Prevention of AIDS

我们的工作重点是使用基于dna的疫苗策略作为预防和免疫治疗方法。我们已经生成了高效的SIV和HIV DNA表达载体。这项工作是基于我们之前的认识，即存在于HIV gag/pol和env编码区的RNA元件（称为INS）负责在没有Rev的情况下转录本的核保留和不稳定性，并且这些元件可以通过改变转录本的核苷酸组成（RNA优化，也称为密码子优化）来消除，而不影响氨基酸序列。通过改变抗原的转运，进一步提高了抗原的免疫原性。引入的蛋白质修饰导致SIV抗原更有效的分泌，从而增加了接种小鼠或恒河猴的细胞和体液免疫反应。这些分子的免疫原性已经在老鼠和猕猴身上进行了测试。在小鼠中进行的研究使我们能够测试不同的DNA载体，并揭示产生不同形式的同一抗原的DNA组合产生更平衡的免疫反应，这是最佳艾滋病疫苗的理想特征。正在测试艾滋病毒/SIV抗原的不同递送方法。艾滋病毒疫苗开发的另一个重要方面是抗原的选择，这必须考虑到不同艾滋病毒进化支的多样性和能够诱导相关免疫反应的关键表位的鉴定。我们正致力于使用保守表位方法或镶嵌分子来优化抗原。使用DNA疫苗接种，我们发现我们优化的DNA疫苗载体能够诱导有效的免疫反应，能够保护恒河猴/SIV攻击模型中的高病毒血症。使用DNA作为疫苗的一个限制是它在肌肉注射时相对低效。改善DNA传递的最新进展包括体内电穿孔。我们报道了电穿孔显著提高了SIV经历和art治疗的幼稚恒河猴的DNA递送效率，导致抗原表达大大增强。我们发现，这种疫苗接种方法可以增强免疫应答，循环siv特异性T细胞的频率较高，多功能T细胞的存在，并诱导效应记忆和中枢记忆CD4和CD8 siv特异性T细胞。我们报道了包含IL-12 DNA作为佐剂导致反应质量的提高。除了全身免疫反应外，使用这种改进的DNA疫苗接种方法也诱导了粘膜反应，尽管只是对一小部分动物。尽管DNA电穿孔提供了一种强大的体液免疫反应，包括在猕猴中中和Ab的产生，但我们最近发现，蛋白质的增加可以诱导更高水平的Ab。重要的是，我们发现，在同一肌肉中同时注射DNA和蛋白质，无论是无佐剂的还是佐剂的，都增加了Ab的产生和粘膜传播。dna和蛋白质联合免疫在引起体液免疫反应方面优于接种两种单独成分中的任何一种。也许更重要的是，疫苗方案还诱导了持久有效的体液免疫，在最后一次接种后4年仍可检测到。接受这种优化疫苗接种方案的动物的攻毒显示出病毒获得的显著延迟和对高致病性SIV攻毒的病毒学控制的改善。因此，有效的DNA递送方法与改进的DNA疫苗混合物以及在疫苗方案中包含蛋白质相结合，在接种了疫苗的恒河猴中大大增强和更平衡的免疫反应。我们报道了全身和粘膜免疫反应与病毒获得的相关性，以及细胞毒性细胞免疫反应与病毒控制的相关性。一种理想的艾滋病毒疫苗应该能够预防所有的艾滋病毒-1变异。HIV序列多样性和潜在的免疫优势“诱饵”表位的存在是开发有效艾滋病疫苗的障碍。为了解决这些问题，我们正在探索将免疫强度和广度最大化的方法，重点关注HIV的高度保守区域，以诱导对几乎不变的蛋白质组片段（对病毒的功能至关重要）的免疫反应，同时排除对可变和潜在的免疫优势“诱饵”表位的反应。我们开发了一种靶向p24gag （p24gagCE DNA疫苗）区域的原型疫苗。在小鼠和猕猴的概念验证研究中，我们证明了用这种DNA免疫可引起针对CE的强大的细胞和体液免疫反应，这是p55gag DNA疫苗无法实现的。重要的是，我们证明了用CE DNA引物和p55gag DNA增强是一种有效的策略，可以最大限度地提高对Gag的反应，为增加疫苗接种的规模和广度提供了一个新的概念。这一新概念的翻译目前正在HVTN/ daids支持的临床试验中进行，目的是测试我们的p24gagCE疫苗是否比优化的gag免疫原（p55gag）产生更好的呼吸和gag反应，p55gag免疫原在HVTN临床试验中显示出最高的免疫反应率。

Our work focuses on the use of DNA-based vaccine strategies both as preventive and immunotherapeutic approaches. We have generated efficient SIV and HIV DNA expression vectors. This work is based on our previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the nucleotide composition of the transcripts (RNA optimization; also referred to as codon optimization) without affecting the amino acid sequence. The immunogencity of the antigens was further improved by modifying the trafficking of the antigens. The introduced modifications of the proteins led to more efficient secretion of the SIV antigens resulting in increased cellular and humoral immune responses in the vaccinated mice or rhesus macaques. The immunogenicity of such molecules has been tested in mice and macaques. Studies in mice allowed us to test different DNA vectors and revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine. Different delivery methods of the HIV/SIV antigens are being tested. Another important aspect of HIV vaccine development is the selection of the antigens, which has to take into consideration the diversity of the different HIV clades and the identification of the critical epitopes able to induce relevant immune responses. We are working on optimizing antigens using approaches that use either a conserved epitope approach or mosaic molecules. Using DNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia in the rhesus macaque/SIV challenge model. A limitation in using DNA as a vaccine is its relative inefficient delivery upon intramuscular injection. Recent developments to improve DNA delivery include in vivo electroporation. We reported that electroporation dramatically increased the efficiency of DNA delivery in both SIV experienced and ART-treated naive rhesus macaques, leading to greatly augmented antigen expression. We found that this vaccination method results in enhanced immune responses with a high frequency of circulating SIV-specific T cells, the presence of multifunctional T cells, and induction of both effector memory and central memory CD4 and CD8 SIV-specific T cells. We reported that the inclusion of IL-12 DNA as adjuvant led to improved quality of the responses. In addition to systemic immune responses, the use of this improved DNA vaccination methodology also induced mucosal responses, albeit only to a subgroup of animals. Although DNA electroporation provides a strong humoral immune response including neutralizing Ab development in macaques, we recently showed that a protein boost can induce higher levels of Ab. Importantly, we showed that injection of DNA and protein either unadjuvanted or adjuvanted in the same muscle at the same time increased Ab production and mucosal dissemination. DNA&Protein co-immunization is superior to vaccination with either of the two individual components in eliciting humoral immune responses. Perhaps more importantly, the vaccine regimen also induced potent long-lasting humoral immunity, detectable for 4 years after the last vaccination. Challenge of animals which received such optimized vaccination regimens showed a significant delay in virus acquisition and improvement in virological control of the highly pathogenic SIV challenge. Thus, efficient DNA delivery methods in combination with improved DNA vaccine cocktails and the inclusion of protein in the vaccine regimen resulted in greatly augmented and more balanced immune responses in vaccinated rhesus macaques. We reported a correlation of systemic and mucosal immune responses and virus acquisition as well as a correlation of cytotoxic cellular immune responses with virus control. An ideal HIV vaccine should provide protection against all HIV-1 variants. HIV sequence diversity and the presence of potential immunodominant "decoy" epitopes are hurdles in the development of an effective AIDS vaccine. To address these problems, we are exploring approaches to maximize immunological strength and breadth focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant "decoy" epitopes. We developed a prototype vaccine targeting regions within the p24gag (p24gagCE DNA vaccine). In proof-of-concept studies in mice and macaques, we demonstrated that immunization with this DNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. Importantly, we demonstrated that priming with CE DNA and boosting with p55gag DNA is an effective strategy to maximize responses against Gag, providing a novel concept to increase the magnitude and breadth of vaccination. The translation of this novel concept is currently being pursued in an HVTN/DAIDS-supported clinical trial with the aim to test whether our p24gagCE vaccine develops superior breath and magnitude of gag responses compared to the optimized gag immunogen (p55gag), which showed the highest immune response rate in HVTN clinical trials.