Laboratory And Preclinical Studies Of Dengue Viruses

登革热病毒的实验室和临床前研究

• 批准号: 7303880
• 负责人: Brian R. Murphy
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7303880
• 关键词: Laboratory; Preclinical; Studies; Dengue; Viruses

The mosquito-borne dengue (DEN) viruses, members of the Flaviviridae family, contain a single-stranded positive-sense RNA genome. A single polypeptide is co-translationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and at least seven non-structural proteins. The genome organization of the DEN viruses is 5?-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3? (UTR ? untranslated region, C ? capsid, prM ? membrane precursor, E ? envelope, NS ? nonstructural). There are four dengue virus serotypes (DEN1, DEN2, DEN3, and DEN4) that circulate in tropical and subtropical regions of the world inhabited by more than 2.5 billion people. Annually, there are an estimated 50-100 million dengue infections and hundreds of thousands of cases of the more severe and potentially lethal dengue hemorrhagic fever/shock syndrome (DHF/DSS) with children bearing much of the disease burden. DEN viruses are endemic in at least 100 countries and cause more human disease than any other mosquito-borne virus. In at least eight Asian countries, the DEN viruses are a leading cause of hospitalization and death in children. Unfortunately, many countries affected by DEN viruses have very limited financial resources for healthcare, and the economic burden of DEN disease is considerable. As such, an economical vaccine that prevents disease caused by the DEN viruses is a global public health priority. The cost-effectiveness, safety, long-term immunity, and efficacy associated with the live attenuated vaccine against yellow fever virus, another mosquito-borne flavivirus, serves as a model for the feasibility of a live attenuated DEN virus vaccine. However, the development of a live attenuated dengue vaccine has been complicated by several factors. First, it has been difficult to develop monovalent vaccines against each of the four serotypes that exhibit a satisfactory balance between attenuation and immunogenicity. Second, an effective live attenuated dengue virus vaccine must consist of a tetravalent formulation of components representing each serotype because multiple serotypes typically co-circulate in a region, each DEN serotype is capable of causing disease, and the introduction of additional serotypes is common. In addition, the association of increased disease severity (DHF/DSS) in previously infected persons undergoing an infection by a different dengue serotype necessitates a vaccine that will confer long-term protection against all four serotypes. Third, it has been difficult to formulate a tetravalent vaccine with low reactogenicity that induces a broad neutralizing antibody response against each DEN serotype. Fourth, a dengue vaccine must confer protection against a wide range of genetically diverse subtypes that are dispersed around the world and can be readily introduced into a new region by international travel. Fifth, a dengue vaccine must be produced economically so that it can be made available to populations that need it most. We have tried to address these issues as part of a program to generate a live attenuated tetravalent dengue virus vaccine. To maximize the likelihood that suitable vaccine candidates would be identified, monovalent vaccine candidates for DEN1-4 were generated by two distinct recombinant methods and found to be attenuated and immunogenic in mouse and rhesus monkey models. In one method, deletion of 30 contiguous nucleotides from the 3? UTR of wild type cDNA clones of DEN1-4 was used to generate vaccine candidates. Specifically, the deletion of nucleotides 10478-10507 of the 3? UTR (del30) of recombinant wild type DEN4 yielded a vaccine candidate, rDEN4del30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans (9). Incorporation of the del30 mutation into infectious cDNA clones of DEN1, but not DEN2 or DEN3, at a site homologous to that in DEN4 attenuated these viruses for rhesus monkeys. Using a second method, antigenic chimeric viruses were generated by replacing wild type M and E structural genes of rDEN4del30 with those from DEN2 or DEN3, and the resulting chimeric viruses were attenuated and immunogenic in rhesus monkeys. Importantly, these vaccine candidates retain wild type structural proteins to maximize infectivity, thereby decreasing the potential for virus interference. In addition, immunity is induced by an authentic wild type E protein that will likely increase the magnitude and breadth of the neutralizing antibody response. Last year we described the successful evaluation of three formulations of a tetravalent vaccine in rhesus monkeys. This year we have focused our efforts on bringing vaccine candidates for each of the serotypes to clinical trials in humans and on the further development of additional attenuating mutants for each serotype to serve as backup candidates in event that the current candidates do not achieve an acceptable balance between attenuation in humans. These efforts include: Studies with DEN1 candidates: Vaccines and INDs Vaccine lot of DEN1del30 manufactured by Charles River Laboratories ? In progress. IND to be filed for this vaccine lot in September 2006 New Vaccine Candidates Two additional attenuated vaccine candidates of DEN1 have been generated and are in the process of evaluation in experimental animals: one is the DEN1del30 virus containing two additional attenuating mutations and the other is an DEN1/4del30 antigenic chimeric virus. Studies with DEN2 candidates: Vaccines and INDs Vaccine lot of DEN2/4del30 manufactured by Charles River Laboratories ? In progress. IND to be filed for this vaccine lot in October 2006. New Vaccine Candidates Two additional attenuated vaccine candidates of DEN2 have been generated and are in the process of evaluation in experimental animals: one is the DEN2/4del30 virus containing two additional attenuating mutations and the other is an attenuated derivative of DEN2. These are currently in preclinical studies. Studies with DEN3 candidates: Vaccines and INDs Vaccine lot of DEN3/4del30 manufactured by Charles River Laboratories - completed. IND to be filed for this vaccine lot in August 2006. New Vaccine Candidates Four additional attenuated vaccine candidates of DEN2 have been generated and are in the process of evaluation in experimental animals. These are attenuated derivatives of DEN3 possessing all proteins of DEN3, i.e., they are not chimeric viruses. These are currently in preclinical studies. Studies with DEN4 candidates: Vaccines and INDs Two vaccine lots of DEN4del30 containing additional attenuating mutations have been manufactured by Charles River Laboratories. Two INDs have been filed for these vaccine candidates and FDA approval for each have been received. A clinical trial with one has been initiated and a trial for the other is planned to initiate in September 2006.

蚊子传播的登革热（DEN）病毒是Flaviviridae家族的成员，其中包含一个单链的阳性RNA基因组。单个多肽是通过生成三种结构蛋白（C，M和E）和至少七个非结构性蛋白质的病毒和细胞蛋白酶共译的。 DEN病毒的基因组组织是5？-UTR-C-PRM-E-NS1-NS2A-NS2B-NS3-NS3-NS4A-NS4B-NS4B-NS5-UTR-3？ （UTR？未翻译的区域，C？capsid，prm？膜前体，e？信封，ns？nonstructural）。有四种登革热病毒血清型（DEN1，DEN2，DEN3和DEN4）在世界的热带和亚热带地区流通，有超过25亿人居住。每年，估计有50-1亿登革热感染，成千上万病例较为严重，可能致命的登革热大量发烧/休克综合征（DHF/DSS），儿童承受着许多疾病负担。在至少100个国家中，DEN病毒是地方性的，并且与任何其他蚊子传播病毒相比，引起更多的人类疾病。在至少八个亚洲国家中，DEN病毒是儿童住院和死亡的主要原因。不幸的是，许多受DEN病毒影响的国家的医疗保健财政资源非常有限，而DEN疾病的经济负担相当大。因此，阻止DEN病毒引起的疾病的经济疫苗是全球公共卫生的重点。 与蚊子 - 传播黄病毒有关的活疫苗与黄热病病毒相关的成本效益，安全性，长期免疫力和功效，是一种模型，可用于实时衰减DEN病毒疫苗的可行性。但是，多种因素使登革热疫苗的活衰减疫苗的发展变得复杂。首先，对于在衰减和免疫原性之间表现出令人满意的平衡的四种血清型中的每一种都很难开发单价疫苗。其次，有效的活死登革热病毒疫苗必须由代表每种血清型的成分的四价配方组成，因为多种血清型通常在一个区域中共同循环，每种DEN血清型都能引起疾病，并且引入其他血清型是常见的。此外，疾病严重程度增加（DHF/DSS）在先前感染的人受到不同登革热血清型感染中的关联需要一种疫苗，这将对所有四种血清型进行长期保护。第三，很难制定具有低反应生成性的四价疫苗，从而诱导针对每种DEN血清型的广泛中和抗体反应。第四，登革热疫苗必须赋予全世界分散的各种遗传多样的亚型的保护，并可以通过国际旅行很容易地引入一个新地区。第五，必须在经济上生产登革热疫苗，以便可以将其提供给最需要它的人群。 我们试图解决这些问题，作为一项计划的一部分，以生成活死的四龙病毒疫苗。为了最大程度地确定合适的候选疫苗的可能性，通过两种不同的重组方法生成了DEN1-4的单价疫苗候选物，并发现在小鼠和恒河猴模型中被衰减和免疫原性。在一种方法中，从3中删除了30个连续的核苷酸？ DEN1-4的野生型cDNA克隆的UTR用于生成候选疫苗。具体而言，3的核苷酸10478-10507的缺失？重组野生型DEN4的UTR（DEL30）产生了疫苗候选者RDEN4DEL30，该疫苗在恒河猴和人类中安全，衰减和免疫原性（9）。将DEL30突变掺入DEN1的传染性cDNA克隆中，而不是DEN2或DEN3，在与Den4中同源的位点掺入了这些病毒的恒河猴。使用第二种方法，通过用DEN2或DEN3的野生型M和E结构基因代替RDEN4DEL30的野生型M和E结构基因来产生抗原性嵌合病毒，并将所得的嵌合病毒衰减并在恒河猴中进行免疫原性。重要的是，这些候选疫苗保留了野生型结构蛋白，以最大程度地提高感染力，从而降低了病毒干扰的潜力。另外，免疫力是由真实的野生型E蛋白诱导的，该蛋白可能会增加中和抗体反应的大小和广度。 去年，我们描述了对恒河猴四甲疫苗疫苗的三种制剂的成功评估。今年，我们将努力集中在将每种血清型的疫苗候选者带到人类的临床试验中，并进一步开发每种血清型的额外衰减突变体的额外衰减突变体，以作为当前候选人在人类减弱之间可接受的平衡的情况下作为备用候选者。这些努力包括： 与DEN1候选人的研究： 疫苗和IND Charles River Laboratories生产的DEN1DEL30的疫苗？进行中。 IND将于2006年9月申请该疫苗。 新的疫苗候选人 已经产生了另外两个DEN1候选疫苗的疫苗，并且正在实验动物的评估过程中：一种是DEN1DEL30病毒，其中含有两个额外的衰减突变，另一个是DEN1/4DEL30抗原嵌合病毒。 与DEN2候选人的研究： 疫苗和IND Charles River Laboratories生产的DEN2/4DEL30的疫苗很多？进行中。 IND将于2006年10月为该疫苗申请。 新的疫苗候选人 已经产生了另外两个DEN2候选疫苗的疫苗，并且正在实验动物的评估过程中：一种是DEN2/4DEL30病毒，其中包含两个额外的衰减突变，另一种是DEN2的衰减衍生物。这些目前正在临床前研究中。 与DEN3候选人的研究： 疫苗和IND Charles River Laboratories生产的DEN3/4DEL30的疫苗 - 完成。 IND将于2006年8月向该疫苗申请。 新的疫苗候选人 DEN2的另外四个候选疫苗候选物已产生，并正在实验动物中进行评估。这些是具有DEN3的所有蛋白质的DEN3的减弱衍生物，即它们不是嵌合病毒。这些目前正在临床前研究中。 与DEN4候选人的研究： 疫苗和IND 查尔斯·河实验室（Charles River Laboratories）生产了两种含有其他衰减突变的DEN4DEL30的疫苗。 已经为这些候选疫苗提交了两个IND，并已收到了FDA的批准。已经开始了一项临床试验，并计划在2006年9月开始对另一个试验进行试验。