Preventive Vaccines for HIV

方法1：人类乳头瘤病毒（HPV）假病毒粒子是我在NCI的合作者Buck、Schiller和Lowy博士开发的一项技术。人乳头瘤病毒自然感染阴道角化细胞；使用HPV作为载体，我们可以将SIV基因直接传递到阴道。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，由于这种疫苗模式的SIV/HIV基因表达随着时间的推移而减弱，可能需要重复免疫来维持阴道中效应t细胞的保护水平。在去年，我们设计了人类乳头瘤病毒样颗粒来表达SIV基因。我们发现阴道内接种携带SIV基因的HPV假病毒粒子可诱导猕猴的体液和细胞介导的免疫反应。这些免疫反应集中在阴道上皮内层和固有层。暴露于SIV后，我们发现疫苗诱导的免疫反应在阴道中迅速扩大，在暴露后几周内阴道引流淋巴结。然而，这种疫苗诱导的二次反应是局灶性的，在阴道内接种疫苗或阴道内SIV攻击后，我们没有观察到直肠免疫反应的扩大。在今年，我们希望测试HPV psv诱导的这些局灶性免疫反应是否足以预防SIV阴道内感染。疫苗诱导的进入门效应反应已被证明存在于抵抗SIV攻击的动物中，然而，在人类中显示边际效力的唯一疫苗是与HIV包膜蛋白联合给药的金丝雀痘载体ALVAC。因此，我们希望测试疫苗诱导的效应反应和全身疫苗接种的结合是否可以防止感染。对于这种双重模式，我们将用ALVAC SIV启动免疫系统，然后用阴道内HPV疫苗增强。我们假设这种方式应该引导系统诱导的免疫反应到阴道。恒河猴模型是HIV感染的最佳动物模型。最近有研究表明，大多数HIV感染是由单一病毒引起的，重复使用低剂量SIVmac251粘膜刺激恒河猴也可以感染单一病毒变体。我们建议使用SIVmac251阴道内重复低剂量攻击作为我们的模型系统，以测试我们的疫苗方法是否可以防止SIV感染和/或高水平的SIV病毒复制和CD4+ T细胞的损失。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。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RT-PCR从血浆中克隆和测序病毒，表征和比较在未接种和接种的rm早期传播的病毒基因型。本研究的科学目标是在DNA启动物/ALVAC-SIV增强中提高ALVAC-SIV疫苗的免疫原性，并通过测试基于sivmac251的疫苗对抗同源和异源病毒SIVSME660的粘膜攻击来评估跨进化的保护作用。ALVAC是金丝雀痘病毒（CPV） Kanapox疫苗株的斑块克隆衍生物。

Approach 1: Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI. The human papilloma virus naturally infects vaginal keratinocytes; using HPV as a vector we can deliver SIV genes directly to the vaginal tract. 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 In the last year we engineered Human Papiloma virus like particles to express SIV genes. We found that intra-vaginal vaccination with HPV Pseudovirions delivering SIV genes induces both humoral and cell mediated immune responses in macaques. These immune responses were focused in the intraepithelial layers and the lamina propria of the vagina. Upon exposure with SIV we found that vaccine induced immune response quickly expanded in the vaginal tract, vaginal draining lymph nodes within weeks post exposure. This vaccine induced secondary response was however focal and we did not observe an expansion of immune responses in the rectum after intravaginal vaccination or intravaginal SIV challenge, In this current year we wish to test if these focal immune responses induced by HPV Psvs are sufficient to prevent SIV intra-vaginal infection.Vaccine induced effector responses at the portal of entry have been shown to be present in animals resistant to an SIV challenge, however, the only vaccine to show marginal efficacy in humans is a systemically administered canarypox vector ALVAC given in combination with the HIV envelope protein. Thus we wish to test if the combination of vaccine induced effector responses and systemic vaccination can protect from infection. For this dual modality we will prime the immune system with ALVAC SIV and then boost with intravaginal HPV vaccination. We hypothesize that this modality should direct the systemically induced immune responses to the vaginal tract. The rhesus macaque model is the best animal model of HIV infection. It has recently been shown that a single virus establishes most HIV infections and using repeated low dose SIVmac251 mucosal challeges rhesus macaques can also be infected with a single viral variant. W propose using a repeated low dose intra-vaginal challenge with SIVmac251 as our model system to test if our vaccine approaches can protect from SIV infection and/or high levels of SIV viral replication and the loss of CD4+ T cells. 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. 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 virus via RT-PCR from the plasma. The scientific goals of this aim are to improve the immunogenicity of an ALVAC-SIV vaccine in a DNA prime /ALVAC-SIV boost and to assess cross-clade protection by testing a SIVmac251-based vaccine against a mucosal challenge with a homologous and heterologous virus, SIVSME660. ALVAC is a plaque-cloned derivative of the Kanapox vaccine strain of the canary poxvirus (CPV).