DNA Vaccines

该项目的一个重要目标仍然是产生和测试最有效的表达载体的特定抗原。我们的假设是，DNA疫苗的剂量是次优的许多人类应用，因此，提高效率是必要的实际人类DNA疫苗。我们已经产生了一组优化的表达载体的HIV和SIV。HIV载体被开发用于最终的人类临床试验。在猕猴中研究了这些载体的免疫原性和保护免受猿猴/人类免疫缺陷病毒杂交病毒（SHIV）攻击的能力。我们的几个载体被用于我们以前的CRADA合作者（惠氏）赞助的临床试验。与此同时，SIV表达载体在最可靠的人类AIDS模型系统中被开发和研究，即，SIV是一种与HIV密切相关的病毒，引起与人类艾滋病非常相似的病理学。我们的结果表明，在没有任何其他形式的疫苗加强的情况下，优化的DNA表达载体能够保护恒河猴在用高致病性SIVmac 251攻击后免于高病毒血症。此外，我们还开发了强大的新DNA和蛋白质联合免疫方案，提高了免疫反应的幅度、速度和寿命。为了进一步提高疫苗效率，我们研究了不同候选抗原的内在特性。我们利用重组DNA技术操纵表达抗原形式的能力。我们已经表明，调节DNA产生的抗原的形式、稳定性和细胞命运对其免疫原性和产生的应答类型具有深远的影响。我们进行比较研究，以开发几种抗原的最佳形式。在恒河猴中的结果证实了表达抗原的形式影响免疫应答的类型和幅度。我们研究了几种不同的抗原形式，以实现最佳的免疫应答，并解决全球流行的HIV毒株的变异性。我们比较了由天然抗原、嵌合体、集中和共识候选物的混合物以及仅含有HIV蛋白的保守元件的抗原产生的免疫应答。这种比较可能会导致保护性免疫反应的进一步优化。我们最近已经表明，用保守元件疫苗构建物进行疫苗接种能够改变免疫应答的层次结构，并将其导向所有HIV进化枝中发现的保守元件。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的免疫应答的能力。用于猕猴DNA疫苗接种的方法和载体已经表明，我们产生了强大、广泛和持久的免疫力，能够抑制病毒复制并预防疾病发展。最近，我们发现，DNA与佐剂蛋白的组合能够在反复低剂量病毒攻击后延迟或预防感染。DNA疫苗接种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，基于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疫苗接种将有许多实际的临床应用。我们利用我们对基因调控的理解开发了非致病性SIV菌株，这些菌株在猕猴中保存了10年以上，但它们不会引起任何疾病。这些动物产生了强烈的保护性免疫应答，即使在用野生型SIV攻击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SIVmac 239的中和抗体，并且CD 8细胞有助于保护作用。我们还表明，这些动物产生高水平的细胞毒性CD 4细胞，这有助于病毒控制。这种猕猴模型对于进一步了解导致艾滋病的致病机制、不同组织中的病毒相互作用以及有助于防止疾病发展的免疫系统组成部分非常重要。除了针对艾滋病的预防性疫苗接种外，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应答的强烈增强和随后的病毒血症控制，表明治疗性疫苗接种可能有助于长期病毒控制。这些结果也有影响的方法的发展，应用DNA疫苗的方法，治疗性癌症疫苗。我们希望将联合收割机治疗性疫苗接种与其他方法结合，以增强免疫应答和细胞毒性细胞的特异性细胞杀伤。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进一步增强细胞毒性细胞应答。我们已经表明，在治疗性疫苗接种中包含hetIL-15导致高水平的细胞毒性细胞。

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.