金葡菌非蛋白成份PGN及LTA模拟肽疫苗候选的保护作用及其机制研究

Staphylococcus aureus (S.aureus) is an important pathogen as the leading cause of community-associated and nosocomial infections in human and animals. Due to the increasing the emergence of methicillin-resistant S.arueus (MRSA) and the MRSA infection, the immunotherapy and prophylaxis have been attracted great attention. Only a few potential vaccines for S.aureus eliciting humoral immune response have been in clinical trials, but none has been approved for clinical application. Peptidoglycon (PGN) and lipoteichoicacid (LTA) are two common and conserved components of the cell wall of many Gram-positive including S.aureus, however these components, as thymus-independent antigen (TI-Ag), can never be considered as vaccine candidates due to their very weak immunogenicity. .In this project , we will attempt to convert the TI-Ag antigenicity of PGN and LTA to be TD-Ag (thymus-dependent antigen) antigenicity by peptide mimic strategy. Particularly, the four or eight-branched multiple-antigen peptides (MAP) will be well designed with different Th epitopes and ajuvant sequence based on peptide mimics to PGN and LTA. The protection against acute and chronical infection in mice with S.aureus will be observed, and secondary humoral immune response, especially T cell mediated immune response will be studied. .In our previous work, several conservative sequences of peptide mimics to PGN were obtained from 12-mer phage displayed peptide library using a mAb against to S.aureus PGN, and a four-branched MAP named as MAP-P31 was synthesized. Immunization of mice with MAP-P31 was able to provoke an effect secondary IgG-type antibody response to PGN and the whole cell lysate of S.aureus, and significantly prolonged the survival and enhanced the bacteria clearance in BLAB/C mice challenged with live S.aureus, demonstrating that MAP-P31, as a peptide mimics to epitope on PGN, can elicit secondary immune response effectively. Most importantly, two MAPs mimic to PGN could stimulate spleen cells from BALB/C mice to produce IFNγ and IL-2，indicating that these peptide mimics vaccine induce succesively T cell effect function, i.e Th1 effect..The significance of this project are as follows ① MAPs mimic to PGN and LTA epitopes are the new attempt for S.aureus vaccine candidates. ② To convert TI-antigen character of PGN and LTA to TD antigen may provok effective Th1/Th17 cell mediated response and secondary antibody response.③ To reveal the regularity of immune response under the application of peptide mimic vaccine and native S.aureus in the same body. ④ It would be expected to accelarete this vaccine candidate used in clinical trial since the carrier protein would not be required for MAP vaccine.

已知金葡菌耐药情况严重，免疫学防治被寄予希望。迄今国外以诱导体液免疫为主的疫苗尚无一被批准应用。PGN与LTA是S.aureus细胞壁保守结构，但因免疫原性弱而不被作为疫苗候选。本项目拟采用短肽模拟PGN与LTA表位，设计多价抗原肽（MAP）并引入不同Th表位与佐剂序列；研究MAP诱导正常和急慢性感染小鼠的免疫应答及其机制。我们初步证实已合成的MAP可模拟PGN表位抗原性，诱导小鼠保护性反应，尤其可刺激小鼠脾细胞分泌IFNγ和IL-2，尚未见国内外同类报导。本项目意义：① 首次尝试以模拟PGN与LTA的MAP作为金葡菌疫苗候选；② 将PGN与LTA由TI抗原性质改变为TD抗原,可望诱导有效的再次抗体应答，尤其是Th1/Th17细胞免疫效应。该策略将为金葡菌疫苗研发另辟蹊径；③ 揭示模拟肽疫苗与金葡菌在同一反应体系中诱导再次免疫应答的规律。④ MAP无需载体蛋白的性质更利于临床实验与应用。

基于金葡菌耐药情况严重，免疫学防治被寄予希望。PGN和LTA是金葡菌的保守结构，但因为TI抗原而不被作为疫苗候选。本项目利用噬菌体肽库筛选技术获得模拟PGN和LTA表位的序列，根据阳性序列合成线性肽和多价抗原肽（MAP），检测合成肽的抗原性及免疫原性，模拟肽免疫后的保护作用及其机制。主要研究结果表明：1. 模拟PGN表位的四分支多价抗原肽MAP27能与抗PGN抗体结合；MAP27免疫小鼠并以灭活菌加强免疫后，诱导抗PGN抗体、抗金葡菌抗体产生。MAP27免疫能降低小鼠脏器中细菌残存量，并在金葡菌致死性攻击后显著延长小鼠生存期。金葡菌感染后，MAP27免疫鼠脾细胞中IFNγ+CD3+T细胞和IL-17+CD4+T细胞百分比明显增高，脾脏和肺脏中IFN-γ、IL-17和CCL3含量明显升高。体外实验表明，灭活菌能刺激MAP27免疫鼠脾细胞分泌IL-2、IFN-γ和IL-17。这一结果说明MAP27作为免疫原所发挥的保护作用与诱导T细胞应答相关。2. 经肽库筛选，获得模拟PGN表位的环肽序列。3. 经筛选肽库，获得一系列模拟LTA表位的序列，阳性序列HSV(G)HWDFRQWWQPSGGGS和HSGHKEDRQWCQHSGG已申请国家发明专利。4. 依据阳性序列GHKEDRQWCQHS合成的肽（命名为MAP2-3）能与抗LTA单抗、抗金葡菌多抗结合，MAP2-3免疫后诱导小鼠产生抗LTA的IgG类抗体，抗体类别鉴定显示MAP2-3免疫后主要诱导IgG1,IgG2a以及IgG2b类抗体，末次免疫后5周仍能检测到抗LTA抗体。MAP2-3免疫鼠能抵抗金葡菌致死性攻击，系统性感染模型中MAP2-3免疫鼠脾脏、肾脏、肺脏中细菌残存量显著降低。体外实验表明，LTA、灭活金葡菌能刺激MAP2-3免疫小鼠脾细胞分泌IL-2和IL-4。提示MAP2-3作为实验性疫苗能有效诱导免疫应答及免疫记忆。5. 依据阳性序列VHWDFRQWWQPS合成MAP3，MAP3能与抗LTA单抗结合，MAP3免疫后能降低感染小鼠组织中的细菌残存量。上述结果说明以TD抗原的MAP替代PGN和LTA作为疫苗候选，能有效诱导特异性再次免疫应答和免疫记忆，MAP免疫后发挥保护作用，这将为金葡菌疫苗研发提供新的思路和依据。