Preventive Vaccines for HIV

方法一：了解并提高ALVAC和gp120疫苗的保护效果。在过去的几年里，我们研究了HIV疫苗的方法，单独基于DNA或与基于痘病毒的疫苗联合使用，发现这些疫苗方式能够引发主要的记忆t细胞，当与gp120蛋白增强剂联合使用时，可以诱导高滴度结合抗体。在泰国最近完成的RV144试验中，使用了类似的策略，将金丝猴痘载体ALVAC与gp120联合使用，显示出边际疗效（31.2%）[Jim Tartalia NejM 2009]。在动物模型中，类似疫苗模式的相对效力因攻毒实验中所用病毒的共受体使用情况、病毒株、动物年龄以及暴露途径和剂量而异。最近的证据表明，SIVmac251（一种CCR5使用者病毒）的攻毒暴露剂量会影响疫苗效力的读数，并且在高剂量的攻毒暴露下，疫苗保护作用减弱，并传播几种病毒变体。据估计，在人类中，艾滋病毒通过不同途径传播的风险在每次接触10到1000之间，当传播发生时，只有少数艾滋病毒变体通过粘膜。我们通过使用与AIDSVAX或RV144试验中使用的疫苗相似的疫苗，以及使用旨在传播少量病毒变体的SIVmac251剂量来更好地模拟人类HIV粘膜传播，探索了SIVmac251猕猴模型与疫苗在人类中的有效性的相关性。我们发现，在这些条件下，动物模型忠实地再现了这些疫苗模式在人类中的功效。现在我们已经定义了一个适当的模型，概括了我们正在设计的研究，旨在确定ALVAC和gp120诱导的保护的相关性。目的：提高ALVAC和gp120的免疫原性，探讨攻毒剂量对疫苗疗效评价的重要性。我们评估了DNA、ALVAC-SIV和gp120联合免疫后SIVmac251攻毒剂量变化对疫苗效力的影响。在非人类灵长类动物艾滋病毒模型中对疫苗效力的评估通常使用单一的高剂量攻击方式进行。虽然现在很清楚，低重复剂量的SIV/SHIV粘膜暴露能更好地再现人类HIV-1的性传播，但仍不清楚病毒接种量与疫苗效力之间是否存在关系。因此，我们研究了粘膜攻击剂量对DNA-SIV/ALVAC-SIV/ gp120蛋白效力的影响，这一策略与泰国试验疫苗类似。我们首次证明，疫苗的效力取决于攻击的剂量。事实上，在单次直肠内注射6100 TCID50疫苗后，疫苗短暂地减少了病毒的复制，但在重复注射低剂量470 TCID50 SIVmac251的12只猕猴中，它保护了3只。在暴露于470 TCID50而不是6100 TCID50 SIVmac251的动物中，接种疫苗还可以延长对高病毒复制的保护时间，减少传播变异的数量以及全身和粘膜CD4+ T细胞再生。有趣的是，虽然在高剂量攻击组中观察到的临时保护仅与CD8 T细胞反应相关，但抗体和CD8细胞对gag和env的反应都参与了对病毒复制的长期保护。我们的数据表明，在评估疫苗效力时，准确地模拟猴体内的艾滋病毒传播非常重要，尤其要注意暴露剂量。方法2：用携带SIV DNA的人乳头瘤病毒假病毒粒子疫苗靶向阴道黏膜大多数HIV感染是通过性传播发生的。艾滋病毒必须在生殖道存活，穿过阴道或直肠上皮，然后传播到全身。目前疫苗战略在提供绝育免疫方面的失败可能是由于疫苗在进入门户时引起的反应的数量、质量或持久性。阻断艾滋病毒的阴道传播可能需要针对女性生殖道并诱导局部免疫的疫苗。基于病毒载体、蛋白质或其组合的艾滋病毒疫苗在人体中进行了三期疫苗功效试验，主要通过肌肉注射接种疫苗诱导全身免疫反应。虽然这些疫苗形式在血液中诱导不同水平的艾滋病毒特异性反应，但对其诱导粘膜反应的能力知之甚少。人乳头瘤病毒（hpv）的一个子集是性传播的粘膜病原体，自然感染宫颈阴道角化细胞。基于HPV- vlp的疫苗是安全且非常有效的，可以预防导致女性宫颈肿瘤的HPV感染。HPV衣壳蛋白L1和L2可以自组装成病毒样颗粒（vlp），当与含有感兴趣基因的质粒共转染时，L1和L2将包裹质粒形成假病毒粒子（psv）。人类乳头状瘤病毒（HPV）假病毒粒子是由我在国家癌症研究所的合作者巴克、席勒和罗伊博士开发的一项技术。在多种动物模型中，HPV psv已被证明能有效地将报告基因传递到女性生殖道。HPV psv感染仅限于角质形成细胞，需要轻微的上皮破坏。转基因的表达是稳定的但短暂的，在小鼠生殖道中持续大约7天。此外，HPV psv可能作为佐剂，参与toll样受体，促进抗原提呈细胞的激活和成熟。我们利用HPV psv靶向女性生殖道的能力，并利用psv作为载体将编码SIV基因的DNA传递到两种非人灵长类动物的SIV传播位点。我们证明，HPV psv作为SIV DNA传递的载体，在食蟹猴和恒河猴的生殖道、生殖道引流淋巴结和血液中诱导了SIVmac251特异性细胞和体液免疫反应。阴道内接种导致CD4+和CD8+ t细胞募集到接种部位，增加了宫颈阴道粘膜中t细胞的绝对数量。此外，HPV psv诱导粘膜免疫反应在阴道暴露于SIVmac251后迅速扩大。我们目前正在研究HPV PsV诱导的免疫反应是否可以保护猕猴免受SIVmac251反复低剂量阴道攻击。方法3：利用热休克gp96- igd重组SIV疫苗在胃肠道诱导效应t细胞。这是与迈阿密大学的Podack博士合作努力的一部分，包括制备293细胞分泌的siv -gp96细胞疫苗。这种基于细胞的gp96-Ig疫苗延长了gp96-Ig肽的体内分泌，可以模仿病毒复制，并提供与减毒病毒相当的免疫刺激。该疫苗由转染的293细胞组成，该细胞分泌er伴侣蛋白gp96-Ig与SIV衍生肽复合物。通过用gp96-Ig-SIV疫苗对猕猴进行ip免疫，可以获得直肠和阴道强烈的CTL反应。在过去的一年中，我们发现重组gp120蛋白与293- gp96sivig疫苗细胞共同注射可显著保护直肠SIV感染。由于gp96-SIV单独或gp120蛋白单独都不能提供对感染的保护，因此我们得出结论，粘膜CTL和抗体都需要保护免受粘膜SIV的攻击。在未来几年，我们将评估提高gp96+ gp120疫苗保护效果的方法。

Approach 1: Understand and improve on the protective efficacy of ALVAC & gp120 vaccination. In the last several years we have investigated vaccine approaches for HIV, based on DNA alone or in combination with poxvirus-based vaccines, and found that these vaccine modalities are able to elicit mainly memory T-cells, and when combined with gp120 protein boost can induce high titer binding antibodies. A similar strategy using the canary pox vector ALVAC in combination with gp120 was used in the recently completed RV144 trial in Thailand that showed marginal efficacy (31.2%) [Jim Tartalia NejM 2009]. The relative efficacy of similar vaccine modalities varied in animal models dependent on co-receptor usage of the virus used in challenge experiments, the viral strain, the age of the animals, and the route and dose of exposure. Recent evidence suggest that the dose of challenge exposure to SIVmac251, a CCR5 user virus, affects the read out of vaccine efficacy and that at high doses of challenge exposure, vaccine protection is diminished and several virus variants are transmitted. It is estimated that in humans, the risk of HIV transmission ranges by different routes between ten and one thousand per encounter and that when transmission occurs, only few HIV variants pass the mucosa. We explored the relevance of the SIVmac251 macaque model to vaccine efficacy in humans by using vaccines similar to those used in the AIDSVAX or the RV144 trials, and by using a dose of SIVmac251 intended to transmit few viral variants to better mimic HIV mucosal transmission in humans. We found that, in these conditions, the animal model faithfully recapitulated the efficacy of these vaccine modalities in humans. Now that we have defined an appropriate model that recapitulates the protection we are designing studies aimed at defining the correlates of protection induced by ALVAC and gp120. In attempt to improve upon the immunogenicity of ALVAC and gp120 and to investigate the importance of challenge dose on evaluating vaccine efficacy. We evaluated the effect of varying the SIVmac251 challenge dose on vaccine efficacy after immunizing with combination of a DNA, ALVAC-SIV, and gp120. The evaluation of vaccine efficacy in the non-human primate model for HIV has been often carried out using a single high dose challenge modality. While it is now clear that low repeated doses mucosal exposure to SIV/SHIV better recapitulate sexual transmission of HIV-1 in humans, it is still not known whether a relationship exists between viral inoculum size and vaccine efficacy. Thus, we investigated the impact of the mucosal challenge dose on the efficacy of DNA-SIV/ALVAC-SIV/ gp120 protein, a strategy similar to the Thai trial vaccine. For the first time, we have demonstrated that vaccine efficacy depends on the dose of challenge. In fact while vaccination transiently decreased viral replication after a single intra-rectal dose of 6100 TCID50, it protected 3 of the 12 macaques challenged with repeated low doses of 470 TCID50 SIVmac251. Vaccination also resulted in prolonged protection from high viral replication, reduction of the number of transmitted variants and systemic and mucosal CD4+ T cell regeneration in the animals exposed to 470 TCID50, but not to 6100 TCID50 SIVmac251. Interestingly, while the temporary protection observed in the high dose challenged group was correlated with CD8 T cell responses only, both antibodies and CD8 cell responses to gag and env participated to the prolonged protection from viral replication. Our data demonstrate the importance of accuratly modeling HIV transmission in macaques then evaluating of vaccine efficacy, with particular attention to the dose of exposure. Approach 2: Targeting the Vaginal Mucosa with Human Papillomavirus Pseudovirion Vaccines delivering SIV DNA The majority of HIV infections occur via sexual transmission. HIV must survive the genital tract, cross the vaginal or rectal epithelium and then disseminate throughout the body. The failure of current vaccine strategies at providing sterilizing immunity may be due to the quantity, quality or durability of vaccine induced responses at the portal of entry. Blocking vaginal transmission of HIV may require vaccines that target the female genital tract and induce local immunity. HIV vaccines based on viral vectors, proteins, or a combination thereof, tested in phase III vaccine efficacy trials in humans, induced mainly systemic immune responses using vaccines delivered by intramuscular inoculation. While these vaccine modalities induce variable levels of HIV-specific responses in the blood, little is known about their ability to induce mucosal responses. A subset of human papillomavirus (HPVs) are sexually transmitted mucosal pathogens that naturally infect cervico-vaginal keratinocytes. HPV-VLP-based vaccines are safe and very effective at preventing the HPV infections that cause cervical neoplasia in women. HPV capsid proteins, L1 and L2, can self assemble into virus like particles (VLPs) and, when co-transfected with a plasmid containing a gene of interest, L1 and L2 will encapsidate the plasmid forming pseudovirions (PsVs). Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI. HPV PsVs have been shown to effectively deliver reporter genes to the female genital tract in multiple animal models. HPV PsVs infection is limited to keratinocytes and requires minor disruption of the epithelium. Expression of the transgene is robust but transient, lasting approximately seven days in the mouse genital tract. Furthermore, HPV PsVs may serve as adjuvants, engaging toll like receptors and facilitating the activation and maturation of antigen presenting cells. We have exploited the ability of HPV PsVs to target the female genital tract and used PsVs as vectors to deliver DNA encoding SIV genes to a site of SIV transmission in two non-human primate species. We demonstrated that HPV PsVs, used as vehicles for the delivery of SIV DNA, induced SIVmac251 specific cellular and humoral immune responses at the genital tract, in the genital draining lymph nodes, and in blood in both cynomolgus and rhesus macaques. Intravaginal vaccination results in the recruitment of both CD4+ and CD8+ T-cells to the site of vaccination, increasing the absolute number of T-cells in the cervico-vaginal mucosa. In addition, HPV PsVs induced mucosal immune responses that rapidly expanded upon vaginal exposure to SIVmac251. We are currently investingating if HPV PsV induced immune responses can protect macaques from a repeated low dose vaginal challenge with SIVmac251. Approach 3: Induce effector T-cells in the gastrointestinal tract using heat shock gp96-Igbased SIV recombinant vaccines. This is part of a collaborative effort with Dr Podack from the University of Miami and includes the preparation of cellular SIV-gp96-vaccines secreted by 293 cells. This cell-based gp96-Ig vaccine has prolonged in vivo secretion of gp96-Ig-peptide that imitates viral replication and provide immune stimuli comparable to attenuated viruses. The vaccine is composed of transfected 293 cells that secrete ER-chaperone gp96-Ig in complex with SIV derived peptides. Strong rectal and vaginal CTL reponses are achieved by i.p. immunization of macaques with the gp96-Ig-SIV vaccine. In the past year we have found that coinjection of recombinant gp120 protein with the 293-gp96SIVIg-vaccine cells provides significant protection from rectal SIV infection. As neither gp96-SIV alone or gp120-protein alone provides protection from infection we conclude that both mucosal CTL and antibodies are required to protect against mucosal SIV challenge. In the upcoming years we will asses methods of improving the protective efficacy of gp96+ gp120 vaccination.