New Approaches To Active Immunoprophylaxis

HVS与GlaxoSmithKline（GSK）（里克森萨尔，比利时）合作开发了几种候选的减毒活HAV疫苗。其中一种候选疫苗经过改良，成为目前获得许可的GSK灭活HAV疫苗。此外，HVS还开发了一种非常有前途的候选重组戊型肝炎疫苗，最近已完成临床试验。在进一步表征该候选戊型肝炎疫苗的研究中，我们进行了广泛的临床前试验，以确定疫苗的效力、保护持续时间、最佳给药方案、其对同源与异源病毒株的保护效力、其预防感染和肝炎的能力以及分别有效预防感染和肝炎的最低抗体滴度。在临床试验中，三剂疫苗预防戊型肝炎的有效性为96%，两剂疫苗的有效性为87%。 这种疫苗没有明显的副作用。 对于疫苗来说，这些结果非常出色。 葛兰素史克目前正在确定其未来生产和分销疫苗的计划。 然而，在2009财政年度，这些计划的进展并不明显，NIAID正在探索戊型肝炎疫苗知识产权许可的其他选择。 几家亚洲制药公司表示有兴趣生产戊型肝炎疫苗，可能与甲型肝炎疫苗结合使用。 HCV的研究，包括疫苗开发，由于病毒的遗传异质性而变得复杂，这导致至少六种主要基因型和许多亚型。 检测和定量病毒需要用于PCR扩增的不同引物组。 我们已经开发了六种主要基因型的实时PCR检测方法，并将其特异性和灵敏度与其他已发表的检测方法进行了比较。 此外，在2009财年，我们通过为每种基因型制备黑猩猩来源的血浆池，并通过反向滴定法在其他黑猩猩中测定它们的感染性滴度，进一步表征了HCV的六种主要基因型。 我们将把这些样本库分发给科学界进行疫苗研究，并预计它们将有助于进一步开发丙型肝炎疫苗。 作为这些研究的延伸，并作为进一步对HCV的基础研究的辅助，我们正在制备这些HCV基因型的感染性cDNA克隆，这些克隆是不可用的（基因型3，4，5和6）。 这些研究是与Jens Bukh博士和William Satterfield博士合作进行的。 对HCV的免疫力知之甚少，并且对先前暴露于病毒的免疫力的完整性和持久性存在争议。 这对预防慢性感染及其后遗症、肝硬化和肝癌具有重要意义。 我们已经证明，对重复暴露于HCV的免疫是基因型特异性的，不完全的和相对短暂的：即使再次暴露于同源病毒也可能导致持续感染。 在其他研究中，进一步研究了GB病毒-B（GBV-B）免疫。GBV-B是丙型肝炎病毒的近亲，丙型肝炎病毒很难研究，因为它只传播给黑猩猩。相比之下，GBV-B可传播给绢毛猴，一种不被认为是濒危的新世界猴。先前感染过GBV-B的绢毛猴再次受到相同病毒的攻击，并表现出对再次感染的免疫力;这种免疫力是长期存在的，显然比黑猩猩和人类对HCV的免疫力更完整。 在2009财政年度进一步表征HCV和GBV-B感染之间相似性的其他研究中，我们检查了绢毛猴急性自限性与慢性GBV-B感染的突变率，并证明了与HCV感染中观察到的相似的遗传变化。 迄今为止，在慢性GBV-B感染中尚未观察到肝癌，可能是因为慢性感染非常罕见，但我们将监测任何其他慢性感染动物的肝癌证据。 2008年，与迈克尔霍顿（前凯龙公司）合作，我们在黑猩猩中测试了来自Chirons HCV疫苗试验的血清。具体地，我们测试了来自接受了从哺乳动物细胞表达的Chirons E1-E2二聚体疫苗（衍生自HCV-1菌株）的黑猩猩的系列血清。 使用携带各主要HCV基因型（HCVpp）包膜糖蛋白的假型逆转录病毒颗粒进行检测。 针对携带异源基因型1a株（H77 C）包膜糖蛋白的HCVpp的抗体应答的幅度与黑猩猩的保护程度成正比：具有高滴度针对HCVpp的中和抗体的动物受到保护，而具有较低滴度的动物则没有。 此外，中和抗体具有广泛的交叉反应性，也主要中和携带HCV基因型4a、5a和6a的包膜糖蛋白的HCVpp以及携带基因型1a的包膜糖蛋白的HCVpp（手稿在制备中）。 我们还在黑猩猩中测试了由表达的HCV E1蛋白组成的HCV疫苗，无论是否表达HCV NS 3蛋白。 该疫苗由Innogenetics N. V.生产，并根据与该公司的合作研究和开发协议（CRADA）进行了测试。 与Chiron疫苗的经验相反，没有一只黑猩猩受到感染或持续感染的保护，并且根据HCVpp测试的测量，没有一只黑猩猩具有HCV中和抗体。 这项研究的决定性结果导致Innogenetics放弃了E1疫苗。

The HVS in collaboration with GlaxoSmithKline (GSK), Rixensart, Belgium, has developed several candidate live attenuated HAV vaccines. One such candidate was modified to become the currently licensed GSK inactivated HAV vaccine. In addition, the HVS has developed a candidate recombinant hepatitis E vaccine that is highly promising and that has recently completed clinical trials. In studies to further characterize this candidate hepatitis E vaccine, we have performed extensive pre-clinical trials to determine the potency of the vaccine, the duration of protection, the optimum regimen for administration, its protective efficacy against homologous versus heterologous virus strains, its ability to prevent infection as well as hepatitis and the minimum antibody titer that was effective in preventing infection and hepatitis, respectively. In the clinical trial, the vaccine was 96% efficacious in preventing hepatitis E following three doses of vaccine and 87% efficacious following two doses. The vaccine had no detectable side effects. These results are outstanding for a vaccine. GSK is currently determining its plans for future manufacture and distribution of the vaccine. However, in FY 2009, progress in such plans have not been apparent and the NIAID is exploring other options for licensing the intellectual property for a hepatitis E vaccine. Several Asian pharmaceutical companies have expressed an interest in making a hepatitis E vaccine, perhaps in conjunction with a hepatitis A vaccine. The study of HCV, including vaccine development, is complicated by the genetic heterogeneity of the virus, which has resulted in at least six major genotypes and many subtypes. Detecting and quantifying the virus requires different sets of primers for PCR amplification. We have developed real-time PCR assays for the six major genotypes and have compared their specificity and sensitivity with other published assays. In addition, in FY 2009, we further characterized the six major genotypes of HCV by preparing chimpanzee-derived plasma pools for each and determining the infectivity titer of them by reverse titration in additional chimpanzees. We will distribute these pools to the scientific community for vaccine studies and anticipate that they will be useful in furthering hepatitis C vaccine development. As an extension of these studies and as an aid to furthering basic research on HCV, we are preparing infectious cDNA clones of those HCV genotypes for which such clones are not available (genotypes 3, 4, 5 and 6). These studies are being performed in collaboration with Dr. Jens Bukh and Dr. William Satterfield. Immunity to HCV is poorly understood and there is controversy over how complete and how long-lasting immunity to prior exposure of the virus is. This has implications for preventing chronic infections and their sequelae, cirrhosis and liver cancer. We have demonstrated that immunity to repeated exposure to HCV is genotype-specific, incomplete and relatively short-lived: even reexposure to the homologous virus can result in persistent infection. In other studies, GB virus-B (GBV-B) immunity was further studied. GBV-B is the closest relative to hepatitis C virus, which is very difficult to study because it is transmissible only to chimpanzees. In contrast, GBV-B is transmissible to tamarins, a species of New World monkey not considered to be endangered. Tamarins that had previously been infected with GBV-B were re-challenged with the same virus and shown to be immune to reinfection; this immunity was long-lived and apparently more complete than immunity to HCV in chimpanzees and humans. In additional studies to further characterize the similarity between infections by HCV and by GBV-B, in FY 2009, we have examined the mutation rate of acute, self-limiting versus chronic GBV-B infections in tamarins and demonstrated genetic changes similar to those observed in HCV infections. To date, liver cancer has not been observed in chronic GBV-B infections, perhaps because chronic infections are so uncommon, but we will monitor any additional chronically infected animals for evidence of liver cancer. In 2008, in collaboration with Michael Houghton (formerly of Chiron Corp.), we tested sera from Chirons HCV vaccine trials in chimpanzees, Specifically, we tested serial sera from chimpanzees that had received Chirons E1-E2 dimer vaccine (derived from the HCV-1 strain) that had been expressed from mammalian cells. Testing was performed with pseudo-typed retrovirus particles bearing the envelope glycoproteins of each of the major HCV genotypes (HCVpp). The magnitude of the antibody response against HCVpp bearing the envelope glycoproteins of a heterologous genotype 1a strain (H77C) paralleled the degree of protection of the chimpanzees: those animals with a high titer of neutralizing antibody against HCVpp were protected, whereas those with a lower titer were not. In addition, the neutralizing antibody was broadly cross-reactive, also neutralizing principally HCVpp bearing envelope glycoproteins of HCV genotypes 4a, 5a and 6a as well as those bearing the envelope glycoproteins of genoytpe 1a (manuscript in preparation). We also tested in chimpanzees an HCV vaccine consisting of expressed HCV E1 protein, either with or without expressed HCV NS3 protein. The vaccine was manufactured by Innogenetics N.V. and was tested under a Cooperative Research and Development Agreement (CRADA) with the company. In contrast to the experience with the Chiron vaccine, none of the chimpanzees was protected from infection or from persistent infection and none had neutralizing antibody to HCV as measured with the HCVpp test. The decisive results of this study led to the abandonment of the E1 vaccine by Innogenetics.