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Studies of human rotavirus candidate non-replicating vaccines and innate immunity in a gnotobiotic pig model of human rotavirus disease .

机译：人轮状病毒候选非复制疫苗和人免疫原性免疫的研究

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摘要

Rotavirus is the major cause of severe dehydrating diarrhea in children and young infants worldwide. The mortality rates reach 600,000 annually, mainly in developing countries and vaccination is an important preventive measure. The first two objectives of my PhD research were to produce and test a combination of replicating and non-replicating human rotavirus (HRV) vaccines or non-replicating HRV vaccines in the gnotobiotic pig model to minimize or avoid the use of more reactogenic live HRV vaccines. The third objective was to assess the mucosal and systemic dendritic cell responses after RV infection because these responses are largely uncharacterized but are important in understanding immunity induced after infection and for design of vaccines. The neonatal gnotobiotic pig is susceptible to HRV for more than 8 weeks and their gnotobiotic status assures that wild type rotavirus infection does not occur during vaccination. Additionally gnotobiotic pigs are optimal for the study of innate immune responses to HRV in-vivo by excluding any confounding factors (e.g. commensal flora, other pathogens etc). For the first objective, gnotobiotic pigs were vaccinated priming with a peroral (PO) live attenuated human rotavirus (AttHRV) and boosting (2x) with a non-replicating 2/6 virus-like particles (VLPs) intranasally (IN) using ISCOM as adjuvant. High protection rates against diarrhea and shedding (71%) were induced which coincided with higher IgA antibody titers in small intestinal contents and serum virus neutralizing (VN) antibody responses. In contrast, vaccination with 2/6VLP alone conferred no protection against diarrhea or shedding suggesting that neutralizing antigens, VP4 and VP7 were needed as part of a non-replicating vaccine formulation to induce protective immune responses in neonatal pigs. Consequently the second objective was to test a non-replicating vaccine that included RV neutralizing antigens. A combination of semipurified VP4 and 2/6/7VLP PO followed by VP4+2/6VLP IN using ISCOM as adjuvant was tested. A 67% protection rate against diarrhea and 33% protection rate against shedding were elicited with high to moderate numbers of IgA antibody secreting cell responses in the gut but low VN antibody titers in serum. Vaccination with 2/6/7VLP PO and 2/6VLP IN (that lacked the semipurified VP4) conferred low protection rates (33% against diarrhea and no protection against shedding) suggesting that VP4 was an essential component of a non-replicating rotavirus vaccine. This study confirmed that RV neutralizing antigens are needed for a non-replicating HRV vaccine formulation to induce protective immune responses in neonatal pigs. Additionally, to improve protection rates against shedding, VN titers likely need to be enhanced by giving more potent adjuvants and/or a higher vaccine concentrations. For the third objective, viral dose effects on DCs ex-vivo and the association with clinical outcome were examined in gnotobiotic pigs after a high or low dose of HRV. We assessed intestinal and splenic activated (CD80/86+) IFNalpha, IL-12, IL-10, IL-6 and TNFalpha producing DCs and their uptake/binding of fluorescent 2/4/6/7VLPs by flow cytometry and serum/intestinal cytokines by ELISA. Because infection with HRV induced mainly intestinal plasmacytoid DCs (pDCs), we studied membrane bound TGFbeta1-latency associated peptide (LAP) CD4+ regulatory T cells known to be induced by pDCs. At post-inoculation day (PID) 2, a high HRV dose induced significantly lower frequencies of intestinal activated IFNalpha + pDCs (and lower IL-12, IL-6 and TNFalpha+ pDCs) than a low dose. The frequencies of intestinal IFNalpha+ pDCs correlated with serum IFNalpha concentrations (r=0.73 p0.01) suggesting that the pDCs were activated in-vivo. Furthermore a high HRV dose induced lower uptake/binding of 2/4/6/7VLP-GFP by intestinal and splenic pDCs and lower frequencies of circulating membrane bound TGFbeta LAP+ CD4+(SWC3-CD8-), T cells compared to a lo
机译：轮状病毒是全世界儿童和幼儿严重脱水腹泻的主要原因。每年主要在发展中国家的死亡率达到60万，疫苗接种是一项重要的预防措施。我的博士研究的前两个目标是在致成生猪模型中生产和测试复制型和非复制型人轮状病毒（HRV）疫苗或非复制型HRV疫苗的组合，以最大程度地减少或避免使用更具反应性的活HRV疫苗。第三个目标是评估RV感染后的粘膜和全身性树突状细胞反应，因为这些反应在很大程度上未表征，但对于了解感染后诱导的免疫力和疫苗设计非常重要。新生的纳奥托猪对HRV敏感超过8周，并且它们的纳奥托状态确保疫苗接种期间不会发生野生型轮状病毒感染。另外，通过排除任何混杂因素（例如共生菌群，其他病原体等），致生性猪最适合用于研究体内对HRV的先天免疫应答。为了实现第一个目标，用ISCOM作为鼻内（IN）的经口（PO）减毒活人轮状病毒（AttHRV）初免接种生猪，并用鼻内（IN）的非复制性2/6病毒样颗粒（VLP）加强免疫（2x）。佐剂。诱导了高的腹泻和脱落保护率（71％），这与小肠内容物中较高的IgA抗体滴度和血清病毒中和（VN）抗体反应相吻合。相反，单独接种2 / 6VLP疫苗不能提供针对腹泻或脱落的保护作用，这表明中和性抗原VP4和VP7需要作为非复制型疫苗制剂的一部分来诱导新生猪的保护性免疫应答。因此，第二个目标是测试一种包含RV中和抗原的非复制型疫苗。测试了使用ISCOM作为佐剂的半纯化VP4和2/6 / 7VLP PO，然后是VP4 + 2 / 6VLP IN的组合。肠道中分泌IgA抗体的细胞反应数量高到中等，而血清中VN抗体滴度低，则导致腹泻的保护率达到67％，腹泻的保护率达到33％。使用2/6 / 7VLP PO和2 / 6VLP IN（缺乏半纯化的VP4）进行的疫苗接种具有较低的保护率（33％的腹泻和无脱落保护），表明VP4是非复制型轮状病毒疫苗的重要组成部分。这项研究证实，非复制性HRV疫苗制剂需要RV中和抗原来诱导新生猪的保护性免疫应答。另外，为了提高针对脱落的保护率，可能需要通过提供更有效的佐剂和/或更高的疫苗浓度来提高VN滴度。对于第三个目标，在高剂量或低剂量的HRV后，在致生性猪中检查病毒对DC体外剂量的影响以及与临床结局的关系。我们通过流式细胞术和血清/肠道评估了肠道和脾脏激活的（CD80 / 86 +）IFNalpha，IL-12，IL-10，IL-6和TNFalpha产生的DC及其对荧光2/4/6 / 7VLP的摄取/结合。 ELISA检测细胞因子。因为HRV感染主要诱导肠道浆细胞样DC（pDC），所以我们研究了已知由pDC诱导的膜结合TGFbeta1潜伏期相关肽（LAP）CD4 +调节性T细胞。在接种后第2天（PID），与低剂量相比，高HRV剂量诱导的肠活化IFNα+ pDC频率（和较低的IL-12，IL-6和TNFalpha + pDC）显着降低。肠道IFNalpha + pDC的频率与血清IFNalpha浓度相关（r = 0.73 p <0.01），表明pDC在体内被激活。此外，高HRV剂量诱导的肠和脾pDC吸收/结合的2/4/6 / 7VLP-GFP较低，与循环膜结合的TGFbeta LAP + CD4 +（SWC3-CD8-）T细胞的频率相比，lo低。

著录项

  • 作者

    Gonzalez, Ana Maria.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Health Sciences Pathology.; Biology Virology.; Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 424 p.
  • 总页数 424
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 病理学;预防医学、卫生学;
  • 关键词

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