丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCV HVR1模拟表位融合蛋白.用Western blot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况.皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应.结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35).融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCV HVR1合成肽发生交叉反应.本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值.     

丙型肝炎病毒复合高变区1模拟表位蛋白的免疫原性分析

将丙型肝炎病毒高变区1(HVR1)模拟表位融合基因插入原核表达载体pGEX-4T-1,在大肠杆菌BL21(DE3)中进行表达,经亲和层析和凝胶过滤层析获得HCVHVR1模拟表位融合蛋白。用Westernblot和ELISA检测融合蛋白与HCV抗体阳性血清的结合情况。皮下注射免疫BALB/c小鼠,用ELISA检测小鼠血清中的抗HCV抗体水平及其与天然HCV高变区1合成肽的交叉反应。结果表明融合蛋白能与HCV抗体阳性血清特异结合,融合蛋白与HCV抗体阳性血清的结合频率为71.6%(25/35)。融合蛋白免疫小鼠后能有效诱导免疫应答,其诱生的特异性抗体最高滴度达104(免疫后第8周),且该抗体能同2条天然HCVHVR1合成肽发生交叉反应。本研究提示,HCV复合HVR1模拟表位融合蛋白在丙型肝炎疫苗的研发中可能具有潜在应用价值。     

丙型肝炎病毒多表位抗原基因的构建与免疫原性研究

丙型肝炎病毒(HCV)基因易发生变异, 尤其是含中和抗原表位的高变区1(HVR1)变异性最大. 模拟HVR1的B细胞表位具有涵盖多种天然表位的抗原特性, 保守的T细胞表位具有各型间的相对保守性. 为解决HCV高变性造成的疫苗研究障碍, 我们选取HCV E2区HVR1(384～410 aa)模拟B细胞表位9条、C区的保守CTL表位2条(35～44 aa, 132～140 aa)、NS3区保守的CTL表位1条(1073～1081 aa)及NS3区保守的Th表位1条(aa 1251～1259), 各表位之间以插入3个氨基酸为连接臂, 人工合成上述13条表位基因串联的HCV多表位抗原基因(mfc). 将mfc与gst基因融合, 表达了多表位抗原蛋白GST-MFC. 同时, 构建了白介素-2信号肽基因、PADRE表位基因和mfc基因串联的候选HCV DNA疫苗, 即质粒pVAX1.0-st-mfc. 以GST-MFC蛋白免疫家兔和质粒pVAX1.0-st-mfc免疫小鼠, 采用ELISA和Western blot方法, 应用10条具有代表性的HCV HVR1合成肽进行检测, 证明有9条HVR1合成肽能与所有免疫动物血清反应, 交叉反应率(cross reactivity, CR)为90%. 应用HCV抗体阳性的感染者血清与多表位抗原GST-MFC蛋白进行反应, 证明其反应识别率(reactivity frequency, RF)为75%. 上述结果表明, 筛选合成的HCV多表位抗原基因mfc具有HCV中和抗原表位的特征, 可作为HCV疫苗研制的候选基因.     

丙型肝炎病毒高变区1中介导感染的关键氨基酸残基鉴定

丙型肝炎病毒( HCV)包膜E2蛋白氨基端的高变区1(HVR1)由27个氨基酸组成,是HCV蛋白中变异频率最高的肽段.HVR1含中和抗体表位,同时对HCV细胞侵入起重要作用,其结构与功能的关系目前尚不清楚.本研究对H77株包膜蛋白基因中的HVR1进行了一系列缺失突变,然后将突变体表达质粒与假病毒包装质粒共转染人胚肾(H...     

整合狂犬病病毒G蛋白的假病毒构建及应用

目的 构建表达狂犬病病毒(rabies virus, RABV)G蛋白的假病毒，为RABV中和抗体检测及疫苗的研发提供有效实验工具。方法 通过无缝克隆技术构建表达RABV G蛋白的假病毒p-RABV-G,通过抗体中和试验评估狂犬疫苗接种者体内的中和抗体水平。结果 成功在293T细胞内进行假病毒p-RABV-G的包装，p-RABV-G可以表达G蛋白，并能高效感染Huh7.5细胞。4位狂犬疫苗接种者21 d的血清对p-RABV-G的抑制效果处于最优水平，在1∶625倍数的稀释条件下对p-RABV-G的抑制率高达95.79%、98.26%、98.42%和98.88%。结论 成功构建表达RABV G蛋白的假病毒p-RABV-G可用于狂犬疫苗的开发与中和抗体的筛选。在狂犬疫苗接种后的111 d,本文调查的4位接种者中的其中3位体内血清仍可对p-RABV-G产生抑制效果。     

三种丙型肝炎包膜感染性假病毒颗粒的制备及初步应用研究

本研究构建携带HCV代表株H77(1a)、中国HeBei株(1b)以及JFH-1株(2a)丙型肝炎病毒完整的E1-E2包膜糖蛋白基因的表达质粒,间接免疫荧光法及Western blot验证了其在细胞膜(293细胞)上的正确表达.三种包膜质粒分别与慢病毒包装质粒pHR'CMV△8.2及携带EGFP报告基因的自灭活(Self-Inactivating,SIN)转移质粒pCS-CG共转染293FT细胞,产生三种不同基因型的丙型肝炎包膜感染性假型(pseudotyped)病毒颗粒,免疫荧光与Western blot分析验证了E1/E2包膜糖蛋白在假病毒颗粒上的表达与掺人,利用p24 ELISA法及感染性实验对HCV假病毒进行滴定.从上清中获得的HCV假病毒可以在体外感染肝癌细胞系Huh7及Huh7-CD81(且后者上的感染效率约为前者上的2～3倍);利用针对HCV E2蛋白的具有广谱交叉中和活性的单克隆抗体AP33建立了基于上述假病毒颗粒的丙肝病毒体外中和抗体滴定方法,并应用于丙型肝炎患者体内的中和抗体水平的研究.本研究成功包装了包括中国流行株在内的3种不同基因型的HCV包膜感染性假病毒颗粒并建立了基于假病毒颗粒的HCV体外中和抗体检测方法,为研究HCV感染早期特性及特异抗病毒药物的体外筛选提供了有效模型,并可用于HCV感染者体内及HCV基因工程疫苗免疫后中和抗体水平的分析.     

逆转录病毒载体介导的HCV囊膜蛋白基因的表达及动物免疫试验

利用DNA重组技术将丙型肝炎病毒(HCV)H77株E1E2囊膜蛋白基因插入逆转录病毒载体pBABE-puro中构建成重组逆转录病毒载体pBABE-puro-E1E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293T细胞中包装逆转录病毒假病毒.用包装的假病毒感染SP2/0细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明HCV ele2基因在SP2/0细胞膜上成功表达.将表达E1E2蛋白的SP2/0细胞腹腔免疫BALB/c小鼠,经FACS分析免疫鼠血清,成功诱导小鼠产生了抗HCVE1E2蛋白的抗体,Western blot表明该抗体能与原核系统表达的E2蛋白结合.     

逆转录病毒载体介导的HCV囊膜蛋白基因的表达及动物免疫试验

利用DNA重组技术将丙型肝炎病毒(HCV)H77株E1E2囊膜蛋白基因插入逆转录病毒载体pBABE-puro中构建成重组逆转录病毒载体pBABE-puro-E1E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293T细胞中包装逆转录病毒假病毒。用包装的假病毒感染SP2／0细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明HCV ele2基因在SP2／0细胞膜上成功表达。将表达E1E2蛋白的SP2／0细胞腹腔免疫BALB／c小鼠,经FACS分析免疫鼠血清,成功诱导小鼠产生了抗HCV E1E2蛋白的抗体,Western blot表明该抗体能与原核系统表达的E2蛋白结合。     

中东呼吸综合征冠状病毒S1蛋白在昆虫细胞中的重组表达及免疫效果评价

目的:利用昆虫杆状病毒表达系统重组表达中东呼吸综合征冠状病毒(MERS-Co V)S1蛋白,并对其免疫效果进行评价。方法:构建含有MERS-Co V S1基因的重组杆状病毒质粒,转染Sf9细胞包装杆状病毒;重组病毒传代3次获得种子病毒,感染Sf9细胞,收获感染上清,通过镍离子亲和层析纯化获得S1重组蛋白;用纯化的S1蛋白免疫BALB/c小鼠,采用ELISA检测免疫小鼠血清抗原特异性的抗体水平;采用假病毒中和试验检测血清中抗体的中和活性。结果:获得了表达MERS-Co V S1蛋白的重组病毒株,在昆虫细胞中表达并纯化了S1重组蛋白;利用重组表达的S1蛋白免疫小鼠3次,血清S1特异性Ig G抗体滴度可达1∶102 400,免疫小鼠血清稀释至1/5120后中和百分比仍达50%以上。结论:利用昆虫细胞重组表达的MERS-Co V S1蛋白具有良好的免疫原性,并能有效诱导产生高滴度中和抗体,为发展MERS-Co V重组蛋白疫苗奠定了基础。     

CpG联合铝佐剂对HCV免疫原体液免疫作用的影响

目的:探讨胞苷酸鸟苷寡脱氧核苷酸(CpG ODN)联合铝佐剂对丙型肝炎病毒(HCV)重组免疫原的体液免疫作用。方法:采用高交叉HCV-HVR1和E1重组蛋白与CpG ODN、铝佐剂组合,免疫BALB/c小鼠后以ELISA、酶联免疫斑点测定、流式细胞术、免疫沉淀等方法检测相关体液免疫指标和免疫血清多抗的交叉反应性。结果:CpG联合铝佐剂激发了最高的特异性抗体滴度;佐剂通过提高抗体分泌细胞数量、增加脾脏中记忆B细胞数量、增加脾淋巴细胞IL-6、IL-10分泌浓度实现体液免疫增效;CpG则能提高免疫效率,联合铝佐剂时显著提高浆细胞数量;12份HCV阳性血清中有10份可与多抗HVR1 IgG发生免疫沉淀。结论:CpG和铝佐剂联合应用具有协同作用,多抗HVR1 IgG具有较好的交叉反应性。     

Allergen mimotopes in food enhance type I allergic reactions in mice.

BIP1is a murine IgG antibody capable of enhancing the IgE binding to Bet v 1, the major birch pollen allergen. We have previously generated a mimotope of BIP1, designated Bet mim 1, from a constrained phage display peptide library. We demonstrated that oral immunization of BALB/c mice with the Bet mim 1 mimotope resulted in the induction of Bet v 1-specific IgG. The aim of this study was to test the influence of such an oral immunization with Bet mim 1 on a subsequent type I allergic response to Bet v 1. Phages displaying Bet mim 1 or control mimotopes, or PBS alone, were delivered to BALB/c mice by intragastric gavages prior to systemic sensitization with recombinant Bet v 1 and Al(OH)(3), an adjuvant inducing preferentially IgE antibody responses. Only mice fed with Bet mim 1-phages displayed substantially enhanced type I allergic skin reactivity to Bet v 1, as compared to mice pretreated with control mimotopes or PBS. A gastric digestion assay indicated that Bet v 1 and its homologue from apple, Mal d 1, were degraded within seconds under physiological conditions. In contrast, phage-displayed mimotopes were resistant to digestion. Our data indicate that allergen mimics in the diet that resist digestion, can induce allergen specific IgG able to enhance an allergic response. We therefore conclude that sensitization via the oral route may represent a mechanism for aggravating type I allergic reactions, probably leading to an earlier onset of symptoms even at lower allergen dosage.     

A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.     

Allergen mimotopes for 3-dimensional epitope search and induction of antibodies inhibiting human IgE.

There is no definite information available on the structural characteristics of IgE binding epitopes on allergenic molecules, although it is widely accepted that most of them are conformational. In the current study we aimed to characterize the IgE epitope of Bet v 1, the major birch pollen allergen, by the application of phage display peptide libraries. We purified IgE specific for Bet v 1 from allergic patients' sera to select mimotopes representing artificial IgE epitopes by biopanning of phage libraries. By linear alignment, it was not possible to attribute mimotope sequences to the primary structure of Bet v 1. We developed a computer-aided, 3-dimensional coarse-grained epitope search. The 3-dimensional search, followed by statistical analysis, revealed an exposed area on the Bet v 1 molecule (located between residues 9-22 and 104-123) as the IgE binding structure. The IgE epitope was located at a 30 A distance from a previously described IgG epitope and the respective mimotope, designated Bet mim E. Such mimotopes could potentially be used for the induction of IgG capable of interfering with the IgE/allergen interaction. To test this hypothesis, we immunized BALB/c mice with the phage-displayed Bet mim E. Immunizations resulted in the induction of Bet v 1-specific IgG, which was able to block the IgE binding to Bet v 1 in vitro. Based on these observations, we propose that immunotherapy with IgE mimotopes generated by biopannings result in formation of blocking IgG. We conclude that mimotope immunotherapy may represent a new and promising concept for treatment of type I allergic disease.     

Identification of peptide mimotopes of gp96 using single-chain antibody library

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96–peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund’s adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4⁺ and CD8⁺) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.     

A mimotope of Pre-S_2 region of surface antigen of viral hepatitis B screened by phage display

INTRODUCTIONHepatitis B virus (HBV) infection is the mostcommon viral infection in humans especially inChina and East Asia. At present, the most effi-cient method to control the disease is vaccinationof new-borns. Both blood derived vaccine and re-combinant vaccine are hepatitis B surface alltigen(HBsAg)-based and useful in the prevention of thedisease[1-3]. HBsAg could evoke protective humoralimmune response in vivo, but the immune memoryonly last for 5 years or so[3, 4]. How to des…     

A mimotope of Pre－S2 region of surface angigen of viral hepatitis B screened by phage display

To acquire the phage-displayed mimotopes which mimic the specificity of hepatitis B virus surface antigen (HBsAg), a random peptide library expressing linear peptide with 12 amino acids in length were used to screen with the serum from a hepatitis B virus infected patient in the recovery phase. After 3 rounds of biopanning, the positive phages were confirmed by competitive ELISA using HBsAg/P33. Two phagotopes were identified and one of them was confirmed as mimotope by competition experiment. Based on the mimotpe, a multiple antigenic peptide with four branches was synthesized by solid phase peptide synthesis. The antiginicity and specificity of the synthesized antigen was tested in BALB/c mice compared with the native epitope-based antigen. The results showed that the mimotope-based antigen could evoke higher titer of antibodies with the same specificity of the epitope-based antigen. Those findings indicate mimotopes can be used in antigen and vaccine design.     

p185, an immunodominant epitope, is an autoantigen mimotope

An immunodominant peptide (p185(378-394)) derived from the c-erbB2 gene product, was recognized by an anti-DNA antibody, B3, and importantly by two classical DNA-binding proteins, Tgo polymerase and Pa-UDG. These reactivities were inhibited by DNA, confirming that the peptide mimicked DNA. BALB/c mice immunized with p185(378-394) developed significant titers of IgG anti-dsDNA antibodies. Screening of 39 human lupus sera revealed that 5% of these sera possessed reactivity toward p185(378-394). Representative mouse and human sera with anti-p185(378-394) reactivity bound intact p185, and this binding was inhibited by dsDNA. This is the first demonstration of a naturally occurring autoantigen mimotope. The present study identifies a potential antigenic stimulus that might trigger systemic lupus erythematosus in a subset of patients.     

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.