MOLECULAR ONTOGENY OF ORAL MUCOSAL RESISTENCE TO SIV

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 流行病学研究表明，成年人的口腔对HIV感染具有相对抵抗力，并且已经假定唾液中存在的许多可溶性因子赋予这种保护。相比之下，由感染艾滋病毒的母亲母乳喂养的婴儿的口腔感染相当常见，这表明口腔对慢病毒的耐药性存在年龄依赖性差异。我们将通过描述恒河猴口腔中的抗SIV先天免疫来研究这些年龄依赖性抗病毒抗性差异的基础。拟议研究的长期目标是描述防御素的抗HIV作用，现在已知防御素是存在于唾液中并在口腔上皮中表达的抗病毒肽。三种人防御素最近被鉴定为抗HIV因子，其由来自长期非进展者的HIV阳性个体的CD-8+ T细胞产生。我们假设防御素有助于唾液的抗SIV/HIV特性和口腔粘膜的先天抗性，2）口腔防御素表达在出生后发展，和3）新生儿口腔对SIV的抗性水平可以通过局部应用一种或多种防御素来增强。为了验证这些假设，我们将追求以下具体目标。 具体目的1是确定婴儿和成年恒河猴唾液中存在的和/或口腔中表达的α、β和θ防御素的水平。具体目标2是合成和/或重组表达特定的α、β和θ-防御素，所述特定的α、β和θ-防御素被证实（在具体目标1中）是成人唾液的组分和/或在口腔组织中表达。将对由此产生的肽进行充分表征并评价其抗SIV功效（具体目标3），并将其用于生产抗肽免疫试剂。具体目标3是确定口服α、β和θ防御素的体外抗SIV和抗HIV活性。将在有和没有新生儿和成人唾液的情况下进行抗SIV测定，以确定这种天然液体对肽活性的影响。还将分析肽的组合以检测加和或协同肽-肽相互作用。具体目标4是确定外源性局部施用防御素是否可以改变新生恒河猴对感染的易感性。 将12只幼年恒河猴分为治疗组（6只）和对照组（6只）。 治疗组接受2 ml口服剂量的恒河猴θ防御素RTD-1（1 μ g/ml）或盐水（载体对照），在用105 TCID 50的SIVmac 239以每周间隔口服攻击之前20分钟施用于口腔粘膜，持续5周。 所有动物在给药第3周时均感染，无论分组如何。 后续免疫学、病毒学和病程研究正在进行中。

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. Epidemiologic studies demonstrate that the oral cavity of human adults is relatively resistant to HIV infection, and a number of soluble factors present in saliva have been postulated to confer this protection. In contrast, oral infection of infants who are breast-fed by HIV-infected mothers is quite common, suggesting that there are age-dependent differences in oral resistance to lentiviruses. We will investigate the basis of these age-dependent differences in antiviral resistance by characterizing anti-SIV innate immunity in the oral cavity of rhesus macaques. The long-term goal of the proposed studies is to delineate the anti-HIV role of defensins, antiviral peptides now known to be present in saliva and expressed in epithelium of the oral cavity. Three human defensins were recently identified as anti-HIV factors produced by CD-8+ T cells from HIV-positive individuals who are longterm non-progressors. We hypothesize that defensins contribute to the anti-SIV/HIV properties of saliva and to the innate resistance of oral mucosa, 2) that oral defensin expression develops postnatally, and 3) that the level of oral resistance to SIV in neonates may be augmented by topical application of one or more defensins. To test these hypotheses, we will pursue the following Specific Aims. Specific Aim 1 is to determine the level of alpha, beta, and theta defensins present in saliva and/or expressed in the oral cavity of infant and adult rhesus macaques. Specific Aim 2 is to synthesize and/or recombinantly express specific alpha, beta, and theta-defensins confirmed (in Specific Aim 1) to be components of adult saliva and/or expressed in oral tissues. Peptides thus produced will be fully characterized and evaluated for their anti-SIV efficacy (Specific Aim 3), and will be used to produce anti-peptide immunologic reagents. Specific Aim 3 is to determine the anti-SIV and anti-HIV activities of oral alpha, beta, and theta defensins in vitro. Anti-SIV assays will be conducted with and without neonatal and adult saliva to ascertain the effect of this natural fluid on peptide activities. Combinations of peptides will also be analyzed to detect additive or synergistic peptide-peptide interactions. Specific Aim 4 is to determine whether exogenous, topically administered defensin can alter the susceptibility to infection of neonatal rhesus macaques. Twelve juvenile rhesus macaques were divided into treatment (6) and control (6) groups. Treatment groups received 2 ml oral doses of rhesus macaque theta defensin RTD-1 (1¿g/ml) or saline (vehicle control) applied to the oral mucosa 20 minutes prior to oral challenge with 105 TCID50 of SIVmac239 at weekly intervals for five weeks. All of the animals became infected by treatment week three regardless of group assignment. Follow-up immunologic, virologic and disease course studies are in progress.