DNA Vaccines

该项目的一个重要目标仍然是生成和测试最有效的特定抗原表达载体。我们的假设是，DNA疫苗剂量对许多人类应用来说是次优的，因此，提高实用的人类DNA疫苗的效率是必要的。我们生成了一组优化的HIV和SIV表达载体。HIV载体被开发用于最终的人体临床试验。在猕猴中研究了这些载体的免疫原性和抵抗猿猴/人类免疫缺陷病毒混合病毒（SHIV）攻击的能力。我们的几个载体被用于由我们的CRADA合作伙伴（惠氏）赞助的临床试验。与此同时，SIV表达载体在人类艾滋病最忠实的模型系统中得到开发和研究。SIV是一种与HIV密切相关的病毒，其引起的病理与人类艾滋病非常相似。我们的研究结果表明，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免受高致病性SIVmac251攻击后的高病毒血症。此外，我们已经开发出强大的新的DNA和蛋白质联合免疫方案，增加了免疫反应的规模，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。恒河猴的结果证实，表达抗原的形式影响免疫反应的类型和强度。我们研究了几种不同的抗原形式，以实现最佳的免疫反应，并解决全球范围内流行的HIV毒株的可变性。我们比较了由天然抗原、嵌合抗原、集中候选抗原和仅含有HIV蛋白保守元素的抗原混合产生的免疫反应。这种比较可能导致保护性免疫反应的进一步优化。用于猕猴DNA疫苗接种的方法和载体表明，我们产生了非常强大、广泛和持久的免疫力，能够遏制病毒复制并预防疾病发展。最近，我们发现DNA与佐剂蛋白结合能够延迟或预防重复低剂量病毒攻击后的感染。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，DNA疫苗接种正在成为人类最强大和最有效的疫苗接种程序。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性SIV毒株，这些毒株在猕猴体内维持了10多年，但它们不会引起任何疾病。这些动物产生强烈的保护性免疫反应，即使在受到野生型SIV的攻击后也能抵抗高病毒血症和疾病的发展。我们发现这些动物产生了针对难以中和的SIVmac239的中和抗体，并且CD8细胞有助于保护作用。我们还表明，这些动物产生了高水平的细胞毒性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免疫系统中有助于预防疾病发展的成分具有重要意义。除了预防艾滋病的疫苗接种外，治疗性疫苗接种方案也使用了同样的方法。在猕猴研究中观察到细胞免疫反应的强烈增强和随后的病毒血症控制，这表明治疗性疫苗可能有助于长期控制病毒。这些结果也对开发将DNA疫苗方法应用于治疗性癌症疫苗的方法产生影响。

Summary 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 CRADA collaborator (Wyeth). In parallel, SIV expression vectors are developed and studied in the most faithful model system for human AIDS, ie., challenge of Rhesus macaques by SIV, a virus closely related to HIV, which causes very similar pathology to human AIDS. Our results show 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. The methodology and vectors used for DNA vaccination of macaques have shown that we produce a very 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 a adjuvanted protein is able to delay or prevent infection after repeated low dose virus challenge. DNA vaccination is emerging as the strongest and mosr effective vaccination procedure 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.