300. Sacha JB, Giraldo-Vela JP, Buechler MB, Martins MA, Maness NJ, Chung C, Wallace LT, Le¿n EJ, Friedrich TC, Wilson NA, Hiraoka A, Watkins DI. Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9791-6. PMID: 19478057 Hessell AJ, Rakasz EG, Poignard P, Hangartner L, Landucci G, Forthal DN, Koff WC, Watkins DI, Burton DR. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog. 2009 May;5(5):e1000433. PMID: 19436712 Wilson NA, Keele BF, Reed JS, Piaskowski SM, MacNair CE, Bett AJ, Liang X, Wang F, Thoryk E, Heidecker GJ, Citron MP, Huang L, Lin J, Vitelli S, Ahn CD, Kaizu M, Maness NJ, Reynolds MR, Friedrich TC, Loffredo JT, Rakasz EG, Erickson S, Allison DB, Piatak M Jr, Lifson JD, Shiver JW, Casimiro DR, Shaw GM, Hahn BH, Watkins DI. Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge. J Virol. 2009 Jul;83(13):6508-21. PMID: 19403685 Greene JM, Lhost JJ, Burwitz BJ, Budde ML, Macnair CE, Weiker MK, Gostick E, Friedrich TC, Broman KW, Price DA, O'Connor S, O'Connor DH. Extra-lymphoid tissue-resident CD8+ T cells from SIVmac239Deltanef-vaccinated macaques suppress SIVmac239 replication ex vivo. J Virol. 2010 Jan 20. Burwitz BJ, Pendley CJ, Greene JM, Detmer AM, Lhost JJ, Karl JA, Piaskowski SM, Rudersdorf RA, Wallace LT, Bimber BN, Loffredo JT, Cox DG, Bardet W, Hildebrand W, Wiseman RW, O'Connor SL, O'Connor DH. Mauritian cynomolgus macaques share two exceptionally common major histocompatibility complec class I alleles that restrict simian immunodeficiency virus specific CD8+ T cells. J Virol. 2009 Jun;83(12):6011-9. PMID: 19339351 Choi KD, Vodyanik MA, Slukvin II. Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors. J Clin Invest. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009 May 8;324(5928):797-801. PMID: 19325077 Rozner AE, Dambaeva SV, Drenzek JG, Durning M, Golos TG.Generation of macrophages from peripheral blood monocytes in the rhesus monkey. J Immunol Methods. 2009 Dec 31;351(1-2):36-40. PMID: 19818793 Bondarenko GI, Dambaeva SV, Grendell RL, Hughes AL, Durning M, Garthwaite MA, Golos TG. Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus. Immunogenetics. 2009 Jun;61(6):431-42. PMID: 19468726 Dambaeva SV, Breburda EE, Durning M, Garthwaite MA, Golos TG. Characterization of decidual leukocyte populations in cynomolgus and vervet monkeys. J Reprod Immunol. 2009 Jun;80(1-2):57-69. PMID: 19398130 Drenzek JG, Vidiguriene J, Vidiguris G, Grendell RL, Dambaeva SV, Durning M, Golos TG. Suppression of Mamu-AG by RNA interference. Am J Reprod Immunol. 2009 Apr 22. PMID: 19392979 Li Q, Skinner PJ, Duan L, Haase AT. A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ. J Vis Exp. 2009 Aug 13;(30). pii: 1561. doi: 10.3791/1561. Salisch NC, Kaufmann DE, Awad AS, Reeves RK, Tighe DP, Li Y, Piatak M Jr, Lifson JD, Evans DT, Pereyra F, Freeman GJ, Johnson RP. Inhibitory TCR coreceptor PD-1 is a sensitive indicator of low-level replication of SIV and HIV-1. J Immunol. 2010 Jan 1;184(1):476-87.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 目的：为在灵长类动物中心进行的艾滋病研究提供专家支持。 1。进展和关注： 2009财年，病毒学服务部（VS）进行了1,700多种SIV病毒负荷确定。这比2008财年少了，因为我们的核心用户回到了他们的研究范围。我们预计，由于已建立的用户被资助以执行更多和更大的实验，因此我们预计2010财年的需求会增加。预计需求的增加，我们使用高通量Roche Lightcycler 480定量PCR平台测试了一个新的工作流程。2010年2月的定量PCR平台。到此工作流程的过渡将有助于我们服务的扩展，并将简化样品的批处理处理，因为我们现在向所有用户收取所有用户，包括内室内和外部的Virus负载负荷确定。我们针对校外调查人员的退款系统有2个用户，并产生了18,255美元的揭晓。 VS还为WNPRC调查人员生产了4,000多瓶高电量SIVMAC239股票和3个自定义SIV突变体。为了响应有兴趣评估多个SIV突变菌株的适应性的研究者，我们部署了“条形码病毒”记者系统。我们使用定位的诱变将无声突变的遗传“条形码”插入我们的标准QRT-PCR分析靶向的SIVMAC239 GAG区域。这些突变不会影响病毒健身，但是它们使用我们的标准测定法的引物和问题消除了检测。因此，这种“条形码”病毒提供了一个标准，可以直接在竞争性共培养测定中直接比较任何病毒变异。 2009年，免疫学服务部门的四聚体核心（IS）生产了约99,000个四聚体测试。在九个不同机构的十位不同调查人员运送了25,508次测试，带来了76,524美元的收款收入。我们还向校园的实验室分发了7,155次测试，包括病理学的David H. O'Connor实验室，兽医学校的Thomas Friedrich实验室和病理学的沃特金斯实验室。这三个实验室也是WNPRC的一部分。我们的FPLC仪器也被非顶级中心研究人员使用，协助他们进行初步研究或何时其FPLC不起作用。我们目前正在追求MHC II类四聚体，并预计将在2010年初生产我们的第一个四聚体。我们扩大了2009年可用于四聚体生产的等位基因，其中包括几个Cynomolgus等位基因。我们将在2010年继续扩大我们的等位基因可用性，因为在SIV研究中发现越来越多的Cyno和Cyno和Rhesus等位基因很重要。此外，我们正在为II类四聚体开发ELISA，以及肽结合的诊断测试。我们的小组将实验室使用的所有P27抗体制造，并将该试剂的200次测试发送给了澳大利亚的Stephen Kent的实验室。 对于校外研究者，处理了1200多个血液样本并运送出来，并进行了1680次IFN-€ELISPOT测试和327个细胞内细胞因子染色测试。 IS还向希望使用我们的流式细胞仪设施的灵长类动物中心，UW-Madison和壁外研究人员提供专家支持。在今年，使用了2500多个小时的流量数据。这些活动共同导致了额外的约55,000美元的退款。 2009年，我们购买了新的定制BD-LSR II机器来扩展我们的服务。我们已经建立或支持了几种多色染色方案的开发。其中包括染色面板，以测试抗原特异性T细胞群体的不同功能能力，以及在黄热病疫苗接种后鉴定先天免疫反应的面板。 为了促进流式细胞术中的更好的数据分析，我们引入了杵和香料数据分析软件的使用。 为了扩大BL-3级分类功能，Rakasz博士将在2月进行Facsaria High Speed Sorter进行培训。她将可以在校园内使用BL-3级分类设施，并为有此要求的调查人员进行细胞排序。 2。资源访问的分配： IV的主要任务是为WNPRC或外部研究人员在灵长类动物中心进行的艾滋病和传染病相关研究提供专家支持。 2009财年，有9个校园内和15个校外实验室，由联邦和非联邦赠款资助。 VS在2009财年支持4个校园和2名校外用户。 3。传播： 博士。弗里德里希（Friedrich），威尔逊（Wilson）和拉卡斯（Rakasz），vs and IS单位的PI，与服务的用户密切协商，有助于设计实验并解释结果。我们要求使用病毒学和免疫学服务的项目确认手稿和演示中的服务。 4。培训： 弗里德里希（Friedrich）博士定期与公认的SIV病毒学和分子生物学领导者进行咨询，以开发和完善我们的技术。例如，自定义SIV诱变方法是与新英格兰灵长类动物中心的Ronald Desrosiers合作开发的。定量RT-PCR技术是与国家癌症研究所Jeffrey Lifson博士协商开发的。 IN员工已经接受了贝克顿·迪金森（Beckton Dickinson）的流式细胞仪技术培训。流式细胞仪设施的成员Rakasz博士和Kim Weisgrau女士定期培训新调查人员，以独立获取其数据，以便于其处置。 出版物： Bonaldo MC, Martins MA, Rudersdorf R, Mudd PA, Sacha JB, Piaskowski SM, Costa Neves PC, Veloso de Santana MG, Vojnov L, Capuano S 3rd, Rakasz EG, Wilson NA, Fulkerson J, Sadoff JC, Watkins DI, Galler R. 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