Novel Carrier for Polysaccharide Conjugates and an EBV Vaccine

描述（由申请人提供）：目前存在对新型抗菌多糖（PS）结合疫苗的新型免疫原性载体蛋白的需求，以及对eb病毒（EBV）感染的保护疫苗的需求。后一种疫苗将对传染性单核细胞增多症、霍奇金淋巴瘤和非霍奇金淋巴瘤、鼻咽癌和淋巴细胞增生性综合征的发病率产生潜在的全球影响。EBV蛋白gp350是EBV中和抗体的主要靶标，也是CD21的配体，CD21是B细胞抗原受体的有效共激活剂。CD21也在滤泡树突状细胞中表达，介导抗原捕获，对诱导生发中心反应很重要。因此，gp350以多聚体gp350- ps偶联物的形式，具有作为ps特异性偶联疫苗和EBV抗体介导疫苗的有效载体蛋白的潜力。在初步研究中，我们有：1)表达并纯化了gp350分子的重组糖基化n端72kDa片段，2)将gp350的多个拷贝偶联到肺炎球菌荚膜多糖血清型14 (PPS14 -gp350) [PPS14-gp350], 3)证明了PPS14-gp350能够特异性结合在恒河猴和人（但不包括小鼠）B细胞上表达的CD21。4)在明矾上吸附0.05 mg的PPS14- gp350免疫后，诱导年轻成年恒河猴血浆IgG抗PPS14和IgG抗gp350抗体增强。该提案的目标是在非人类灵长类动物中建立原理验证，以便在临床上使用gp350作为PS结合疫苗的新型组合载体蛋白和EBV保护性抗体疫苗。在这个提议中，我们将利用年轻的成年恒河猴：1)直接比较铝吸附的未偶联或PPS14偶联的单体或未偶联的二聚体gp350引发高滴度和高亲和力的ebv中和性抗gp350抗体和gp350特异性T细胞启动的能力；2)直接比较PPS14偶联的白喉类毒素（DT）[一种已建立的结合疫苗载体蛋白]与PPS14偶联的单体gp350引发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PPS14载体蛋白的佐剂性中的作用。这些临床前研究将成为直接进行人体临床试验的基础。eb病毒与感染性单核细胞增多症、鼻咽癌、伯基特淋巴瘤、非霍奇金淋巴瘤和免疫抑制患者的淋巴细胞增生性综合征的发病机制有关，目前尚无针对eb病毒的预防性疫苗。此外，由于交叉抑制现象，多糖结合疫苗还需要新的蛋白质载体，以引起抗体介导的对细胞外细菌的保护。拟建的研究以恒河猴为体内模型，将确定使用EBV包膜蛋白gp350（一种内在免疫刺激分子）作为基于多糖的结合疫苗的新型组合载体和作为有效抗体介导的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.