STRATEGIES FOR INDUCTION OF MUCOSAL IMMUNITY TO HIV

世卫组织估计，全世界75%的艾滋病毒感染病例是以 性交的结果。因此，针对HIV的疫苗的开发 能够提供保护性的粘膜免疫，尤其是局部的 生殖器免疫将是控制这一流行病的关键。这个 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，这可能会带来有效的， 针对艾滋病毒感染的持久和保护性粘膜免疫。这 目标将通过以下具体目标来实现。1) 确定最佳口服佐剂/HIV免疫原方案。这个问题 将通过口服三种HIV组合免疫小鼠来解决 亚单位免疫原(gp120蛋白，代表初级 Gp120和Tat蛋白的中和决定簇)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。用酶联免疫吸附试验检测分泌型抗体反应 和Elispot，将被用来测量粘膜对这些物质的免疫反应 养生法。2)测定分泌抗体的中和能力 爱滋病毒。重要的是要确定口服免疫是否 所采用的方案能够产生针对艾滋病毒的中和抗体。 为了确定这一点，将在体外对分泌的抗体进行艾滋病毒检测。 病毒中和(防止p24表达和病毒细胞病变 效果)和抑制细胞间病毒传播。3)确定 人类免疫缺陷病毒能否产生细胞毒反应(CTL和ADCC) 口服免疫后的粘膜组织。抗HIV的细胞毒性T细胞 很可能在对艾滋病毒的粘膜免疫保护中发挥关键作用。 因此，来自粘膜组织的淋巴细胞将被检测CTL和ADCC 体外检测表达HIV免疫原的靶细胞的活性。 4)确定口服免疫产生局部粘膜的能力 对艾滋病毒的免疫反应。生殖器的局部粘膜免疫反应 将通过检测艾滋病毒抗体来评估肠道或直肠对艾滋病毒的影响 (包括中和抗体)在这些部位的分泌物中。5) 确定对艾滋病毒的分泌性抗体应答的持续时间和 记忆B细胞和T细胞反应的长度。对于口服疫苗来说， 有效的，分泌物中的抗体必须长期存在。 口服免疫的小鼠将在不同的时间间隔收集分泌物， 免疫后12个月，进行HIV抗体检测。 记忆反应将通过收集粘膜中的淋巴细胞来评估 口服免疫后不同时间点的组织学检测 对HIV抗原的体外反应。6)确定口服免疫是否 使用HIV免疫原会导致口服对这些免疫原的耐受性。这 将通过系统地重新免疫以前有过 用相同的HIV免疫原口服免疫，并评估 随后的系统免疫反应(即血清抗体反应和 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).