DNA Vaccines

该项目的一个重要目标仍然是产生和测试针对特定抗原的最有效的表达载体。我们的假设是，DNA疫苗的剂量对于许多人类应用来说是次优的；因此，提高效率对于实用的人类DNA疫苗是必要的。我们已经生成了一组针对HIV和SIV的优化表达载体。HIV载体是为最终的人类临床试验而开发的。这些载体在猕猴身上进行了免疫原性和抵御猿猴/人类免疫缺陷病毒杂交病毒(SIV)攻击的能力研究。我们的几个载体被用于由我们之前的CRADA合作者(惠氏)赞助的临床试验。同时，在人类艾滋病最忠实的模型系统中开发和研究了SIV表达载体，即SIV对恒河猴的攻击。SIV是一种与HIV密切相关的病毒，其致病机制与人类AIDS非常相似。我们的结果表明，在没有任何其他形式的疫苗增强的情况下，优化的DNA表达载体能够保护恒河猴在高致病性SIVmac251攻击后免受高病毒血症的影响。此外，我们还开发了强大的DNA和蛋白质联合免疫方案，以提高免疫反应的幅度、速度和寿命。为了进一步提高疫苗的效率，我们研究了不同候选抗原的内在属性。我们利用重组DNA技术来操纵表达的抗原的形式。我们已经证明，调节DNA产生的抗原的形式、稳定性和细胞命运对它们的免疫原性和产生的反应类型有深远的影响。我们进行比较研究，以开发几种抗原的最佳形式。在恒河猴身上的结果证实，表达的抗原的形式影响免疫反应的类型和大小。我们研究了几种不同的抗原形式，以实现最佳的免疫反应，并解决全球流行的艾滋病毒毒株的变异性。我们比较了天然抗原、嵌合体、集中候选抗原和一致候选抗原，以及只包含HIV蛋白保守元件的抗原所产生的免疫反应。这样的比较可能导致保护性免疫反应的进一步优化。我们最近已经证明，用保守元件疫苗构建的疫苗能够改变免疫反应的层次结构，并将其导向保守元件，这些保守元件在所有HIV分支中都存在。在这些数据的基础上，我们提出了一项临床试验，以测试保守元件载体在人类中提供更广泛免疫反应的能力。用于猕猴DNA疫苗接种的方法和载体表明，我们产生了一种强大、广泛和持久的免疫力，能够遏制病毒复制和防止疾病发展。最近，我们发现DNA与佐剂蛋白的结合能够延缓或预防反复低剂量病毒攻击后的感染。DNA疫苗正在成为人类细胞免疫发展中最强大和最有效的疫苗接种程序，其基础是使用我们为猕猴共同开发的相同方法和载体的临床试验。这些结果有力地表明，DNA疫苗将有许多实际的临床应用。我们已经利用我们对基因调控的理解开发了非致病的SIV毒株，这种毒株在猕猴体内保持了10多年，但它们不会导致任何疾病。这些动物产生了强大的保护性免疫反应，即使在挑战野生型SIV之后，也能够抵抗高病毒血症和疾病发展。我们发现，这些动物产生了针对难以中和的SIVmac239的中和抗体，CD8细胞参与了保护作用。我们还表明，这些动物会产生高水平的细胞毒性CD4细胞，这有助于病毒控制。这一猕猴模型对于进一步了解导致艾滋病的致病机制、病毒在不同组织中的相互作用以及有助于防止疾病发展的免疫系统组件具有重要意义。除了预防接种艾滋病疫苗外，治疗性疫苗接种方案也采用了同样的方法。在猕猴研究中观察到细胞免疫反应和随后的病毒血症控制的强烈促进，这表明治疗性疫苗接种可能有助于长期的病毒控制。这些结果也对开发将DNA疫苗方法学应用于治疗性癌症疫苗的方法具有指导意义。我们希望将治疗性疫苗接种与其他方法相结合，以增强免疫反应和细胞毒细胞对特定细胞的杀伤。因此，基于我们完成的证明hetIL-15在感染猕猴中的安全性的研究，我们将使用hetIL-15进一步增强细胞毒细胞反应。我们已经证明，在治疗性疫苗接种中加入hetIL-15会导致高水平的细胞毒细胞。

An important goal of this project continues to be the generation and testing of maximally efficient expression vectors for specific antigens. Our hypothesis is that the DNA vaccine dose is suboptimal for many human applications; therefore, increased efficiency is necessary for practical human DNA vaccines. We have generated a set of optimized expression vectors for HIV and SIV. HIV vectors are developed for eventual human clinical trials. These vectors are studied in macaques for immunogenicity and ability to protect against challenge with Simian/Human Immunodeficiency Virus hybrid viruses (SHIV). Several of our vectors were used in clinical trials sponsored by our previous CRADA collaborator (Wyeth). In parallel, SIV expression vectors are developed and studied in the most faithful model system for human AIDS, i.e., challenge of Rhesus macaques by SIV, a virus closely related to HIV, which causes very similar pathology to human AIDS. Our results have shown that optimized DNA expression vectors in the absence of any other form of vaccine boosting are able to protect rhesus macaques from high viremia after challenge with a highly pathogenic SIVmac251 challenge. In addition, we have developed powerful new DNA and protein co-immunization protocols that increase the magnitude, rapidity and longevity of immune responses. To further improve vaccine efficiency we study the intrinsic properties of the different candidate antigens. We take advantage of the ability to manipulate the form of expressed antigen by recombinant DNA technology. We have shown that modulating the form, stability and cellular fate of the DNA-produced antigens has profound effects on their immunogenicity and the type of response generated. We perform comparative studies to develop optimal forms of several antigens. Results in rhesus macaques verified that the form of expressed antigen affects the type and magnitude of immune response. We study several different antigen forms to achieve optimal immune response and to address the variability of HIV strains circulating worldwide. We compare the immune response generated by either mixes of native antigens, mosaics, centralized and consensus candidates, and also antigens containing only conserved elements of HIV proteins. Such comparisons may lead to further optimization of a protective immune response. We have recently shown that vaccination with conserved elements vaccine constructs have the ability to alter the hierarchy of immune response and to direct it towards conserved elements, which are found in all HIV clades. On the basis of these data, we have proposed a clinical trial to test the ability of Conserved Element vectors to provide broader immune response in humans. The methodology and vectors used for DNA vaccination of macaques have shown that we produce a strong, broad and long-lasting immunity, which is able to contain virus replication and prevent disease development. More recently, we showed that DNA in combination with an adjuvanted protein is able to delay or prevent infection after repeated low dose virus challenge. DNA vaccination is emerging as the strongest and most effective vaccination procedure for the development of cellular immunity in humans, based on clinical trials using the same methods and vectors we co-developed for macaques. These results strongly suggest that DNA vaccination will have many practical clinical applications. We have used our understanding of gene regulation to develop non-pathogenic strains of SIV, which are maintained in macaques for more than 10 years, yet they do not cause any disease. These animals develop a strong protective immune response and are able to resist high viremia and disease development even after challenge with wild-type SIV. We showed that these animals develop neutralizing antibodies against difficult-to-neutralize SIVmac239, and that CD8 cells contribute to the protective effect. We have also shown that these animals develop high levels of cytotoxic CD4 cells, which contribute to viral control. This macaque model is important for the further understanding of the pathogenic mechanisms leading to AIDS, the virus interactions in different tissues and the components of the immune system contributing to protection from disease development. In addition to prophylactic vaccination against AIDS, the same methodologies were used in therapeutic vaccination protocols. A strong boost of cellular immune responses and subsequent control of viremia was observed in macaque studies, suggesting that therapeutic vaccination may contribute to long-term virus control. These results have also implications for the development of methods to apply DNA vaccine methodology to therapeutic cancer vaccines. We wish to combine therapeutic vaccination with additional methods to boost immune response and specific cell killing by cytotoxic cells. We therefore will use hetIL-15 to further boost cytotoxic cells responses based on our completion of studies demonstrating the safety of hetIL-15 in infected macaques. We have shown that inclusion of hetIL-15 in therapeutic vaccination results in high levels of cytotoxic cells.