摘要
肾综合征出血热(HFRS)是一种由汉坦病毒引起的急性传染病,临床上以发热、出血和急性肾功能损害为主要特征。我国是HFRS危害最严重的国家,年发病人数为5~10万人,其流行范围广、发病人数多、病死率较高。临床上尚缺乏特异有效的治疗方法。国内外近年来虽已研制出HFRS的灭活疫苗,但从部分人群试用的情况来看,该类疫苗还存在明显不足,尤其是不能有效地刺激细胞免疫应答。因此仍需针对灭活疫苗存在的不足,进一步研究更为有效的HFRS新型疫苗。
汉坦病毒是一种有囊膜的单股负链RNA病毒,基因组分为L、M、S三个节段,分别编码病毒的RNA依赖的RNA聚合酶、包膜糖蛋白(G1和G2)以及核衣壳蛋白(NP)。以往研究表明,汉坦病毒的M基因编码的包膜糖蛋白(GP)可刺激机体产生中和抗体,而对感染动物和人体起到保护作用;但GP免疫原性较弱,刺激产生的抗体出现晚,滴度不高。NP是该病毒结构蛋白中免疫原性最强的,其刺激机体产生的特异性抗体出现早、滴度高、维持时间长,并且还有诱导机体细胞免疫应答的作用。由于汉坦病毒中和抗原表位主要存在于病毒GP上,目前HFRS基因工程疫苗的基础研究主要集中于GP。该方面的研究虽已取得较大进展,但由于GP相对较弱的免疫原性,故免疫效果仍不理想。国内外研究表明,汉坦病毒GP和NP在诱导机体免疫应答中可能均起重要作用,因此,如何发挥其不同结构蛋白在诱导机体免疫应答中的优势互补作用,是HFRS基因工程疫苗研究中亟待解决的问题。
本室前期曾将汉滩病毒(HTNV)76-118株的M基因分别与编码NP氨基端aa1~247的S基因片段(S0.7,编码NP主要抗原区段)拼接,利用杆状病毒和腺病毒表达系统进行融合表达以及表达产物的免疫学分析。结果证实HTNV 76-118株G1S0.7和G2S0.7嵌合基因均能刺激机体的体液免疫和细胞免疫应答。然而在研究中我们也发现了一些问题,如尽管免疫小鼠后各表达系统均能刺激机体产生体液及细胞免疫应答,且腺病毒表达系统的效果要优于其他系统,但是整体的免疫水平还不十分理想,特别是刺激机体细胞免疫应答的水平不高。国内外已证实汉坦病毒感染后以CTL为主的T细胞应答对于机体保护有重要作用,因此利用汉坦病毒CTL表位结合结构蛋白构建疫苗可能是一种可行的方案。
本研究在前期工作基础上,利用基因重组技术,构建了含有HTNV M基因(编码糖蛋白G1、G2)和部分S基因(编码NP主要抗原区段)以及S基因的多个CTL表位的多种重组腺病毒,在对这些重组腺病毒进行系统鉴定的基础上,观察了其刺激小鼠体液免疫和细胞免疫应答的能力。
1、将HTNVG1S0.7和G2S0.7嵌合基因片段分别与合成的CTL表位串联基因CTL1(表位间加间隔序列AAY)及CTL2(表位间不加间隔序列AAY)以不同组合方式克隆到腺病毒转移载体pShuttle中,CTL表位拼接于S0.7 3’端,通过酶切鉴定选取阳性克隆,分别命名为pG1S0.7CTL1、pG1S0.7CTL2、pG2S0.7CTL1、pG1S0.7CTL2。
2、通过特异性的I-Ceu I和PI-Sce I酶切后将重组转移载体与Adeno-X载体病毒DNA相连,电转化E.coli JM109,并用PCR方法进行了筛选和鉴定,获得重组腺病毒的DNA,转染HEK293细胞得到重组腺病毒原种,分别命名为AG1S0.7CTL1、AG1S0.7CTL2、AG2S0.7CTL1、AG1S0.7CTL2。
3、将各重组腺病毒经CsCl密度梯度离心纯化及测定滴度后分别感染HEK293和Vero-E6细胞。IFA和ELISA检测结果显示,各重组腺病毒均可在细胞中表达相应HTNV抗原,表明CTL表位的插入并没有影响相应抗原的表达及其与特异性单克隆抗体(mAb)的结合活性。Western-blot分析结果显示,各重组腺病毒组在HEK293细胞中均可表达能与特异性mAb产生反应、相对分子质量(Mr)分别约为97KDa和80KDa的融合蛋白,与预期融合蛋白大小相符,表明重组腺病毒在HEK293细胞中表达的为完整的融合蛋白。
4、以各重组腺病毒分别经腹腔注射免疫Balb/c小鼠,通过IFA、ELISA、微量细胞中和试验、T淋巴细胞增殖试验、CTL杀伤试验、细胞因子检测等方法观察免疫效果。结果表明,各重组腺病毒免疫小鼠均可刺激机体产生低滴度的抗NP(1∶160~1∶320)和抗GP抗体(1∶20~1∶40),同时也可产生效价约1∶5~1∶40的低滴度中和抗体。各重组腺病毒均可诱导小鼠体内特异的细胞免疫应答,其中AG2S0.7CTL1免疫组小鼠脾细胞对NP及GP的增殖指数明显高于对照组;AG1S0.7CTL2、AG2S0.7CTL1、AG2S0.7CTL2组增殖指数也都略高于AG1S0.7和AG2S0.7组,表明加有CTL多表位的重组腺病毒能更有效地刺激小鼠的细胞免疫应答。CTL杀伤试验的结果表明,各重组腺病毒免疫组在不同的效/靶比条件下均可诱导针对靶细胞的特异性杀伤效应,并随着效/靶比的提高,杀伤效应也相应提高;其中含有CTL多表位的四种重组腺病毒免疫所诱导的CTL杀伤效应明显高于不含CTL表位的两种重组腺病毒。对在CTL多表位间加入或不加入间隔序列AAY的各重组腺病毒的比较来看,在CTL多表位间加入间隔序列AAY的重组腺病毒免疫小鼠能产生更高的细胞免疫应答,表明间隔序列AAY对于各CTL表位的分隔能提高重组腺病毒的免疫效果。对各重组腺病毒免疫小鼠血清中细胞因子检测的结果显示,各重组腺病毒均可刺激Th1类细胞因子(INF-γ、IL-2、IL-12)水平的提高,而Th2类细胞因子水平变化不明显。说明重组腺病毒免疫小鼠后可以刺激机体Th1类细胞的优势应答,从而有助于提高小鼠的细胞免疫应答能力。
Hemorrhagic fever with renal syndrome (HFRS), which is caused byhantavirus, is an acute infectious disease characterized by fever, vascularhemorrhage and kidney dysfunction. HFRS is more severely in China. About50,000-100,000 cases of HFRS are reported annually and HFRS has a highmorbidity and mortality. Until now, there are still no effective therapeutical drugson HFRS. Several types of inactivated vaccine for HFRS have been developed athome and abroad. However, these inactivated vaccines have displayed severalshortcomings, especially on poor stimulation of effective cellular immunity. Sothere is a desperate need to develop more efficacious vaccines to improve vaccineefficiencyfor HFRS.
Hantavirus (HTNV) is a single-stranded negative-sense RNA virus, whosegenome is composed of L, M and S segments encoding RNA-dependent RNApolymerase (RDRP), envelope glycoprotein G1 and G2, and nucleoprotein (NP)respectively. It is indicated that the glycoprotein (GP), encoded by M segment,could elicit neutralization antibodies and thus protect infected animals and humanfrom HTNV lethal infection. On contrast, the immunogenicity of GP is muchweak. The antibodies stimulated by GP are produced later with lower titers thanNP. Researches have shown that NP, another structural protein, is more immunogenic and thus stimulate an early, high-titer and long-lasting antibodyresponse, which plays an important role in inducing cellular immunity againstHTNV infection in vivo. As neutralization epitopes are principally resided onenvelope glycoprotein (GP) of Hantavirus, envelope glycoprotein is currently themain target protein in genetic engineering vaccine for HFRS. However, GP ismuch less immunogenic than NP because the antibodies stimulated by GP weregenerated later and the titers were low. Research indicated that GP and NP mightboth play important role in stimulating humoral or cellular immune response. Soa prominent problem in genetic engineering vaccine development for HFRS ishow to take both advantages from GP and NP in inducing immune response invivo.
In previous work in our laboratory, G1 and G2 fragment of M segment, and0.7 kb fragment of S segment 5’terminal of the HTNV76-118 strain were spliced,expressed in baculovirus and adenovirus expression system respectively, and theresults showed that the HTNV 76-118 strain G1S0.7 and G2S0.7 chimeric geneproducts elicited both humoral and cellular immune response in mice. Evenadenovirus express system was better than baculovirus system, the overallimmunity in mice was far from satisfaction, particularlly on cellular immunity.There is evidence that T cell immunity (mainly CTLs) is crucial for host defenseagainst many virus infections (including HTNV) especially in the clearance ofvirus infected cells. In this case, it is reasonable to combine both CTL epitopesand neutralization epitopes on structural proteins of HTNV to enhence bothhumoral and cellular responses.
On the basis of our previous work, several recombinant adenovirusescontaining HTNV M segment (encoding glycoprotein G1 and G2), partial Ssegment (encoding the major antigen component of NP), and multi-CTLepitopes on S segment, were generated, and their immune response in mice were studied.
1. Two synthetic CTL epitope genes in tandem, with (CTL1) or withoutinterval sequence AYY (CTL2), were inserted into the 3’terminal of chimericgene of HTNV G1S0.7 and G2S0.7 respectively, subcloned into adenovirustransfer vector pShuttle followed by restriction enzyme analysis for positiveclone selection, and designated pG1S0.7CTL1, pG1S0.7CTL2, pG2S0.7CTL1,and pG2S0.7CTL2, respectively.
2. Recombinant adenovirus transfer vector pShuttle, after I-Ceu I and PI-SceI digestion respectively, was connected with Adeno-X DNA, elctroporated into E.coli JM109, and identified by PCR. Positive clones of recombinant adenoviruseswere then harvested after transfection of package cell line HEK293, anddesignated AG1S0.7CTL1, AG1S0.7CTL2, AG2S0.7CTL1 and AG2S0.7CTL2respectively.
3. Recombinant adenoviruses were purified by double cesium chloridegradient centrifugation, titered by plaque formation assay, and infected HEK293and Vero-E6 cells. IFA and ELISA results showed that the recombinantadenoviruses expressed HTNV antigens in both HEK293 and Vero-E6 cells,indicating that the insertion of CTL epitopes did not affect the expression ofHTNV antigen and the binding activity to specific mAbs. Western blot analysisalso demonstrated that recombinant adenovirus expressed the fusion proteins inHEK293 cells, with 97 kDa and 80 kDa specifically reacted with mAb, indicatingthe integrityof expressed fusion proteins.
4. BALB/c mice were inoculated with recombinant adenoviruses, andassayed by IFA, ELISA, microcell-culture neutralization test, T lymphocyteproliferation (MTT assay), cell-mediated cytotoxicity, and cytokine detection.The results showed that low titer antibodies against NP (1:160~1:320) and GP (1:20~1:40) were generated by administration of recombinant adenovirus, andlow neutralization antibodies (1:5~1:40) were observed. The lymphocyteproliferation index of group AG2S0.7CTL1 was significantly higher than thecontrol group, while other groups of AG1S0.7CTL2, AG2S0.7CTL1 andAG2S0.7CTL2 were little higher than the control group without statasticalsignificance. Recombinant adenoviruses containing CTLepitopes induced a moreeffective cellular immune response than the group without CTL epitopes.Cell-mediated cytotoxicity assay results indicated that recombinant adenovirusesproduce specific cytotoxic effects on target cells, in a proportionate manner withthe rise of E/T rations. Meanwhile, recombinant adenoviruses containing CTLepitopes elicited stronger cellular immune response than the groups without CTLepitopes. Recombinant adenoviruses with CTL epitops intervalled by AYYsequence (CTL1) exhibited a more effective cellular immune response than thegroup without AYY (CTL2), among which recombinant adenovirus groupAdnoG2s0.7CTL1 produced the strongest cellular immunity compared with allother groups, suggesting that the recombinant adenovirus containing CTLepitopes stimulated more efficient cellular immune response in mice. Cytokinedetection test results demonstrated a Th1-type response with rise of INF-γ, IL-2, and IL-12, while Th2-type cytokines such as IL-4, IL-10 did not changeremarkedly, indicating that recombinant adenoviruses elicited more efficientcellular immune response in immunized BALB/c mice.
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