GP350 AS A NOVEL VACCINE PROTEIN CARRIER

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 目前需要一种针对EB病毒（EBV）感染具有保护性的疫苗。这种疫苗将对传染性单核细胞增多症、霍奇金和非霍奇金淋巴瘤、鼻咽癌和淋巴组织增生综合征的发病率产生潜在的全球影响。EBV蛋白，gp350具有满足这一需求的潜力。因此，1）gp350与B细胞和滤泡树突细胞（FDC）上的补体受体2型（CD21）结合，并因此可以作为有效的佐剂，2）gp350是抗体介导的EBV中和的主要靶，3）PS骨架上gp350的多聚化本身将增强抗gp350应答，和4）gp350是免疫原性外源蛋白，其将募集CD4 + T细胞帮助诱导抗gp350抗体。在发展中国家，EBV感染发生在婴儿群体中。相反，由于发达国家的EBV感染高峰发生在青少年中，因此gp350也可用作后者人群的抗EBV疫苗。 在初步研究中，我们有：1）表达并纯化gp350分子的重组糖基化N-末端72kDa片段，2）将gp350的多个拷贝缀合至肺炎球菌荚膜多糖血清型14（PPS 14）[PPS 14-gp350]，3）证明PPS 14-gp350特异性结合在恒河猴和人而非鼠上表达的CD21的能力，B细胞，和4）在年轻成年恒河猴中诱导加强的血浆IgG抗PPS 14和IgG抗gp 350抗体。用吸附在明矾上的少至0.05mg PPS 14-gp350免疫。然而，对数据的仔细分析和血浆样品的EBV中和能力使我们假设含有强T细胞表位的新的基因工程多聚体gp350将是上级疫苗。 本提案的目的是扩展上述研究，以确定使用恒河猴，一种新的基因工程多聚体gp350作为一种高效EBV疫苗的潜在能力。这些临床前研究将成为直接进入人体临床试验的基础。在本提案中，我们将利用年轻的成年恒河猴： 直接比较两种不同剂量的明矾吸附的单体或遗传工程化的多聚体gp350的能力， a. 通过ELISA测定，引发一级、二级和三级血浆IgG抗gp350滴度并诱导亲和力成熟。 B. 通过体外B细胞转化和ELISA中和试验确定诱导EB病毒中和抗体。 C. 如通过体外再刺激测定所确定的，引发特异性CD4 + T细胞。 总的来说，这些研究旨在建立非人灵长类动物中的原理验证，相对于单体gp350，临床上使用gp350作为EBV的保护性抗体疫苗。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There currently exists a need for a vaccine that is protective against Epstein-Barr virus (EBV) infection. Such a vaccine would have a potential global impact on the incidence of infectious mononucleosis, Hodgkin's and non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and lymphoproliferative syndrome. The EBV protein, gp350 has the potential to meet this need. Thus, 1) gp350 binds to the complement receptor type 2 (CD21) on B cells and follicular dendritic cells (FDCs) and thus may act as a potent adjuvant, 2) gp350 is the main target for antibody-mediated EBV neutralization, 3) multimerization of gp350 on a PS backbone will itself enhance anti-gp350 responses, and 4) gp350 is an immunogenic foreign protein that will recruit CD4+ T cell help for induction of anti-gp350 antibody. In the developing world infection with EBV, occurs in the infant population. Since, in contrast, peak EBV infection in the developed world occurs in adolescents, gp350 would also be of use as an anti-EBV vaccine in this latter population. In preliminary studies we have: 1) expressed and purified a recombinant glycosylated N-terminal 72kDa fragment of the gp350 molecule, 2) conjugated multiple copies of gp350 to pneumococcal capsular polysaccharide, serotype 14 (PPS14) [PPS14-gp350], 3) demonstrated the ability of PPS14-gp350 to specifically bind to CD21 expressed on rhesus and human, but not murine, B cells, and 4) induced boosted plasma IgG anti-PPS14 and IgG anti-gp350 antibodies in young adult rhesus monkeys following i.m. immunization with as little as 0.05 mg of PPS14-gp350 adsorbed on alum. However, careful analysis of the data, and EBV neutralizing ability of the plasma samples led us to hypothesize that a new, genetically engineered multimeric gp350 containing strong T cell epitopes would be a superior vaccine. The goal of this proposal is to extend the above studies to determine, using rhesus monkeys, the potential ability of a new genetically engineered multimeric gp350 to serve as a highly potent EBV vaccine . These pre-clinical studies will form the basis for progressing directly to human clinical trials. In this proposal we will utilize young adult rhesus monkeys to: Directly compare the ability of two distinct doses of alum-adsorbed monomeric or genetically engineered multimeric gp350 to a. elicit primary, secondary, and tertiary plasma IgG anti-gp350 titers and induce affinity maturation as determined by ELISA. b. induce EBV-neutralizing antibody as determined by both an in vitro B cell transformation and ELISA neutralization assay. c. prime specific CD4+ T cells as determined by an in vitro re-stimulation assay. Collectively, these studies are designed to establish a proof-of-principle in non-human primates, for using gp350 clinically as a protective, antibody-based vaccine for EBV relative to monomeric gp350.