金葡菌肽聚糖模拟肽疫苗候选保护性作用的初步研究
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摘要
金黄色葡萄球菌(Staphylococcus aureus, S.aureus)是一种可寄居在人体皮肤和粘膜部位的革兰阳性菌(G+菌),是社区和院内感染的主要病原体。当各种原因导致免疫低下或缺陷、异位寄生、菌群失调时可引起如高死亡率的脓毒血症等严重后果。自20世纪60年代首次发现耐甲氧西林的菌株以来(MRSA),各种耐药菌株陆续出现,使临床用药面临着严峻的挑战；在应对上述挑战中,人们对兴起于20世纪60年代的金葡菌免疫防治寄予希望。但真正将免疫治疗、预防及应急预防用于临床仍有很多的难题需解决。目前国内外用于研发金葡菌疫苗的主要成份包括：金葡菌减毒活疫苗、荚膜多糖—蛋白连接疫苗(StaphVAX, PentaStaph)和毒素(a-hemolysin、PVL、TSST-1等)疫苗；葡糖胺(PNAG、PNSG)和表面蛋白(IsdA,IsdB,SdrD and SdrE)疫苗；和DNA疫苗FnBPA-CIfA等。虽部分被批准进入临床实验,但尚无一被批准临床应用。
肽聚糖(Peptidoglycan, PGN)是金葡菌细胞壁的重要成份,近年更是被称为是细菌的“阿喀琉斯之踵”,意为强者的致命弱点,是免疫防治的靶抗原。但至今未被作为疫苗候选靶点的主要原因在于：PGN为非蛋白抗原,即TI抗原(胸腺非依赖抗原),免疫原性弱,即便是感染个体其抗体滴度也不高。本研究尝试用短肽模拟PGN抗原表位,将其抗原性质由TI抗原改变为TD(胸腺依赖抗原)抗原,以诱导有效的再次免疫应答,探索以模拟肽作为疫苗候选的可能性。
已知,短肽可模拟蛋白与非蛋白(糖、脂等)表位,常用于构象性表位特性以及糖脂类抗原表位研究。近十余年来,已在研究病原生物及肿瘤的非蛋白成份方面做了大量探索,并可用于分析蛋白质折叠中形成非连续性表位的特点。合成肽疫苗的优势是对有效与非必要抗原表位的组合与取舍,而缺点则是免疫原性弱,需设计合适的载体与佐剂。但近年国内的多价抗原肽(Multiple Antigenic Peptide,MAP)技术有了长足的发展,其优势体现在：不需交联载体,可不用佐剂；除抗体反应,还可诱导CTL与Th细胞反应；尤其在不同的分枝上可有不同序列的肽链,可用于构成或模拟更为复杂或不同功能的表位。国外近年已有数个多价肽疫苗进入临床观察。
本课题的主要研究思路是：利用噬菌体肽库筛选获得的短肽模拟PGN的保守性结构表位,将其非蛋白抗原表位的性质改变为短肽,由此使TI-Ag改变为TD-Ag；并以所获模拟肽序列合成四分枝多价抗原肽(MAP)作为疫苗候选诱导机体内产生有效的再次抗体应答和保护性免疫。
本课题组用抗PGN商品化单抗从噬菌体随机展示肽库中筛选了十余个模拟位克隆,结合生物信息学分析选择三个PGN表位的序列进行修饰与合成,并进行体外间接ELISA与抑制ELISA鉴定,从中选择效果较好的SP31进行四分枝多价抗原肽,并对其免疫原性及免疫效果进行评价。
一、应用噬菌体展示技术筛选金葡菌PGN模拟肽克隆
从噬菌体线性肽库中筛选可模拟PGN表位的噬菌体克隆以商品化抗PGN单克隆抗体为靶分子对噬菌体线性12线性肽库进行了3轮筛选,富集率达为2千倍以上,显示了明显的富集效果；从第三轮随机挑选49噬菌体克隆中用夹心ELISA鉴定出可与抗PGN单克隆抗体结合的14个克隆。
阳性噬菌体测序及序列分析将上述14个阳性噬菌体克隆进行DNA测序,并对其氨基酸序列进行分析,其第31、28、43号克隆具有共同序列：ATWxHxLxSAGL,并与27、39、1序列有保守序列xHx或Hx,除噬菌体克隆1号外与anti-PGN McAb均有高反应性(OD450),第5、23、38、44虽具有共同序列GWWPYAALRALS,但多次重复后第5序列非特异性结合较高,可能含有吸板序列：因此,选择31,27,39三种噬菌体作为后续研究对象。
二、PGN模拟肽的设计、合成与体外鉴定
阳性克隆展示序列抗原特性分析通过在线软件www.syfpeithi.de, http://bio.dfci.harvard.edu/Tools/antigenic.pl, http://www.darrenflower.info/mhcpred预测和T细胞表位预测软件(DNAstar)分析发现,三种阳性噬菌体展示序列均具有人和鼠MHC结合表位；其中No.31可能的抗原表位为-TWxHxLx-序列,在两端分别加上SG-、GG后可能具有T细胞表位；No.27可能的抗原表位为—SPHxH000RS-,在两端加上SG/G GG的不同组合后均不具有T细胞表位；No.39可能的抗原表位为-WxHxVxW-;在两端加上不同SG/A GG氨基酸后可能具有T细胞表位,序列分析结果表明三种序列具有构成Th、B细胞表位的特性。但是否真正模拟PGN的抗原表位,还需进一步的生物实验验证。
PGN模拟肽抗原性鉴定体外ELISA鉴定结果表明,以生物素标记三种线性合成肽链(SP31,SP27,SP39)能特异结合抗PGN单抗及兔抗S.aureus全菌多抗(FSP31=1139.058, PSP31=0.000; FSP27=196.091, PSP27=0.000; FSP39=90.811,PSP39=0.000),其中SP31、SP27与抗PGN单抗的反应性优于抗S.aureus多抗,而SP39则反之；抑制实验表明,PGN能剂量依赖地抑制SP31、SP27与抗PGN单克隆抗体结合(FPGN抑制SP31=729.036,PPGN抑制SP31=0.027；FSP31抑制PGN=12.286,P SP31抑制PGN=0.039；FPGN抑制SP27=74.776,PPGN抑制SP27=0.001),抑制效果在40%-60%,而39号合成肽抑制效果较差,量效关系不显著(数据未在文中显示)。上述结果提示SP31,SP27,SP39线性肽可不同程度地模拟PGN表位的抗原性。基于SP31,SP27为模板合成四分枝多价抗原肽(MAP)鉴定表明,MAP-P31剂量依赖地与抗PGN单抗(F=2021.727,P=0.000)和抗S.aureus多抗反应(F=178.344,P=0.000),并能与PGN相互抑制与抗PGN McAb的反应(FPGN抑制MAP-P31=64.032,PPGN抑制MAP-P31=0.015,FMAP-P31抑制PGN=17.532,PMAP-P31抑制PGN=0.053),而MAP-P27仅与抗S.aureus多抗反应；由此我们以MAP-P31作后续的免疫原性研究。
PGN模拟肽对小鼠腹腔巨噬细胞(M9)刺激作用实验结果表明MAP-P31模拟肽能显著刺激小鼠腹腔巨噬细胞产生炎症因子TNFα、IL-6(FrNFα=1265.415,PTNFα=0.000；FIL_6=248.353,PIL-6=0.000),并呈一定的剂量依赖关系,但低于PGN刺激的细胞因子产生水平。该结果提示MAP-P31不但可模拟PGN表位的抗原性,亦可模拟其刺激巨噬细胞并产生细胞因子的生物学活性。
三、PGN抗原模拟肽MAP-P31主动免疫效果评价
不同免疫方式的效果评价以MAP-P31(无交联载体)为免疫原,采用不同免疫方案免疫小鼠,二次免疫后一周检测免疫血清效价。结果显示：MAP-P31加弗氏佐剂乳化组效果显著优于基础免疫组(弗氏佐剂提前一周注射,后续均用不含佐剂的MAP-P31进行免疫)、无佐剂组(P=0.001vs FCA prior to MAP-P31;P=0.005vsMAP-P31)。结果提示,MAP-P31免疫接种可以不需要交联其他的蛋白载体,但仍需佐剂。该条件用于后续实验。
抗血清免疫反应性评价合成肽MAP-P31加弗氏佐剂免疫小鼠的抗血清含可与PGN、MAP-P31以及S.aureus结合的IgG型抗体,并持续8周以上。提示MAP-P31可诱导小鼠产生了针对PGN和S.aureus的再次抗体应答与免疫记忆(FPGN=70.122,PPGN=0.000；FSaureus=379.255,PSaureus=0.000)。此外,抗MAP-P31抗血清尚可与金葡菌磷壁酸(LTA)有微弱反应,但与LPS不反应；并与表皮葡萄球菌(S.epidermidis)、大肠杆菌(E.coli)和铜绿假单胞菌(P. aeruginosus)的超声裂解物反应(P<0.05 vs anti-unrelated MAP),表现出一定的交叉反应性。提示MAP-P31可能模拟不同细菌PGN的共同或相似抗原表位。
抗血清的体外杀菌/抑菌及调理吞噬作用实验结果表明,在补体存在或补体灭活情况下,抗MAP-P31抗血清对甲氧西林敏感模式菌株(ATCC25923)均具有杀菌／抑制作用；并对肠球菌、人葡萄球菌、大肠杆菌、绿脓杆菌和嗜麦芽黄单胞菌均表现出一定的抑制／杀灭作用,然而对MRSA(ATCC43300)反而促进其生长作用,该结果提示血清杀菌作用及抗菌谱具有一定局限性,需进一步优化序列结构,以期能产生广谱的抗菌作用。研究结果亦证实在灭活补体后,抗MAP-P31血清仍能显著增强小鼠腹腔巨噬细胞对FITC标记的甲氧西林敏感模式菌株(ATCC25923)的吞噬作用(F=981.862,P=0.000)。
MAP-P31诱导的小鼠体内保护作用小鼠尾静脉注射亚致死剂量的甲氧西林敏感模式菌株(ATCC25923)后,MAP-P31免疫组小鼠肝、脾、肾等主要脏器中细菌载量显著低于无关MAP免疫对照组(Piver=0.018,Pspleen=0.015,Pkindey=0.020)；腹腔注射致死剂量的甲氧西林敏感模式菌株(ATCC25923)后,MAP-P31免疫组2周后的生存率为90%,而PBS免疫组和无关MAP免疫组小鼠生存率分别为0%和20%,提示免疫MAP-P31免疫后小鼠产生了针对甲氧西林敏感模式菌株(ATCC25923)的保护性免疫。
四、PGN模拟肽改造及其被动免疫效果评价
PGN模拟肽序列的改造鉴于MAP-P31免疫血清对MSSA菌杀菌作用明显,而对MRSA则有促进其生长作用,我们对其进行了序列改造的尝试。根据I)NAstar软件预测,我们在MAP-P31的两端延长序列中改变个别氨基酸使其形成Th细胞识别表位,合成四分枝多价抗原肽并命名为MAP-P31.1,以期获得对MRSA有杀伤/抑制作用的序列。
MAP-P31.1免疫反应性鉴定以此MAP-P31.1加弗氏佐剂为免疫原免疫家兔和Balb/C小鼠,5次免疫后,其免疫血清可结合胰酶消化后PGN(?)S.aureus,提示其可能针对的是PGN的多糖表位,该模拟肽与PGN有相同或相似的抗原表位。MAP-P31.1免疫后,用热灭活S.aureus做加强免疫可增强抗体效价10倍(P=0.002vs no boost),其可能的临床应用价值在于：用合成肽疫苗接种后,自然感染S.aureus可能发挥加强免疫的作用。
MAP-P31.1抗血清杀菌/抑菌作用抗MAP-P31.1小鼠血清在有/无补体作用下对MRSA与MSSA均有显著杀菌/抑菌作用(FMSSA(inactive complements)=16.490, PMSSA(inactive complements)=0.001;FMRSA (inactive complements)=13.213,PMRSA(inactive complements) =0.006;FMRSA (complements)=14.150,PMRSA(complements)=:0.005),且均显著优于anti-S.aureus多抗血清与抗MAP-P31血清(P<0.001vs anti-S.aureus组；t=-8.302,P=0.001)。
MAP-P31.1免疫兔IgG的被动保护作用与正常兔IgG、抗S.aureus多抗对照组比较,纯化抗MAP-P31.1 IgG抗体被动输入能延长甲氧西林敏感金葡菌(MSSA)攻击小鼠的生存时间与生存率,MAP31.1保护组小鼠一周后生存率为60%,抗S.aureus与NS组均为0%,NRS组为20%；显著增强小鼠主要脏器对MSSA清除率(Pliver=0.006 vs NRS, Pspleen=0.013 vs NRS,Pkidey=0.010 vs NRS)。该结果提示MAP-P31.1免疫兔IgG对S.aureus具有一定的保护效果；遗憾的是该抗MAP-P31.1纯化IgG对甲氧西林耐药菌株(MRSA)仍无保护作用。随后的清除实验表明正常兔IgG和MAP-P31.1免疫纯化IgG与MRSA(ATCC43300)混合静脉注射后,抗MAP-P31.1组脾脏中细菌载量显著降低(P=0.030vsNRS),肝脏中无显著差异,该结果提示抗MAP-P31.1 IgG对MRSA可能具有一定保护作用。对于MSSA所致脓肿,MAP-P31.1注射组脓肿数量或大小明显少于其余各组,提示MAP-P31.1有抑制MSSA型脓肿的形成；而对于MRSA所致脓肿,anti-S.aureus组脓肿数最多呈弥散分布,其余各组集中在边缘；抗MAP-P31.1 IgG组脓肿数或面积虽然低于正常IgG和抗S.aureus IgG组,但与NS组比较无显著差别,提示抗MAP-P31.1IgG对MRSA所致脓肿无明显抑制作用。上述结果提示,对MAP-P31序列改造虽然显示了一定的效果,如抗血清的体外杀菌活性,但对MRSA体内攻击仍不能形成有效的保护。
Staphylococcus aureus, a gram-positive bacterium, is a leading cause of community-associated and nosocomial infections which colonizatied in human skin and mucosa, particularly the anterior nares. However, it often causes bacteraemia with high morbidity and mortality in immunocompromised hosts, surgical patients and in those with indwelling medical devices in spite of adequate antibiotic therapy. Since appearance of multidrug resistant strains especially methicillin-resistant S.aureus (MRSA) in past 50 years, which indicates S.aureus cannot always be controlled by commonly used antibiotics, additional control strategies are greatly needed. Therefore, the immunotherapy and prevention have been explored and emphasised since the 1960s, especially, the development with vaccine has been expected and been made great efforts. However, about ten vaccine candidates with different components of S.aureus including StaphVAX (the polysaccharide-carrier conjugates, failed in Trial III), PentaStaph (containing CP8/5, a-toxoid and PVL) a-hemolysin, Panton-Valentine leukocidin (PVL), Toxic shock syndrome toxin-1 (TSST-1), S. aureus attenuated toxin strain and Poly-N-acetyl-glucosamine (PNAG), poly-N-succinyl beta-1-6 glucosamine (PNSG) protein or capsule polysaccharide-protein conjugates, surface protein vaccines (IsdA, IsdB, SdrD and SdrE), and a DNA vaccine prepared from FnBPA-CIfA, have not been approved to be used in clinical.
PGN is a vital component on cell wall of many kinds of bacteria, especially in Gram positive bacteria such as S.aureus. Recently, PGN was considered as 'the bacterial Achille' sheel, providing the weak point for targeting. However, PGN has never been used as vaccine candidate because PGN as nonprotein component, belongs to thymus independent antigen (TI-Ag), and neither induces the production of memory B cells nor effective secondary antibody response. Our strategy is to convert the PGN, a TI-Ag, to becoming peptide mimics to PGN as thymus dependent antigen (TD-Ag) that can provoke secondary immune responese.
The work on peptide mimics to PGN has been not reported before, however, this strategy and technique have been widely used in research of non-protein or protein vaccines, as well as single conformation-dependant antigenic determinant and antigenic epitopes mapping, These reports include the peptide mimics to provoke the production of protective antibodies against Cryptococcus neoformans, group B meningococcal, Haemophilus influenzae, Streptococcus pneumoniae, Vibrio cholerae, Brucella, Entamoeba histolytica, as well as the production of cytotoxic antibody against tumor cells.
In recent years, multiple antigen peptide (MAP) has been considered and used as a valuable approach to amplify peptide immunogens. The advantages of MAP are as follows:MAP is consisted of nearly pure antigenic sequences without protein carrier to make conjugate; and MAP can be used an immunogen without adjuvant; additionally, MAP can provoke T cell mediated immune response.
We screened the peptide mimics to PGN from a random phage display peptide library using an anti-PGN monoclonal antibody (McAb) and synthesised the four-branch multiple antigenic peptides that mimics epitopes on PGN. These MAPs were used as vaccine candidates to immunize the mice, and antigenicity and immunogenicity were identified by serial of experiments in vivo and in vitro. We also studied whether these peptide mimics can induce an effective protection immune against S.aureus or not.
This research can be divided into the following four parts.
PartⅠ. Screening of the peptide mimics to PGN from phage displayed peptide libraries
The PGN mimotopes were screened from 12-mers linear phage display peptide library by using monoclonal antibody against PGN and the antigenicity of selected clones was identified by ELISA. The screening of phage clones showed an enrichment process from first round to third round, and 14 of 49 phage clones were defined as positive clones that bound with anti-PGN McAb identified by indirect ELISA. The amino acid sequences deduced from DNA sequences were analysed and were shown that three phage clones as:No.31, No.28 and No.43, had the consensus sequence ATWxHxLxSAGL, and showed higher reactivity with anti-PGN McAb than other clones. Phage clones No.39, No.27 and No.1 shared the conserved sequence xHx or Hx found in clone No.31, these phages may display the mimotopes of PGN.
PartⅡ. The design, synthesis and identification to peptide mimics of PGN in vitro
In this part, three peptide mimics sequences'SP31, SP27, and SP39'were redesigned and synthesized by adding S (serine) A (alanine) and G G (glycine) on the two sides of origin sequence for stability of the conformation as well as adding biotin on the N- terminal as a tag. Four branch mutilgen peptides (MAP) were also synthesized in order to enhance antigenicity and immunogenicity of the peptides SP31 and SP27. Finally, the antigenicity of peptide mimics or MAPs was identified.
In the design of peptide mimic for systhesis, we estimated or predicted the antigenic epitopes, T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares (http://bio.dfci.harvard.edu/Tools/antigenic.pl, www.syfpeithi.de, http://www.darrenflower.info/mhcpred), indicating that three peptides contain sites bound both mice and human MHC. The Sequences-TWxHxLx-' and 'WxHxVxW-' may be antigenic epitope as SP31 and SP39 respectively, which contains a T cell epitope by adding 'S' 'A' and 'G' 'G' on the two sides. However, SP27 does not contain a T cell epitope even if was added different assemblies of amino acid residue 'S A G G' and the sequence '-SPHxH000RS-'as an antigenic epitope.
According to above design, the sequence of clone No.31,27 and No.39 were synthesized and termed as SP31, SP27 and SP39, respectively. To enhance the immunogenicity, a four-branch MAP based on the sequence SP31 and SP27 were synthesized and termed as MAP-P31 and MAP-P27, respectively, Then the antigenicity of peptide mimics or MAPs was identified by indirect and competitive ELISA. Our results show that both SP31 and MAP-P31 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus in a dose-dependent manner (FSP31 binding to anti-PGN McAb=367.090, P SP31 binding to anti-PGN McAb=0.000; FSP31 binding to anti-S.aureus PcAb=367.090,PSP31 binding to anti-S.aureus PcAb=0.000; FMAP-P31 to anti-PGN McAb=2021.727, PMAP-P31 to anti-PGN McAb=0.000; FMAP-P31 to binding to anti-S.aureus PcAb=178.344,PMAP-P31 to binding to anti-S.aureus PcAb=0.000, respectively). Moreover, both SP31 and MAP-P31 could inhibit the binding of PGN to anti-PGN McAb in a dose-dependent manner(F SP31=12.286, P SP31=0.039; FMAP-P31=17.532, PMAP-P31=0.053; respectively); conversely, PGN could inhibit the binding of SP31 and MAP-P31 to the anti-PGN McAb. However, MAP-P27 could not bind to McAb against PGN even if SP27 better bound to both PcAb and McAb against PGN. Additionally, SP39 could bind to antibodies against PGN or S.aureus but not in a dose-dependent manner. These data indicate that both SP31 and MAP-P31 mimic to epitopes on PGN.
Since PGN can stimulate murine peritoneal macrophage (pM0) to produce some proinflammatory cytokines such as TNF-αand IL-6, we have tested whether the peptide mimics to PGN likde MAP-P31 could stimulate the production of proinflammatory cytokines or not. Polymyxin B was used to exclude the influence of lipopolysaccharide (LPS) if any LPS presented in the supernatant. The results show that TNF-a and IL-6 were produced by pMΦstimulated with MAP-P31 in dose-dependent manner(FTNF-α=1265.415,PTNF-α=0.000, FIL-6=248.353, PIL-6=0.000, respectively), suggesting that MAP-P31 can also mimic the biological activity of PGN despite the weaker activity of MAP-P31 to stimulate cytokine production than that of PGN.
PartⅢ. The characteristization of active immunity in mice immunized with peptide mimics to PGN
In order to characterize MAP-P31 mimics to PGN as candidate vaccine, six-eight week-old Balb/C mice were randomly divided into five groups to be immunised as follows:MAP-P31 emulsified with FCA for the first immunisation, followed by boost with Freund incomplete adjuvant; FCA as basic immunization for one week prior to MAP-P31; MAP-P31 alone; and unrelated MAP emulsified with FCA and PBS were used as controls. The antisera were collected for identification of antibodies to MAP-P31, PGN and S.aureus, S.epidermidis, E.coli and P.aeruginosa by indirect ELISA. The results show that the better antibody response was provoked in mice immunised with MAP-P31 emulsified with Freund adjuvant(P=0.001 vs FCA prior to MAP-P31; P=0.005 vs MAP-P31), and the antiserum can bind with PGN well(FPGN=70.122, PPGN=0.000), but bind with LTA and LPS weakly. Consistent with our expectations, the antiserum can also bind to ultrasonic cell lysates of S.aureus, S.epidermidis, E.coli, and P.aeruginosus (P<0.05 vs anti-unrelated MAP). In addition, the serum IgG type antibodies against PGN and S.aureus endured in mice, which demonstrates that the MAP-P31 as peptide mimics to epitopes on PGN, provokes an effective secondary or recall antibody response that can only be induced by a TD-Ag and protects against infection with S.aureus.
We estimated the bactericidal/acteriostatic activity as well as opsonophagocytosis activity of the antiserum. The results show that anti-MAP-P31 serum possess a more potent bactericidal/bacteriostatic activity than that of anti-unrelated MAP serum or normal serum in a dose-dependent manner with or without complements, and enchance phagocytosis of murine peritoneal macrophages (pMΦ)to FITC-labeled s.aureus (ATCC 25923)(F=981.862, P=0.000). In addition, anti-MAP-P31 serum also has complement-independent bactericidal activity against F.enterococcus, S.hominis, P.aeruginosa and E.coli but not against clinic isolated and standard methicillin resistant S.aureus (MRSA).
Since the most important evaluation for a vaccine should be protection of mice from lethal bacterial attack, the bacterial clearance and protection against infection with lethal live S.aureus were observed in 6 to 8 week-old Balb/c mice immunised with MAP-P31 or an unrelated MAP and PBS as controls. The results show that bacterial clearance in spleen, liver and kidney tissues was enhanced significantly in mice immunised with MAP-P31 compared to that in mice immunised with an unrelated MAP(Pliver=0.018 vs unrelated MAP,Pspieen=0.015 vs unrelated MAP and Pkindey=0.020 vs unrelated MAP). Moreover, mice were challenged with 6×108 live S.aureus by intraperitoneal injection (ip) at two weeks after the fifth immunisation, the survival of the mice immunised with MAP-P31 was notably longer than that of mice immunised with an unrelated MAP or PBS control, and mice never died after one week of attack. Consequently, we conclude that MAP-P31 can provoke protection against a lethal infection with S.aureus (MSSA), which may be associated with enhancing the bacterial clearance in vivo. However, MAP-P31 could not protect mice infected with MRSA, which would be a serious problem and challenge for development of vaccine.
Part IV. The estimation of passive immunity by antiserum to modified MAP-P31
In order to make PGN peptide mimics induce a protective immunity to MRSA, we have modified MAP-P31 by changing a few of amino acide to form a Th epitope, the modified MAP-P31 was named as MAP-P31.1. All of MAP-P31.1, MAP-P31 and MRSA with Freund ajuvant were used to immunize 6-8 week-old Balb/C or rabbits.
The ten folds increasing antibody titer was found in mice immunized with MAP-P31.1 and followed by boost with heat-killed S.aureus, which suggests that natural infection with S.aureus could become a vaccine boost to enhance the specific immunity to infection.
The estimation in vitro for bactericidal/bacteriostatic activity show that anti-MAP-P31.1 antisera had a more potent bactericidal/bacteriostatic activity against MSSA (ATCC 25923) than that of anti-MAP-P31 serum or anti-S.aureus serum with or without complements(FMSSA(inactive complements)16.490,PMSSA(inactive complements)=0.001); importantly, anti-MAP-P31.1 antisera also show a bactericidal/bacteriostatic activit on MRSA such as standard (ATCC43300) or clinic isolation MRSA strains (317177, identifying by clinical labs) (FMRSA(inactive complements)=13.213, PMRSA(inactive complements)=O.006; FMRSA(complements)=14.150 and PMRSA(complements)=0.005), whereas anti-MAP-P31 antisera could not. The antisera to S.aureus were not shown the bactericidal or bacteriostatic activity against MSSA and MRSA without complements, although it showed a better binding to PGN and S.aureus than antisera against MAP-P31 and MAP-P31.1. These results suggest that MAP31.1 can provoke an antibody response with bactericidal or bacteriostatic activity against both MSSA and MRSA in vitro.
For the evaluation of passive immunity by injecting anti-MAP-P31.1 rabbit IgG antisera into mice with S.aureus infection, we found that anti-MAP-P31.1 purified IgG notably prolonged living time and survival of mice infected with MSSA but not MRSA. Additionally, bacterial clearance in spleen, liver and kidney tissues was enhanced in mice challenging with MSSA after administering anti-MAP-P31.1, compared to that of administering normal rabbit IgG(Pliver=0.006 vs NRS, Pspleen=0.013 vs NRS,Pkidey=0.010 vs NRS); and the inhibition of renal abscess causing by MSSA (ATCC25923) but not MRSA (ATCC43300) were also observed.
In this work, the modified peptide mimics MAP-P31.1 could induce an improved antibody response to MRSA in vitro, compare to MAP-P31, but it still could not provoke an effective protection against infecton with MRSA.
肽聚糖(Peptidoglycan, PGN)是金葡菌细胞壁的重要成份,近年更是被称为是细菌的“阿喀琉斯之踵”,意为强者的致命弱点,是免疫防治的靶抗原。但至今未被作为疫苗候选靶点的主要原因在于：PGN为非蛋白抗原,即TI抗原(胸腺非依赖抗原),免疫原性弱,即便是感染个体其抗体滴度也不高。本研究尝试用短肽模拟PGN抗原表位,将其抗原性质由TI抗原改变为TD(胸腺依赖抗原)抗原,以诱导有效的再次免疫应答,探索以模拟肽作为疫苗候选的可能性。
已知,短肽可模拟蛋白与非蛋白(糖、脂等)表位,常用于构象性表位特性以及糖脂类抗原表位研究。近十余年来,已在研究病原生物及肿瘤的非蛋白成份方面做了大量探索,并可用于分析蛋白质折叠中形成非连续性表位的特点。合成肽疫苗的优势是对有效与非必要抗原表位的组合与取舍,而缺点则是免疫原性弱,需设计合适的载体与佐剂。但近年国内的多价抗原肽(Multiple Antigenic Peptide,MAP)技术有了长足的发展,其优势体现在：不需交联载体,可不用佐剂；除抗体反应,还可诱导CTL与Th细胞反应；尤其在不同的分枝上可有不同序列的肽链,可用于构成或模拟更为复杂或不同功能的表位。国外近年已有数个多价肽疫苗进入临床观察。
本课题的主要研究思路是：利用噬菌体肽库筛选获得的短肽模拟PGN的保守性结构表位,将其非蛋白抗原表位的性质改变为短肽,由此使TI-Ag改变为TD-Ag；并以所获模拟肽序列合成四分枝多价抗原肽(MAP)作为疫苗候选诱导机体内产生有效的再次抗体应答和保护性免疫。
本课题组用抗PGN商品化单抗从噬菌体随机展示肽库中筛选了十余个模拟位克隆,结合生物信息学分析选择三个PGN表位的序列进行修饰与合成,并进行体外间接ELISA与抑制ELISA鉴定,从中选择效果较好的SP31进行四分枝多价抗原肽,并对其免疫原性及免疫效果进行评价。
一、应用噬菌体展示技术筛选金葡菌PGN模拟肽克隆
从噬菌体线性肽库中筛选可模拟PGN表位的噬菌体克隆以商品化抗PGN单克隆抗体为靶分子对噬菌体线性12线性肽库进行了3轮筛选,富集率达为2千倍以上,显示了明显的富集效果；从第三轮随机挑选49噬菌体克隆中用夹心ELISA鉴定出可与抗PGN单克隆抗体结合的14个克隆。
阳性噬菌体测序及序列分析将上述14个阳性噬菌体克隆进行DNA测序,并对其氨基酸序列进行分析,其第31、28、43号克隆具有共同序列：ATWxHxLxSAGL,并与27、39、1序列有保守序列xHx或Hx,除噬菌体克隆1号外与anti-PGN McAb均有高反应性(OD450),第5、23、38、44虽具有共同序列GWWPYAALRALS,但多次重复后第5序列非特异性结合较高,可能含有吸板序列：因此,选择31,27,39三种噬菌体作为后续研究对象。
二、PGN模拟肽的设计、合成与体外鉴定
阳性克隆展示序列抗原特性分析通过在线软件www.syfpeithi.de, http://bio.dfci.harvard.edu/Tools/antigenic.pl, http://www.darrenflower.info/mhcpred预测和T细胞表位预测软件(DNAstar)分析发现,三种阳性噬菌体展示序列均具有人和鼠MHC结合表位；其中No.31可能的抗原表位为-TWxHxLx-序列,在两端分别加上SG-、GG后可能具有T细胞表位；No.27可能的抗原表位为—SPHxH000RS-,在两端加上SG/G GG的不同组合后均不具有T细胞表位；No.39可能的抗原表位为-WxHxVxW-;在两端加上不同SG/A GG氨基酸后可能具有T细胞表位,序列分析结果表明三种序列具有构成Th、B细胞表位的特性。但是否真正模拟PGN的抗原表位,还需进一步的生物实验验证。
PGN模拟肽抗原性鉴定体外ELISA鉴定结果表明,以生物素标记三种线性合成肽链(SP31,SP27,SP39)能特异结合抗PGN单抗及兔抗S.aureus全菌多抗(FSP31=1139.058, PSP31=0.000; FSP27=196.091, PSP27=0.000; FSP39=90.811,PSP39=0.000),其中SP31、SP27与抗PGN单抗的反应性优于抗S.aureus多抗,而SP39则反之；抑制实验表明,PGN能剂量依赖地抑制SP31、SP27与抗PGN单克隆抗体结合(FPGN抑制SP31=729.036,PPGN抑制SP31=0.027；FSP31抑制PGN=12.286,P SP31抑制PGN=0.039；FPGN抑制SP27=74.776,PPGN抑制SP27=0.001),抑制效果在40%-60%,而39号合成肽抑制效果较差,量效关系不显著(数据未在文中显示)。上述结果提示SP31,SP27,SP39线性肽可不同程度地模拟PGN表位的抗原性。基于SP31,SP27为模板合成四分枝多价抗原肽(MAP)鉴定表明,MAP-P31剂量依赖地与抗PGN单抗(F=2021.727,P=0.000)和抗S.aureus多抗反应(F=178.344,P=0.000),并能与PGN相互抑制与抗PGN McAb的反应(FPGN抑制MAP-P31=64.032,PPGN抑制MAP-P31=0.015,FMAP-P31抑制PGN=17.532,PMAP-P31抑制PGN=0.053),而MAP-P27仅与抗S.aureus多抗反应；由此我们以MAP-P31作后续的免疫原性研究。
PGN模拟肽对小鼠腹腔巨噬细胞(M9)刺激作用实验结果表明MAP-P31模拟肽能显著刺激小鼠腹腔巨噬细胞产生炎症因子TNFα、IL-6(FrNFα=1265.415,PTNFα=0.000；FIL_6=248.353,PIL-6=0.000),并呈一定的剂量依赖关系,但低于PGN刺激的细胞因子产生水平。该结果提示MAP-P31不但可模拟PGN表位的抗原性,亦可模拟其刺激巨噬细胞并产生细胞因子的生物学活性。
三、PGN抗原模拟肽MAP-P31主动免疫效果评价
不同免疫方式的效果评价以MAP-P31(无交联载体)为免疫原,采用不同免疫方案免疫小鼠,二次免疫后一周检测免疫血清效价。结果显示：MAP-P31加弗氏佐剂乳化组效果显著优于基础免疫组(弗氏佐剂提前一周注射,后续均用不含佐剂的MAP-P31进行免疫)、无佐剂组(P=0.001vs FCA prior to MAP-P31;P=0.005vsMAP-P31)。结果提示,MAP-P31免疫接种可以不需要交联其他的蛋白载体,但仍需佐剂。该条件用于后续实验。
抗血清免疫反应性评价合成肽MAP-P31加弗氏佐剂免疫小鼠的抗血清含可与PGN、MAP-P31以及S.aureus结合的IgG型抗体,并持续8周以上。提示MAP-P31可诱导小鼠产生了针对PGN和S.aureus的再次抗体应答与免疫记忆(FPGN=70.122,PPGN=0.000；FSaureus=379.255,PSaureus=0.000)。此外,抗MAP-P31抗血清尚可与金葡菌磷壁酸(LTA)有微弱反应,但与LPS不反应；并与表皮葡萄球菌(S.epidermidis)、大肠杆菌(E.coli)和铜绿假单胞菌(P. aeruginosus)的超声裂解物反应(P<0.05 vs anti-unrelated MAP),表现出一定的交叉反应性。提示MAP-P31可能模拟不同细菌PGN的共同或相似抗原表位。
抗血清的体外杀菌/抑菌及调理吞噬作用实验结果表明,在补体存在或补体灭活情况下,抗MAP-P31抗血清对甲氧西林敏感模式菌株(ATCC25923)均具有杀菌／抑制作用；并对肠球菌、人葡萄球菌、大肠杆菌、绿脓杆菌和嗜麦芽黄单胞菌均表现出一定的抑制／杀灭作用,然而对MRSA(ATCC43300)反而促进其生长作用,该结果提示血清杀菌作用及抗菌谱具有一定局限性,需进一步优化序列结构,以期能产生广谱的抗菌作用。研究结果亦证实在灭活补体后,抗MAP-P31血清仍能显著增强小鼠腹腔巨噬细胞对FITC标记的甲氧西林敏感模式菌株(ATCC25923)的吞噬作用(F=981.862,P=0.000)。
MAP-P31诱导的小鼠体内保护作用小鼠尾静脉注射亚致死剂量的甲氧西林敏感模式菌株(ATCC25923)后,MAP-P31免疫组小鼠肝、脾、肾等主要脏器中细菌载量显著低于无关MAP免疫对照组(Piver=0.018,Pspleen=0.015,Pkindey=0.020)；腹腔注射致死剂量的甲氧西林敏感模式菌株(ATCC25923)后,MAP-P31免疫组2周后的生存率为90%,而PBS免疫组和无关MAP免疫组小鼠生存率分别为0%和20%,提示免疫MAP-P31免疫后小鼠产生了针对甲氧西林敏感模式菌株(ATCC25923)的保护性免疫。
四、PGN模拟肽改造及其被动免疫效果评价
PGN模拟肽序列的改造鉴于MAP-P31免疫血清对MSSA菌杀菌作用明显,而对MRSA则有促进其生长作用,我们对其进行了序列改造的尝试。根据I)NAstar软件预测,我们在MAP-P31的两端延长序列中改变个别氨基酸使其形成Th细胞识别表位,合成四分枝多价抗原肽并命名为MAP-P31.1,以期获得对MRSA有杀伤/抑制作用的序列。
MAP-P31.1免疫反应性鉴定以此MAP-P31.1加弗氏佐剂为免疫原免疫家兔和Balb/C小鼠,5次免疫后,其免疫血清可结合胰酶消化后PGN(?)S.aureus,提示其可能针对的是PGN的多糖表位,该模拟肽与PGN有相同或相似的抗原表位。MAP-P31.1免疫后,用热灭活S.aureus做加强免疫可增强抗体效价10倍(P=0.002vs no boost),其可能的临床应用价值在于：用合成肽疫苗接种后,自然感染S.aureus可能发挥加强免疫的作用。
MAP-P31.1抗血清杀菌/抑菌作用抗MAP-P31.1小鼠血清在有/无补体作用下对MRSA与MSSA均有显著杀菌/抑菌作用(FMSSA(inactive complements)=16.490, PMSSA(inactive complements)=0.001;FMRSA (inactive complements)=13.213,PMRSA(inactive complements) =0.006;FMRSA (complements)=14.150,PMRSA(complements)=:0.005),且均显著优于anti-S.aureus多抗血清与抗MAP-P31血清(P<0.001vs anti-S.aureus组；t=-8.302,P=0.001)。
MAP-P31.1免疫兔IgG的被动保护作用与正常兔IgG、抗S.aureus多抗对照组比较,纯化抗MAP-P31.1 IgG抗体被动输入能延长甲氧西林敏感金葡菌(MSSA)攻击小鼠的生存时间与生存率,MAP31.1保护组小鼠一周后生存率为60%,抗S.aureus与NS组均为0%,NRS组为20%；显著增强小鼠主要脏器对MSSA清除率(Pliver=0.006 vs NRS, Pspleen=0.013 vs NRS,Pkidey=0.010 vs NRS)。该结果提示MAP-P31.1免疫兔IgG对S.aureus具有一定的保护效果；遗憾的是该抗MAP-P31.1纯化IgG对甲氧西林耐药菌株(MRSA)仍无保护作用。随后的清除实验表明正常兔IgG和MAP-P31.1免疫纯化IgG与MRSA(ATCC43300)混合静脉注射后,抗MAP-P31.1组脾脏中细菌载量显著降低(P=0.030vsNRS),肝脏中无显著差异,该结果提示抗MAP-P31.1 IgG对MRSA可能具有一定保护作用。对于MSSA所致脓肿,MAP-P31.1注射组脓肿数量或大小明显少于其余各组,提示MAP-P31.1有抑制MSSA型脓肿的形成；而对于MRSA所致脓肿,anti-S.aureus组脓肿数最多呈弥散分布,其余各组集中在边缘；抗MAP-P31.1 IgG组脓肿数或面积虽然低于正常IgG和抗S.aureus IgG组,但与NS组比较无显著差别,提示抗MAP-P31.1IgG对MRSA所致脓肿无明显抑制作用。上述结果提示,对MAP-P31序列改造虽然显示了一定的效果,如抗血清的体外杀菌活性,但对MRSA体内攻击仍不能形成有效的保护。
Staphylococcus aureus, a gram-positive bacterium, is a leading cause of community-associated and nosocomial infections which colonizatied in human skin and mucosa, particularly the anterior nares. However, it often causes bacteraemia with high morbidity and mortality in immunocompromised hosts, surgical patients and in those with indwelling medical devices in spite of adequate antibiotic therapy. Since appearance of multidrug resistant strains especially methicillin-resistant S.aureus (MRSA) in past 50 years, which indicates S.aureus cannot always be controlled by commonly used antibiotics, additional control strategies are greatly needed. Therefore, the immunotherapy and prevention have been explored and emphasised since the 1960s, especially, the development with vaccine has been expected and been made great efforts. However, about ten vaccine candidates with different components of S.aureus including StaphVAX (the polysaccharide-carrier conjugates, failed in Trial III), PentaStaph (containing CP8/5, a-toxoid and PVL) a-hemolysin, Panton-Valentine leukocidin (PVL), Toxic shock syndrome toxin-1 (TSST-1), S. aureus attenuated toxin strain and Poly-N-acetyl-glucosamine (PNAG), poly-N-succinyl beta-1-6 glucosamine (PNSG) protein or capsule polysaccharide-protein conjugates, surface protein vaccines (IsdA, IsdB, SdrD and SdrE), and a DNA vaccine prepared from FnBPA-CIfA, have not been approved to be used in clinical.
PGN is a vital component on cell wall of many kinds of bacteria, especially in Gram positive bacteria such as S.aureus. Recently, PGN was considered as 'the bacterial Achille' sheel, providing the weak point for targeting. However, PGN has never been used as vaccine candidate because PGN as nonprotein component, belongs to thymus independent antigen (TI-Ag), and neither induces the production of memory B cells nor effective secondary antibody response. Our strategy is to convert the PGN, a TI-Ag, to becoming peptide mimics to PGN as thymus dependent antigen (TD-Ag) that can provoke secondary immune responese.
The work on peptide mimics to PGN has been not reported before, however, this strategy and technique have been widely used in research of non-protein or protein vaccines, as well as single conformation-dependant antigenic determinant and antigenic epitopes mapping, These reports include the peptide mimics to provoke the production of protective antibodies against Cryptococcus neoformans, group B meningococcal, Haemophilus influenzae, Streptococcus pneumoniae, Vibrio cholerae, Brucella, Entamoeba histolytica, as well as the production of cytotoxic antibody against tumor cells.
In recent years, multiple antigen peptide (MAP) has been considered and used as a valuable approach to amplify peptide immunogens. The advantages of MAP are as follows:MAP is consisted of nearly pure antigenic sequences without protein carrier to make conjugate; and MAP can be used an immunogen without adjuvant; additionally, MAP can provoke T cell mediated immune response.
We screened the peptide mimics to PGN from a random phage display peptide library using an anti-PGN monoclonal antibody (McAb) and synthesised the four-branch multiple antigenic peptides that mimics epitopes on PGN. These MAPs were used as vaccine candidates to immunize the mice, and antigenicity and immunogenicity were identified by serial of experiments in vivo and in vitro. We also studied whether these peptide mimics can induce an effective protection immune against S.aureus or not.
This research can be divided into the following four parts.
PartⅠ. Screening of the peptide mimics to PGN from phage displayed peptide libraries
The PGN mimotopes were screened from 12-mers linear phage display peptide library by using monoclonal antibody against PGN and the antigenicity of selected clones was identified by ELISA. The screening of phage clones showed an enrichment process from first round to third round, and 14 of 49 phage clones were defined as positive clones that bound with anti-PGN McAb identified by indirect ELISA. The amino acid sequences deduced from DNA sequences were analysed and were shown that three phage clones as:No.31, No.28 and No.43, had the consensus sequence ATWxHxLxSAGL, and showed higher reactivity with anti-PGN McAb than other clones. Phage clones No.39, No.27 and No.1 shared the conserved sequence xHx or Hx found in clone No.31, these phages may display the mimotopes of PGN.
PartⅡ. The design, synthesis and identification to peptide mimics of PGN in vitro
In this part, three peptide mimics sequences'SP31, SP27, and SP39'were redesigned and synthesized by adding S (serine) A (alanine) and G G (glycine) on the two sides of origin sequence for stability of the conformation as well as adding biotin on the N- terminal as a tag. Four branch mutilgen peptides (MAP) were also synthesized in order to enhance antigenicity and immunogenicity of the peptides SP31 and SP27. Finally, the antigenicity of peptide mimics or MAPs was identified.
In the design of peptide mimic for systhesis, we estimated or predicted the antigenic epitopes, T cell epitopes and scores binding to MHC of these peptides by using DNASTAR and online softwares (http://bio.dfci.harvard.edu/Tools/antigenic.pl, www.syfpeithi.de, http://www.darrenflower.info/mhcpred), indicating that three peptides contain sites bound both mice and human MHC. The Sequences-TWxHxLx-' and 'WxHxVxW-' may be antigenic epitope as SP31 and SP39 respectively, which contains a T cell epitope by adding 'S' 'A' and 'G' 'G' on the two sides. However, SP27 does not contain a T cell epitope even if was added different assemblies of amino acid residue 'S A G G' and the sequence '-SPHxH000RS-'as an antigenic epitope.
According to above design, the sequence of clone No.31,27 and No.39 were synthesized and termed as SP31, SP27 and SP39, respectively. To enhance the immunogenicity, a four-branch MAP based on the sequence SP31 and SP27 were synthesized and termed as MAP-P31 and MAP-P27, respectively, Then the antigenicity of peptide mimics or MAPs was identified by indirect and competitive ELISA. Our results show that both SP31 and MAP-P31 could bind to both anti-PGN McAb and a polyclonal antibody against S.aureus in a dose-dependent manner (FSP31 binding to anti-PGN McAb=367.090, P SP31 binding to anti-PGN McAb=0.000; FSP31 binding to anti-S.aureus PcAb=367.090,PSP31 binding to anti-S.aureus PcAb=0.000; FMAP-P31 to anti-PGN McAb=2021.727, PMAP-P31 to anti-PGN McAb=0.000; FMAP-P31 to binding to anti-S.aureus PcAb=178.344,PMAP-P31 to binding to anti-S.aureus PcAb=0.000, respectively). Moreover, both SP31 and MAP-P31 could inhibit the binding of PGN to anti-PGN McAb in a dose-dependent manner(F SP31=12.286, P SP31=0.039; FMAP-P31=17.532, PMAP-P31=0.053; respectively); conversely, PGN could inhibit the binding of SP31 and MAP-P31 to the anti-PGN McAb. However, MAP-P27 could not bind to McAb against PGN even if SP27 better bound to both PcAb and McAb against PGN. Additionally, SP39 could bind to antibodies against PGN or S.aureus but not in a dose-dependent manner. These data indicate that both SP31 and MAP-P31 mimic to epitopes on PGN.
Since PGN can stimulate murine peritoneal macrophage (pM0) to produce some proinflammatory cytokines such as TNF-αand IL-6, we have tested whether the peptide mimics to PGN likde MAP-P31 could stimulate the production of proinflammatory cytokines or not. Polymyxin B was used to exclude the influence of lipopolysaccharide (LPS) if any LPS presented in the supernatant. The results show that TNF-a and IL-6 were produced by pMΦstimulated with MAP-P31 in dose-dependent manner(FTNF-α=1265.415,PTNF-α=0.000, FIL-6=248.353, PIL-6=0.000, respectively), suggesting that MAP-P31 can also mimic the biological activity of PGN despite the weaker activity of MAP-P31 to stimulate cytokine production than that of PGN.
PartⅢ. The characteristization of active immunity in mice immunized with peptide mimics to PGN
In order to characterize MAP-P31 mimics to PGN as candidate vaccine, six-eight week-old Balb/C mice were randomly divided into five groups to be immunised as follows:MAP-P31 emulsified with FCA for the first immunisation, followed by boost with Freund incomplete adjuvant; FCA as basic immunization for one week prior to MAP-P31; MAP-P31 alone; and unrelated MAP emulsified with FCA and PBS were used as controls. The antisera were collected for identification of antibodies to MAP-P31, PGN and S.aureus, S.epidermidis, E.coli and P.aeruginosa by indirect ELISA. The results show that the better antibody response was provoked in mice immunised with MAP-P31 emulsified with Freund adjuvant(P=0.001 vs FCA prior to MAP-P31; P=0.005 vs MAP-P31), and the antiserum can bind with PGN well(FPGN=70.122, PPGN=0.000), but bind with LTA and LPS weakly. Consistent with our expectations, the antiserum can also bind to ultrasonic cell lysates of S.aureus, S.epidermidis, E.coli, and P.aeruginosus (P<0.05 vs anti-unrelated MAP). In addition, the serum IgG type antibodies against PGN and S.aureus endured in mice, which demonstrates that the MAP-P31 as peptide mimics to epitopes on PGN, provokes an effective secondary or recall antibody response that can only be induced by a TD-Ag and protects against infection with S.aureus.
We estimated the bactericidal/acteriostatic activity as well as opsonophagocytosis activity of the antiserum. The results show that anti-MAP-P31 serum possess a more potent bactericidal/bacteriostatic activity than that of anti-unrelated MAP serum or normal serum in a dose-dependent manner with or without complements, and enchance phagocytosis of murine peritoneal macrophages (pMΦ)to FITC-labeled s.aureus (ATCC 25923)(F=981.862, P=0.000). In addition, anti-MAP-P31 serum also has complement-independent bactericidal activity against F.enterococcus, S.hominis, P.aeruginosa and E.coli but not against clinic isolated and standard methicillin resistant S.aureus (MRSA).
Since the most important evaluation for a vaccine should be protection of mice from lethal bacterial attack, the bacterial clearance and protection against infection with lethal live S.aureus were observed in 6 to 8 week-old Balb/c mice immunised with MAP-P31 or an unrelated MAP and PBS as controls. The results show that bacterial clearance in spleen, liver and kidney tissues was enhanced significantly in mice immunised with MAP-P31 compared to that in mice immunised with an unrelated MAP(Pliver=0.018 vs unrelated MAP,Pspieen=0.015 vs unrelated MAP and Pkindey=0.020 vs unrelated MAP). Moreover, mice were challenged with 6×108 live S.aureus by intraperitoneal injection (ip) at two weeks after the fifth immunisation, the survival of the mice immunised with MAP-P31 was notably longer than that of mice immunised with an unrelated MAP or PBS control, and mice never died after one week of attack. Consequently, we conclude that MAP-P31 can provoke protection against a lethal infection with S.aureus (MSSA), which may be associated with enhancing the bacterial clearance in vivo. However, MAP-P31 could not protect mice infected with MRSA, which would be a serious problem and challenge for development of vaccine.
Part IV. The estimation of passive immunity by antiserum to modified MAP-P31
In order to make PGN peptide mimics induce a protective immunity to MRSA, we have modified MAP-P31 by changing a few of amino acide to form a Th epitope, the modified MAP-P31 was named as MAP-P31.1. All of MAP-P31.1, MAP-P31 and MRSA with Freund ajuvant were used to immunize 6-8 week-old Balb/C or rabbits.
The ten folds increasing antibody titer was found in mice immunized with MAP-P31.1 and followed by boost with heat-killed S.aureus, which suggests that natural infection with S.aureus could become a vaccine boost to enhance the specific immunity to infection.
The estimation in vitro for bactericidal/bacteriostatic activity show that anti-MAP-P31.1 antisera had a more potent bactericidal/bacteriostatic activity against MSSA (ATCC 25923) than that of anti-MAP-P31 serum or anti-S.aureus serum with or without complements(FMSSA(inactive complements)16.490,PMSSA(inactive complements)=0.001); importantly, anti-MAP-P31.1 antisera also show a bactericidal/bacteriostatic activit on MRSA such as standard (ATCC43300) or clinic isolation MRSA strains (317177, identifying by clinical labs) (FMRSA(inactive complements)=13.213, PMRSA(inactive complements)=O.006; FMRSA(complements)=14.150 and PMRSA(complements)=0.005), whereas anti-MAP-P31 antisera could not. The antisera to S.aureus were not shown the bactericidal or bacteriostatic activity against MSSA and MRSA without complements, although it showed a better binding to PGN and S.aureus than antisera against MAP-P31 and MAP-P31.1. These results suggest that MAP31.1 can provoke an antibody response with bactericidal or bacteriostatic activity against both MSSA and MRSA in vitro.
For the evaluation of passive immunity by injecting anti-MAP-P31.1 rabbit IgG antisera into mice with S.aureus infection, we found that anti-MAP-P31.1 purified IgG notably prolonged living time and survival of mice infected with MSSA but not MRSA. Additionally, bacterial clearance in spleen, liver and kidney tissues was enhanced in mice challenging with MSSA after administering anti-MAP-P31.1, compared to that of administering normal rabbit IgG(Pliver=0.006 vs NRS, Pspleen=0.013 vs NRS,Pkidey=0.010 vs NRS); and the inhibition of renal abscess causing by MSSA (ATCC25923) but not MRSA (ATCC43300) were also observed.
In this work, the modified peptide mimics MAP-P31.1 could induce an improved antibody response to MRSA in vitro, compare to MAP-P31, but it still could not provoke an effective protection against infecton with MRSA.
引文
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