STRATEGIES FOR INDUCTION OF MUCOSAL IMMUNITY TO HIV

据世卫组织估计，全世界75%的艾滋病毒感染病例是在 性交的结果。 因此，研制艾滋病毒疫苗 能够赋予保护性粘膜免疫，特别是局部 生殖器免疫将是控制这一流行病的关键。 的 该项目的目标是确定一种口服免疫方案， 使用小动物（小鼠）作为模型，其可能赋予有效的， 对HIV感染的持久和保护性粘膜免疫。 这 将通过以下具体目标实现这一目标。第一章 确定最佳口服佐剂/ HIV免疫原方案。 这个问题 将通过口服免疫小鼠与三种艾滋病毒的组合来解决 亚单位免疫原（gp 120蛋白，代表主要免疫原的肽） GP 120和达特蛋白上的中和决定簇）和三种口服佐剂 （霍乱毒素，iscoms和微球）在不同剂量和 不同的免疫方案。 分泌性抗体应答（ELISA法） 和ELISPOT，将用于测量粘膜免疫应答，这些 养生法2)确定分泌的抗体中和 艾滋病。 重要的是要确定口服免疫是否 所采用的治疗方案能够产生HIV的中和抗体。 为了确定这一点，将在体外测定分泌的抗体中的HIV 病毒中和（预防p24表达和病毒性细胞病变） 作用）和抑制细胞间病毒传播。3)确定 是否可以产生对HIV的细胞毒性反应（CTL和ADCC）， 口服免疫后的粘膜组织。 针对HIV的细胞毒性T细胞是 可能在保护粘膜免疫HIV中发挥关键作用。 因此，将对粘膜组织中的淋巴细胞进行CTL和ADCC检测 使用表达HIV免疫原的靶细胞的体外测定来测定活性。 4)确定口服免疫产生局部粘膜免疫的能力 对艾滋病毒的免疫反应。 生殖器局部粘膜免疫反应 将通过测量HIV抗体来评估肠道或直肠对HIV的感染 （包括中和抗体）。第五章） 确定对HIV的分泌性抗体应答的持续时间， 记忆B和T细胞应答的长度。 对于口服疫苗来说 有效，分泌物中的抗体必须持续很长一段时间。 口服免疫的小鼠将在不同的时间间隔收集分泌物， 直至免疫后12个月，并测定HIV抗体。 记忆反应将通过从粘膜中收集淋巴细胞来评估。 在口服免疫后的不同时间点， 对HIV抗原的体外反应。6)确定口服免疫是否 与HIV免疫原接触导致对这些免疫原的口服耐受。 这 将通过全身再免疫小鼠来评估，所述小鼠先前已经 用相同的HIV免疫原口服免疫，并评估 随后的全身免疫应答（即，血清抗体应答， 细胞毒性）。

WHO estimates that 75% of the cases of HIV infection worldwide occur as a result of sexual intercourse. Thus, development of a vaccine against HIV capable of conferring protective mucosal immunity, and especially local genital immunity, will be crucial for control of this epidemic. The objective of this project will be to identify an oral immunization regimen, using small animals (mice) as a model, which is likely to confer effective, long-lasting and protective mucosal immunity against HIV infection. This objective will be achieved through the following specific aims. 1) Determine an optimal oral adjuvant / HIV immunogen regimen. This question will be addressed by orally immunizing mice with combinations of three HIV subunit immunogens (gp120 protein, a peptide representing the primary neutralizing determinant on gp120 and tat protein) and three oral adjuvants (cholera toxin, iscoms and microspheres) in different doses and with different immunization regimens. Secretory antibody responses, by ELISA and ELISPOT, will be used to measure the mucosal immune response to these regimens. 2) Determine the ability of secreted antibodies to neutralize HIV. It will be important to determine whether the oral immunization regimens employed are capable of generating neutralizing antibodies to HIV. To determine this, secreted antibodies will be assayed in vitro for HIV virus neutralization (prevention of p24 expression and viral cytopathic effects) and inhibition of cell to cell virus transmission. 3) Determine whether cytotoxic responses (CTL and ADCC) to HIV can be generated in mucosal tissues after oral immunization. Cytotoxic T cells against HIV are likely to play a key role in protective mucosal immunity to HIV. Therefore, lymphocytes from mucosal tissues will be tested for CTL and ADCC activity using in vitro assays with HIV immunogen-expressing target cells. 4) Determine the ability of oral immunization to generate a local mucosal immune response to HIV. Local mucosal immune responses in the genital tract or rectum to HIV will be assessed by measuring antibodies to HIV (including neutralizing antibodies) in secretions from these sites. 5) Determine the duration of secretory antibody responses to HIV and the length of memory B and T cell responses. For an oral vaccine to be effective, antibodies in secretions must persist for long periods of time. Orally immunized mice will have secretions collected at various, intervals, up to 12 months after immunization, and assayed for antibodies to HIV. Memory responses will be assessed by collecting lymphocytes from mucosal tissues at various time points after oral immunization and assaying for in vitro responses to HIV antigens. 6) Determine whether oral immunization with HIV immunogens results in oral tolerance to these immunogens. This will be evaluated by systemically reimmunizing mice which have previously been orally immunized with the same HIV immunogen and evaluating the subsequent systemic immune response (ie. serum antibody response and cytotoxicity).