Preventive Vaccines for HIV

方法1：人乳头瘤病毒（HPV）假病毒粒子是由我在NCI的合作者Buck、Schiller和Lowy博士开发的一种技术，它将DNA传递到阴道粘膜上皮的角化细胞。因此，通过这种方法，有可能在女性生殖道中引发效应t细胞。然而，限制可能是，由于这种疫苗方式的SIV/HIV基因表达随着时间的推移而减弱，可能需要重复免疫以维持阴道内效应t细胞的保护水平。用含有SIV gag基因的HPV 16和HPV 45构建物对恒河猴进行阴道免疫，并在粘膜和全身部位评估免疫反应。对照组给予全身黄体酮治疗，肌注provera 30mg/kg，使阴道上皮变薄。动物分别用10%的N9凝胶或12%的N9泡沫处理。一组动物也用细胞刷轻轻擦伤阴道壁。如果HPV疫苗在阴道中诱导并维持多功能记忆反应，那么将产生表达早期SIV基因（Retanef）和包膜的额外HPV构建体。SIV包膜DNA序列将从阴道攻击暴露后传播给RM的SIV基因型序列中获得。这个信封会被设计成保密的。第二阶段的总体目标是评估粘膜和全身部位对SIV基因诱导的t细胞反应对抵抗同源SIV阴道攻击的影响。此外，将评估包膜抗体结合和/或中和抗体的作用，特别是如果存在于阴道分泌物中。方法2：在胃肠道诱导效应t细胞，并使用热休克gp96- igd重组SIV疫苗。这是与迈阿密大学的Podack博士合作努力的一部分，包括制备293细胞分泌的siv -gp96细胞疫苗。SIV-gp96疫苗在非人灵长类动物中的最佳剂量是由特异性CD8反应决定的。这些基于细胞的gp96-Ig疫苗，通过在体内长时间分泌gp96-Ig肽，应该可以模仿病毒复制并提供与减毒病毒相当的免疫刺激。在小鼠模型系统中，Podack博士已经表明，gp96-Ig转染，抗原表达的肿瘤细胞在体内分泌gp96-Ig，刺激先天DC和NK以及适应性，同源细胞CD8 CTL免疫反应，并在没有淋巴结的情况下独立于CD4帮助产生特异性CD8记忆。gp96-Ig疫苗可在上皮内、固有层和Peyers贴片中产生全身和强粘膜免疫CD8 CTL。由于其独特的性质，我们现在计划在非人灵长类动物模型中评估gp96疫苗在粘膜和全身部位的免疫原性（R21）。此外，我们将检验siv -gp96疫苗对后续病毒攻击的保护能力（R33）。为了最大限度地提高成功的机会，迈阿密的团队，波达克博士和帕瓦博士与我的实验室合作，汇集了具有人类和非人类灵长类动物艾滋病毒/SIV发病机制专业知识的基础免疫学家。我们构建了编码Gag、Env和嵌合Retanef蛋白的gp96-Ig疫苗，并在293细胞的上清液中证明了这些蛋白的分泌（见上图左图）。我们已经完成了R21期，并证明了3次腹腔免疫50 mg分泌的gp96-Ig蛋白可诱导NHP直肠黏膜上皮内淋巴细胞发生siv特异性功能反应（见左图）。该资助的R33部分的目的是用分泌的gp96-Ig疫苗产生粘膜和全身SIV免疫，并通过直肠内途径将猕猴暴露于SIV，以测试这些疫苗的相对功效。方法3：疫苗方法基于减毒HTLV-II载体。HTLV-II感染树突状细胞，持续存在于宿主t细胞中，并在肠道相关淋巴组织（GALT）和淋巴结中低水平表达。目的是产生在宿主体内持续存在的HTLV-II载体SIV疫苗，并研究肠道中低水平、持久的效应t细胞和淋巴结中的中枢记忆细胞的激发是否会防止SIV获取和传播到远端部位。这些方法在NHP中应告知CD4+和CD8+记忆细胞的质量、数量和位置，这是防止SIV早期播种所必需的。方法4：这不是一种新方法，但我们仍在调查ALVAC作为艾滋病毒疫苗交付平台的相对优点。ALVAC是一种减毒的金丝雀痘衍生载体，不能在哺乳动物细胞中有效复制。ALVAC HIV-1疫苗是一种候选HIV-1疫苗，目前正在进行三期临床试验，试验结果将于2009年9月底公布。我们设计了一项研究，以提高ALVAC SIV疫苗的免疫原性，目的是平行测试其对高剂量或低剂量重复攻击暴露的效力。过去，我们在猕猴婴儿模型中测试了一种ALVAC-SIV候选HIV疫苗，以评估该疫苗平台是否可以减少SIV通过母乳喂养的传播。研究人员在猕猴出生后的前3周内对其进行了多次免疫，接种了表达猴免疫缺陷病毒（SIV）结构蛋白Gag、Pol和Env的重组痘病毒疫苗（ALVAC-SIV或修饰痘苗病毒安卡拉[MVA]-SIV）。与未接种alvac - siv疫苗的婴儿相比，每天重复口服低剂量的SIVmac251后，alvac - siv免疫婴儿的感染显著减少。在婴儿期未受口腔攻击感染的猴子在16个月或更大时通过每周重复的口腔SIV暴露再次受到感染；未接种SIV疫苗的动物比接种ALVAC-SIV疫苗的动物感染SIV的次数少。当感染时，ALVAC-SIV疫苗接种的动物与未接种的动物相比，病毒血症也减少。这些结果表明，人类婴儿接种基于痘病毒的HIV候选疫苗可能对早期和晚期HIV感染提供保护，这与这些疫苗在成年猕猴中的较差效果形成对比。目前尚不清楚在这两项研究中观察到的不同程度的保护是由年龄还是攻击方式造成的。因此，我们进一步研究这种方法的原因如下：1)ALVAC是一种鸟类媒介；因此，预先存在的免疫力不是一个可能增强艾滋病毒传染性的因素。鉴于最近基于ad5的艾滋病毒疫苗试验（STEP试验）的结果，这一点尤其重要。在该试验中，观察到对媒介具有高水平免疫力的个体（未行包皮环切术）的艾滋病毒传播增加了五倍。2)ALVAC-HIV单独可能没有足够的免疫原性或保护性。3)我们将利用这项研究同时测试高剂量和重复低剂量SIVmac251对粘膜攻击的保护程度。希望通过将这项研究的结果与泰国试验的结果进行比较，可以了解这两种挑战模型在预测疫苗对人类的效力方面的相关性。我们还计划通过克隆和测序vi来表征和比较在未接种和接种的rm早期传播的病毒基因型[摘要截断为7800个字符]。

Approach 1: Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI, delivers DNA to keratinocytes in the vaginal mucosal epithelium. So with this approach, it may be possible to elicit effector T-cells in the female genital tract. However, a limitation could be that,as the expression of SIV/HIV genes by this vaccine modality wanes with time,repeated immunizations may be required to maintain protective levels of effector T-cells in the vagina. Rhesus macaques have been immunized intravaginally with HPV 16 and HPV 45 constructs containing the SIV gag gene and immune response evaluated at mucosal and systemic sites. RMs were given systemic progesterone treatment, intramuscular Depo-Provera 30mg/kg, to thin the vaginal epithelium. Animals were treated with either 10% N9 gel or 12% N9 foam. A group of animals were also treated with a cytobrush to lightly abrade the vaginal wall. If the HPV vaccination induces and sustains polyfunctional memory responses in the vagina, then additional HPV constructs expressing early SIV genes (Retanef) and envelope will be generated. The SIV envelope DNA sequence will be derived from the sequence of SIV genotype transmitted to RM following vaginal challenge exposure. The envelope will be engineered such that it will be secreted. The overall goals of the second phase are to evaluate the impact of T-cell responses to SIV genes induced at the mucosal and systemic sites in the protection against a homologous SIV vaginal challenge. In addition, the role of binding and/or neutralizing antibodies to the envelope, particularly if present in vaginal secretions, will be evaluated. Approach 2: Induce effector T-cells in the gastrointestinal tract and will use heat shock gp96-Igbased SIV recombinant vaccines. This is part of a collaborative effort with Dr Podack from the University of Miami and includes the preparation of cellular SIV-gp96-vaccines secreted by 293 cells. Optimal dose-finding for SIV-gp96 vaccines in non-human primates was determined by specific CD8 responses. These cell-based gp96-Ig vaccines, by prolonged in vivo secretion of gp96-Ig-peptide, should imitate viral replication and provide immune stimuli comparable to attenuated viruses. In model systems in mice, Dr. Podack has shown that gp96-Ig transfected, antigen expressing tumor cells secrete gp96-Ig in vivo and stimulate the innate DC and NK as well as adaptive, cognate cellular CD8 CTL immune response and generate specific CD8 memory independent of CD4 help and in the absence of lymph nodes. Both systemic and strong mucosal immunity in intraepithelial, lamina propria, and Peyers patch CD8 CTL is generated by gp96-Ig vaccines. Because of their unique properties, we now plan to evaluate the gp96-vaccines in nonhuman primate models for SIV for their immunogenicity at mucosal and systemic sites (R21). In addition, we will examine the protective power of SIV-gp96-vaccines against subsequent viral challenge (R33). To maximize the chances of success, the team in Miami, Dr. Podack and Dr. Pahwa have entered into collaboration with my lab bringing together basic immunologists with expertise in human and non human primate HIV/SIV pathogenesis. We have constructed gp96-Ig vaccines encoding Gag, Env, and a chimeric Retanef protein and demonstrated the secretion of these proteins in the supernatant of 293 cells (see picture above on the left). We have completed the R21 phase and demonstrated that 3 intraperitoneal immunizations with 50 mg of secreted gp96-Ig protein induce SIV-specific functional responses in intra epithelium lymphocytes of the rectal mucosa of NHP (see picture on left). The aims of the R33 part of this grant are to generate mucosal and systemic SIVimmunity with secreted gp96-Ig vaccines and expose macaques to SIV by the intrarectal route to test the relative efficacy of these vaccines. Approach 3: Vaccine approach is based on an attenuated HTLV-II vector. HTLV-II infects dendritic cells, persists in the host T-cells, and is expressed at low level in the gut associated lymphoid tissues (GALT) and lymph nodes. The goal is to generate HTLV-II vectored-SIV vaccines that persist in the host and investigate whether the elicitation of low level, durable, effector T-cells in the gut, and central memory cells in lymph nodes will protect from SIV acquisition and dissemination to distal sites. These approaches in NHP should inform on the quality, quantity, and location of CD4+ and CD8+ memory cells necessary to prevent early seeding of SIV. Approach 4: This is not a new approach, but we are still investigating the relative merit of the ALVAC as a deliver platform for an HIV vaccine. ALVAC is an attenuated canarypox derived vector that cannot replicate productively in mammalian cells. ALVAC HIV-1 vaccine is a candidate HIV-1 vaccine, which is now in Phase III clinical trials and the results of this trail will become available at the end of September 2009. We have designed a study to improve the immunogenicity of an ALVAC SIV vaccine with the goal to test in parallel its efficacy against the high dose or the low dose repeated challenge exposure. In the past, we have tested an ALVAC-SIV vaccine candidate for HIV in an infant macaque model to assess whether this vaccine platform could reduce SIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral exposure to low doses of virulent SIVmac251 significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV. When infected, ALVAC-SIV -vaccinated animals also had reduced viremia compared with unimmunized animals. These results suggested that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection and were in contrast with the poor efficacy of these vaccines in adult macaques. It is unclear whether the age or the mode of challenge is responsible for the different degree of protection observed in these two studies. Thus, we are further investigating this approach for the following reasons: 1) ALVAC is an avian vector; therefore, pre-existing immunity is not a factor that could enhance HIV infectivity. This is particularly important in light of the recent results with the Ad5-based HIV vaccine trial (STEP trial) wherein a five fold increase in HIV transmission was observed in individuals (not circumcised)that had high levels of pre-existing immunity to the vector. 2)ALVAC-HIV alone may not be sufficiently immunogenic or protective.3)We will use this study to test in parallel the degree of protection from a mucosal challenge exposure to high and repeated low doses of SIVmac251. Hopefully, the results of this study compared to the results of the Thailand Trial, will inform on the relevance of the two challenge models in predicting the efficacy of vaccines in humans. We also plan to characterize and compare the viral genotypes that are transmitted early in nave and vaccinated RMs by cloning and sequencing the vi [summary truncated at 7800 characters]