COMBINATION OF VACCINE MODALITIES TO PREVENT HIV-I INFECTION AND MODELING OF EARL

第一部分研究计划的第一部分是在恒河猴模型中评估高度减毒的重组痘病毒疫苗候选物。这项工作是与Pasteur Merieux康诺特的合作努力，康诺特与NCI有一个CRADA，并涉及与NIAID的机构内合作。正在测试两种载体：基于金丝雀痘的载体（ALVAC）和遗传减毒牛痘菌株（哥本哈根菌株的衍生物）NYVAC。两种载体都能够完成其复制周期，并在早期或早期/晚期启动子的控制下表达异源基因。工作假设是，因为这两种痘病毒载体，即使在没有完整的生命周期的情况下，仍然表达大量的蛋白质，对异源抗原的反应可能是有限的。因此，我们正在研究这样的假设，即传统的DNA质粒（针对表达进行了优化）或DNA复制（基于塞姆利基森林病毒复制子）可以引发足够的免疫应答，然后可以通过进一步加强携带相同SIV基因的重组痘病毒来扩增。此外，我们还测试了在这些疫苗模式中将所有辅助/调节基因添加到SIV/HIV结构基因中的重要性。一旦实现了模式的最佳组合，可以通过研究免疫途径的重要性和免疫调节分子的帮助来进一步优化其他免疫方案。第2部分鉴于在恒河猴中使用预防性NYVAC-SIV-gag-pol-env候选疫苗获得的令人鼓舞的结果（1），我们假设这样的疫苗在SIV感染后的免疫干预方案中也是有效的。因此，我们在猕猴中开发了一种模型，以测试在存在或不存在抗逆转录病毒治疗的情况下疫苗接种的效果。该研究旨在评估以下内容：（1）延长抗逆转录病毒治疗是否对治疗暂停后的病毒反弹有任何影响，（2）猕猴接种疫苗是否可以增强SIV特异性CD 4 + T辅助细胞和CD 8 + T细胞的细胞毒性应答，以及（3）接种疫苗是否增加宿主在抗逆转录病毒治疗暂停后维持低水平病毒血症的能力。选择高度减毒的痘病毒NYVAC-SIV-gag-pol-env活重组疫苗候选物作为疫苗方法来测试这些概念，因为其在猕猴中作为预防性疫苗候选物的有效性已得到证实。在研究中使用了三组八只猕猴，每组包含三只Mamu-A*01-Macaca Mulata;恒河猴MHC-动物的命名法。所有动物静脉内接种10个感染剂量的高致病性SIV 251，在SIV 251感染后第15天，（A和B组）开始抗逆转录病毒治疗（HAART），皮下接种PMPA 20 mg/kg/d，静脉接种去羟肌苷（DDI）10 mg/kg/d，口服司他夫定（d4 T），每日分两次给药（总计2.4 mg/kg/天）。在感染后10、19和23周，B组中的所有猕猴（HAART处理）和C用10至八分之一pfu的NYVAC-SIV-gag-pol-env疫苗肌内免疫A组动物（未处理的），而A组动物（未处理的）用10至八分之一pfu的NYVAC-SIV-gag-pol-env疫苗肌内免疫，（HAART处理的）接受10至8 pfu的模拟NYVAC亲本病毒。1.建立了一种动物模型，在该模型中，可以以受控的方式评估免疫和免疫干预。2.证明了CD 4+和CD 8 + T细胞应答都被激发。在接受HAART治疗的受感染动物中，这一点在目前HAART可能沉默免疫应答而损害宿主的情况下是非常重要的。3.在具有活跃病毒复制的动物（C组）以及HAART治疗的猕猴中使用这种减毒活载体是安全的（A组和B组）。4. HAART的早期干预改变了疾病的自然病程。5.免疫接种可能有效果，尽管如此，我们观察到一个明显的趋势，由于样本量的原因，我们无法证明这一发现的统计学意义。由于这些结果令人鼓舞，明年将开始使用基于NYVAC的HIV-I疫苗在HIV-I感染者中进行安全性和免疫原性试验。

Part 1The first part of the research plan is to assess highly attenuated recombinant poxvirus vaccine candidates in the rhesus macaque model. This work is a collaborative effort with Pasteur Merieux Connaught, which has a CRADA with the NCI, and involves intra-agency collaboration with the NIAID. Two vectors are being tested: a canarypox-based vector (ALVAC) and the genetically attenuated vaccinia strain (a derivative of the Copenhagen strain) NYVAC. Both vectors are able to complete their replication cycle and express heterologous genes under the control of early or early/late promoters. The working hypothesis is that, because both poxvirus vectors, even in the absence of a complete life cycle, still express a large number of their proteins, the response against the heterologous antigens may be limited. Thus we are investigating the hypothesis that conventional DNA plasmid (optimized for expression) or DNA replication (based on the Semliky forest virus replicon) may prime a sufficient immune response that may then be amplified by further boosting with recombinant poxviruses carrying the same SIV genes. Furthermore, we are also testing the importance of the addition of all the accessory/regulatory genes to the SIV/HIV structural genes in these vaccine modalities. Once an optimal combination of modalities is achieved, other regimens of immunization may be further optimized by investigating the importance of the route of immunization and the help of immunomodulating molecules. Part 2In view of the encouraging results obtained with a preventive NYVAC-SIV-gag-pol-env vaccine candidate in rhesus macaques (1), we hypothesized that such a vaccine could also be effective in a regimen of immune intervention following SIV infection. Thus, we develop a model in macaques whereby to test the effect of vaccination in the presence or absence of antiretroviral therapy. The study was designed to assess the following:(1) whether prolonged antiretroviral therapy had any effect on viral rebound following therapy suspension, (2) whether vaccination of macaques could enhance SIV-specific CD4+ T-helper and CD8+ T-cell cytotoxic responses, and (3) whether vaccination increased the ability of the host to maintain viremia at low levels following antiretroviral therapy suspension. The highly attenuated poxvirus NYVAC-SIV-gag-pol-env live recombinant vaccine candidate was chosen as a vaccine approach to test these concepts because of its demonstrated effectiveness as a preventive vaccine candidate in macaques. Three groups of eight macaques were used in the study, each group containing three Mamu-A*01--Macaca Mulata; nomenclature for rhesus MHC--animals. All animals were inoculated intravenously with ten infectious doses of highly pathogenic SIV251 and, on day 15 after SIV251 infection, sixteen animals (groups A and B) were started on antiretroviral therapy (HAART) with subcutaneous inoculation of 20 mg/kg/day of PMPA, intravenous inoculation of 10 mg/kg/day of Didanosine (DDI), and oral administration of Stavudine (d4T) divided into two doses daily (total of 2.4 mg/kg/day). At weeks 10, 19, and 23 after infection, all macaques in groups B (HAART-treated) and C (untreated) were immunized intramuscularly with 10 to the eighth pfu of NYVAC-SIV-gag-pol-env vaccine, whereas animals in group A (HAART-treated) received 10 to the eighth pfu of mock NYVAC parental virus.In summary, we have achieved the following:1.Establishment of an animal model in which immunization and immune intervention can be assessed in a controlled fashion.2.Demonstration that both CD4+ and CD8+ T-cell responses are elicited/boosted by a vaccine in infected animals treated with HAART. This point is very important in the context of the present concern that HAART may silence the immune response to the detriment of the host.3.The use of this attenuated live vector is safe in animals with active viral replication (group C) as well as in HAART-treated macaques (groups A and B).4.Early intervention with HAART changes the natural course of disease.5.Immunization may have an effect, and we observe a clear trend although, because of the sample size, we were unable to demonstrate the statistical significance of this finding. Because of the encouraging nature of these results, a safety and immunogenicity trial in HIV-I-infected individuals will be initiated the coming year using a NYVAC-based HIV-I vaccine.