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

研究计划的第一部分是在恒河猴模型中评估高减毒重组痘病毒候选疫苗。这项工作是与巴斯德梅里埃康诺（Pasteur Merieux Connaught）合作开展的，该公司与NCI拥有CRADA，并涉及与NIAID的机构内合作。目前正在测试两种载体：基于金丝雀痘的载体（ALVAC）和基因减毒牛痘株（哥本哈根株的衍生物）NYVAC。这两种载体都能在早期启动子或早/晚启动子的控制下完成其复制周期并表达异源基因。目前的假设是，由于这两种痘病毒载体，即使在没有完整的生命周期的情况下，仍然表达大量的它们的蛋白质，对异源抗原的反应可能是有限的。因此，我们正在研究一种假设，即传统的DNA质粒（对表达进行了优化）或DNA复制（基于Semliky森林病毒复制子）可能引发足够的免疫反应，然后通过携带相同SIV基因的重组痘病毒进一步增强免疫反应。此外，我们还在测试在这些疫苗模式中向SIV/HIV结构基因添加所有辅助/调节基因的重要性。一旦实现了最佳的模式组合，通过研究免疫途径的重要性和免疫调节分子的帮助，其他免疫方案可能会进一步优化。鉴于在恒河猴中获得的令人鼓舞的NYVAC-SIV-gag-pol-env候选预防性疫苗(1)，我们假设这种疫苗也可以有效地用于SIV感染后的免疫干预方案。因此，我们在猕猴中建立了一个模型，以此来测试在存在或不存在抗逆转录病毒治疗的情况下接种疫苗的效果。该研究旨在评估：(1)长期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CD4+ T-helper和CD8+ t细胞的细胞毒性反应，以及(3)接种疫苗是否增加了宿主在抗逆转录病毒治疗暂停后将病毒血症维持在低水平的能力。选择高度减毒痘病毒NYVAC-SIV-gag-pol-env活重组候选疫苗作为疫苗方法来测试这些概念，因为它在猕猴中被证明是一种有效的预防性候选疫苗。研究中使用了三组猕猴，每组8只，每组含有3只Mamu-A*01—Macaca Mulata；恒河MHC的命名法——动物。所有动物静脉注射10剂高致病性SIV251，在SIV251感染后第15天，16只动物（A组和B组）开始抗逆转录病毒治疗（HAART），皮下注射PMPA 20 mg/kg/天，静脉注射二丹参（DDI） 10 mg/kg/天，口服司他夫定（d4T），分两剂每日（共2.4 mg/kg/天）。在感染后第10、19和23周，B组（经haart治疗）和C组（未经haart治疗）的所有猕猴均肌肉注射10 ^ 8 pfu的NYVAC- siv -gag-pol-env疫苗，而A组（经haart治疗）的动物则注射10 ^ 8 pfu的模拟NYVAC亲本病毒。总的来说，我们取得了以下成绩：建立一种动物模型，使免疫和免疫干预能够以可控的方式进行评估。证明在接受HAART治疗的感染动物中，疫苗可激发/增强CD4+和CD8+ t细胞反应这一点非常重要，因为目前人们担心HAART可能会抑制免疫反应，从而损害宿主。在病毒复制活跃的动物（C组）以及经haart治疗的猕猴（A组和B组）中使用这种减毒活载体是安全的。早期干预HAART可以改变疾病的自然进程。免疫接种可能有影响，我们观察到一个明显的趋势，尽管由于样本量的原因，我们无法证明这一发现的统计意义。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.