Vitamin A adjuvant to enhance gut immunity and rotavirus vaccines in neonates

描述（由申请方提供）：轮状病毒（RV）导致全球婴儿腹泻，非洲和亚洲的死亡人数最多。新批准的口服RV疫苗在贫困国家的婴儿中的效力未经证实或较低。它们的高成本仍然是普遍采用的障碍。在发展中国家，普遍存在的维生素A缺乏症（VAD）损害了儿童的健康、肠道免疫反应和疫苗的有效性。世卫组织已将婴儿补充维生素A与免疫接种计划联系起来，以减少VAD并提高疫苗覆盖率。维生素A补充剂对RV疫苗的影响尚不清楚。我们的创新和新颖的策略是使用补充维生素A作为佐剂与口服疫苗的新生儿。新的发现表明，维生素A是肠道免疫反应的关键调节剂，而VAD会损害肠道免疫反应。肠道树突状细胞（DC）将饮食中的维生素A转化为视黄酸（RA），并且DC和/或RA与关键信号分子（细胞因子、炎症介质）协同作用，在T和B细胞上印记肠道归巢（CCR 9，a4 <$7），促进同种型转换为伊加并增强伊加产生。在婴儿和仔猪中，粪便或血清伊加抗体或肠道中的伊加抗体分泌细胞与针对RV腹泻的保护相关。肠道DC协调对肠道病原体的肠道免疫应答的关键上调（通过T效应细胞、B细胞和介体）或对食物抗原和唾液酸的下调（通过免疫调节性T细胞和介体如TGF β）。我们假设，“危险信号”组成的炎症或TLR信号是必不可少的激活树突状细胞克服肠道下调，并增加口服疫苗的免疫反应。为了测试这一点，我们将使用在肠上皮细胞中复制的减毒RV（增加促炎性细胞因子，减少TGF-β细胞因子）作为口服疫苗与维生素A佐剂以增强肠道伊加应答。或者，非复制型轮状病毒样颗粒（VLP）疫苗可能需要TLR信号（聚肌苷-聚胞苷酸合成dsRNA，TLR 3配体）与VitA来模拟RV dsRNA的作用。使用维生素A作为佐剂，我们将比较减毒活疫苗（目标1）和VLP（目标2）口服RV疫苗的免疫应答。将在VAD与用强毒人RV攻毒的VitA充足的新生无菌（Gn）猪中检测疫苗效力（目标3）。新生Gn仔猪在其胃肠道生理学、粘膜免疫应答和对人RV腹泻的易感性方面模仿婴儿。猪像人类一样代谢维生素A，并且可以操纵维生素A水平以模拟婴儿的VAD。为了阐明免疫机制，我们将评估先天性（DC、NK/NKT细胞、IFNa、炎性细胞因子）和适应性T（Teff/Treg比率）和B细胞（伊加）肠道免疫应答，包括肠道DC和细胞因子谱及其与保护的相关性。了解VAD对肠道免疫的影响对于提高发展中国家VAD婴儿口服疫苗的有效性至关重要。如果成功的话，在现有的RV口服疫苗的基础上战略性地在婴儿中补充维生素A（如世卫组织所建议的）可能是可行的，并可在发展中国家提供更具成本效益的疫苗（剂量更低、更少）。一个创新的结果将是维生素A佐剂的普遍应用，不仅对RV疫苗，而且对其他粘膜疫苗，如艾滋病毒。 轮状病毒（RV）每年在全世界造成66万儿童死亡，其中大多数（90%）在非洲和亚洲。虽然口服轮状病毒活疫苗已获得许可，但在最需要这种疫苗的贫困国家，这种疫苗未经证实或对婴儿的有效性较低，而且成本高昂，仍然是普遍采用的障碍。在发展中国家，普遍存在的营养不良、微量营养素缺乏症（维生素A等）和多重并发感染损害了儿童的健康、肠道免疫反应和疫苗的有效性。新的发现表明，维生素A是肠道免疫反应的关键调节剂，维生素A缺乏会损害肠道免疫反应。我们将在维生素A缺乏和维生素A充足的新生无菌猪中测试补充维生素A作为活的或非复制性口服轮状病毒疫苗的佐剂。我们的研究将阐明免疫反应和保护的相关性，以攻击与强毒的人轮状病毒。新生的无菌仔猪将被用作疾病模型，因为它们是唯一对人轮状病毒腹泻易感的动物。此外，他们在解剖学、胃肠道生理学、营养需求/代谢和粘膜免疫发育方面与婴儿相似。此外，猪的视黄醇代谢与人类相当。因此，维生素A对猪免疫系统的影响最接近于人类，而仔猪最能反映婴儿对轮状病毒腹泻和疫苗的反应。了解维生素A缺乏对肠道免疫反应的影响对于提高发展中国家维生素A缺乏婴儿口服疫苗的有效性至关重要。如果成功，立即在现有RV疫苗的基础上对婴儿补充维生素A（世卫组织建议）可能是可行的，并可在发展中国家提供更具成本效益的疫苗（剂量更低、更少）。维生素A佐剂可能普遍适用，不仅适用于轮状病毒疫苗，而且适用于其他人类粘膜疫苗，如HIV。

DESCRIPTION (provided by applicant): Rotavirus (RV) causes diarrhea in infants worldwide with most deaths in Africa and Asia. Newly licensed oral RV vaccines have unproven or lower efficacies in infants in impoverished countries. Their high costs remain obstacles to universal adoption. In developing countries, widespread vitamin A deficiencies (VAD) compromise children's health, gut immune responses and vaccine effectiveness. WHO has linked vitamin A (VitA) supplementation in infants to immunization programs to reduce VAD and increase vaccine coverage. The impact of VitA supplementation on RV vaccines is unknown. Our innovative and novel strategy is to use supplemental VitA as an adjuvant with oral vaccines in neonates. New discoveries have revealed that VitA is a key regulator of intestinal immune responses and that VAD impairs gut immune responses. Gut dendritic cells (DCs) convert dietary VitA to retinoic acid (RA) and DCs and/or RA in concert with key signaling molecules (cytokines, inflammatory mediators), imprint gut homing (CCR9, a4¿7) on T and B cells, promote isotype switching to IgA and enhance IgA production. In infants and piglets, fecal or serum IgA antibodies or IgA antibody secreting cells in the gut correlate with protection against RV diarrhea. Gut DCs orchestrate critical upregulation of intestinal immune responses (via Teffector, B cells and mediators) to enteric pathogens or downregulation (via immunoregulatory T cells and mediators like TGF¿) to food antigens and commensals. We hypothesize that "danger signals" consisting of inflammatory or TLR signaling are essential to activate DCs to overcome intestinal downregulation and increase immune responses to oral vaccines. To test this, we will use attenuated RV that replicates in enterocytes, (increase proinflammatory, decrease TGF¿ cytokines) as oral vaccine with VitA adjuvant to enhance gut IgA responses. Alternatively nonreplicating rotavirus-like-particle (VLP) vaccines may require TLR signals (polyinosine-polycytidylic acid synthetic dsRNA, TLR3 ligand) with VitA to mimic the effect of RV dsRNA. Using VitA as adjuvant, we will compare immune responses to live attenuated (Aim 1) with VLP (Aim 2) oral RV vaccines. Vaccine efficacy will be tested in VAD versus VitA replete neonatal gnotobiotic (Gn) pigs challenged with virulent human RV (Aim 3). Neonatal Gn piglets mimic infants in their gastrointestinal physiology, mucosal immune responses and susceptibility to human RV diarrhea. Pigs metabolize VitA like humans and VitA levels can be manipulated to mimic VAD in infants. To elucidate immune mechanisms, we will assess innate (DCs, NK/NKT cells, IFNa, inflammatory cytokines) and adaptive T (Teff/Treg ratio) and B cell (IgA) intestinal immune responses, including gut DC and cytokine profiles and their correlation with protection. Understanding the impact of VAD on gut immunity is critical to improve oral vaccine efficacy in VAD infants in developing countries. If successful, strategic implementation of supplemental VitA in infants (as recommended by WHO) with existing RV oral vaccines may be feasible and could provide more cost-effective vaccines (lower, fewer doses) in developing countries. An innovative outcome would be universal application of VitA adjuvants not only to RV vaccines, but also to other mucosal vaccines such as for HIV. RELEVANCE: Rotavirus (RV) causes 660,000 childhood deaths annually worldwide, with most (90%) in Africa and Asia. Although live oral rotavirus vaccines are licensed, their unproven or lower effectiveness in infants in impoverished countries, where they are most needed, and high costs remain obstacles to their universal adoption. In developing countries, widespread malnutrition with micronutrient deficiencies (vitamin A, etc) and multiple concurrent infections compromise children's health, gut immune responses and vaccine effectiveness. New discoveries have revealed that vitamin A is a key regulator of intestinal immune responses and that vitamin A deficiency impairs gut immune responses. We will test supplemental vitamin A as adjuvant with live or nonreplicating oral rotavirus vaccines in vitamin A deficient versus vitamin A replete neonatal gnotobiotic pigs. Our studies will elucidate the immune responses and correlates of protection to challenge with virulent human rotavirus. Neonatal gnotobiotic piglets will be used as a disease model because they are the only animal susceptible to human rotavirus diarrhea. Moreover they are similar to infants in anatomy, gastrointestinal physiology, nutrient requirements/metabolism, and development of mucosal immunity. Also swine metabolize retinol comparable to humans. Thus the effects of vitamin A on the porcine immune system most closely mimic that in humans, and piglets best mirror responses of infants to rotavirus diarrhea and vaccines. An understanding of impacts of vitamin A deficiency on gut immune responses is critical to improve oral vaccine efficacy in vitamin A compromised infants in developing countries. If successful, immediate implementation of supplemental vitamin A in infants (as recommended by WHO) with the existing RV vaccines may be feasible and could provide more cost-effective vaccines (lower, fewer doses) in developing countries. Vitamin A adjuvants may be universally applicable, not only to rotavirus vaccines, but also to other mucosal vaccines for humans such as HIV.