Novel Carrier for Polysaccharide Conjugates and an EBV Vaccine

描述(由申请人提供)：目前需要新的免疫原性载体蛋白用于新的抗细菌多糖(PS)结合疫苗，以及一种对EB病毒(EBV)感染具有保护作用的疫苗。后一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、鼻咽癌和淋巴增殖综合征的发病率产生潜在的全球影响。EBV蛋白gp350是EBV中和抗体的主要靶点，也是CD21的配体，CD21是B细胞抗原受体的有效辅助激活因子。CD21也由滤泡树突状细胞表达，在那里它介导抗原捕获，这对生发中心反应的诱导很重要。因此，多聚体gp350-PS结合物形式的gp350既有可能作为PS特异性结合疫苗的有效载体蛋白，也有可能作为EBV抗体介导的疫苗。在初步研究中，我们已经：1)表达并纯化了gp350分子N端72 kDa的重组片段，2)将多个拷贝的gp350与肺炎链球菌囊膜多糖14(PPS14)[PPS14-gp350]结合，3)证实了PPS14-gp350能够与表达于恒河猴和人而不是小鼠B细胞的CD21特异性结合，以及4)诱导免疫增强的年轻成年恒河猴血浆中抗PPS14的抗体和抗gp350的抗体。仅0.05 mg的PPS14-gp350吸附在明矾上进行免疫。这项建议的目的是在非人类灵长类动物中建立一种原则证明，将gp350用于临床，作为PS结合疫苗的新型组合载体蛋白，以及作为EBV的保护性抗体疫苗。在这项建议中，我们将利用年轻的成年恒河猴来：1)直接比较明胶吸附的未结合或PPS14结合的单体或非结合二聚体gp350诱导高滴度和高亲和力的EBV中和抗gp350抗体和gp350特异性T细胞激发的能力，以及2)直接比较与PPS14结合的白喉毒素(DT)[已确定的结合疫苗的载体蛋白]与PPS14结合的单体gp350诱导高滴度和高亲和力的保护性抗PPS14抗体的能力，并确定CD21结合在gp350作为PSP14载体蛋白佐剂可变性中的作用。这些临床前研究将为直接进入人体临床试验奠定基础。目前，还没有针对EB病毒(EBV)的预防性疫苗，EBV与传染性单核细胞增多症、鼻咽癌、Burkitt淋巴瘤、非霍奇金淋巴瘤和免疫抑制患者的淋巴增殖综合征的发病机制有关。此外，由于交叉抑制现象，还需要用于多糖结合疫苗的新型蛋白载体，其诱导抗体介导的对胞外细菌的保护。这项拟议的研究以恒河猴为活体模型，将确定使用EBV包膜蛋白gp350作为基于多糖的结合疫苗的新载体和有效抗体介导的EBV感染预防性疫苗的靶抗原的可行性。

DESCRIPTION (provided by applicant): There currently exists a need for novel, immunogenic carrier proteins for new anti-bacterial polysaccharide (PS) conjugate vaccines, as well as a vaccine that is protective against Epstein-Barr virus (EBV) infection. The latter 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, is the major target for EBV neutralizing antibody, as well as a ligand for CD21, a potent co-activator of the B cell antigen receptor. CD21 is also expressed by follicular dendritic cells where it mediates antigen trapping, important for induction of the germinal center reaction. Thus, gp350 in the form of a multimeric gp350-PS conjugate, has the potential to serve as both a potent carrier protein for PS-specific conjugate vaccines as well as an antibody-mediated vaccine for EBV. 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. The goal of this proposal is to establish a proof-of-principle in non-human primates, for using gp350 clinically, as a combined novel carrier protein for PS conjugate vaccines and as a protective, antibody-based vaccine for EBV. In this proposal we will utilize young adult rhesus monkeys to: 1) directly compare the ability of alum-adsorbed unconjugated or PPS14-conjugated monomeric or unconjugated dimeric gp350 to elicit high titer and high affinity, EBV-neutralizing anti-gp350 antibody and gp350-specific T cell priming, and 2) directly compare the ability of diphtheria toxoid (DT) [an established carrier protein for conjugate vaccines], conjugated to PPS14, with PPS14- conjugated monomeric gp350 to elicit high titer and high affinity, protective anti-PPS14 antibody, and to determine the role of CD21 binding in the adjuvanticity of gp350 as a carrier protein for PPS14. These pre-clinical studies will form the basis for progressing directly to human clinical trials. Currently, there is no prophylactic vaccine for the Epstein-Barr virus (EBV), which is implicated in the pathogenesis of infectious mononucleosis, nasopharyngeal carcinoma, Burkitt lymphoma, non-Hodgkin's lymphoma, and lymphoproliferative syndrome in immunosuppressed patients. Further, there is also a need for novel protein carriers for polysaccharide conjugate vaccines, which elicit antibody-mediated protection against extracellular bacteria, due to the phenomenon of cross-inhibition. The proposed studies, using the rhesus macaque as an in vivo model, will determine the feasibility of using the EBV envelope protein, gp350, an intrinsically immunostimulatory molecule, as a combined novel carrier for polysaccharide-based conjugate vaccines and as a target antigen for a potent antibody-mediated prophylactic vaccine against EBV infection.