Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16

Both the Human papillomavirus (HPV) major (L1) and minor (L2) capsid proteins have been well investigated as potential vaccine candidates. The L1 protein first oligomerizes into pentamers, and these capsomers assemble into virus-like particles (VLPs) that are highly immunogenic. Here we examine the potential of using HPV type 16 (HPV-16) L1 subunits to display a well-characterized HPV-16 L2 epitope (LVEETSFIDAGAP), which is a common-neutralizing epitope for HPV types 6 and 16, in various regions of the L1 structure. The L2 sequence was introduced by PCR (by replacing 13 codons) into sequences coding for L1 surface loops D-E (chideltaC-L2), E-F (chideltaA-L2), and an internal loop C-D (chideltaH-L2); into the h4 helix (chideltaF-L2); and between h4 and beta-J structural regions (chideltaE-L2). The chimeric protein product was characterized using a panel of monoclonal antibodies (MAbs) that bind to conformational and linear epitopes, as well as a polyclonal antiserum raised to the L2 epitope. All five chimeras reacted with the L2 serum. ChideltaA-L2, chideltaE-L2, and chideltaF-L2 reacted with all the L1 antibodies, chideltaC-L2 did not bind H16:V5 and H16:E70, and chideltaH-L2 did not bind any conformation-dependent MAb. The chimeric particles elicited high-titer anti-L1 immune responses in BALB/c mice. Of the five chimeras tested only chideltaH-L2 did not elicit an L2 response, while chideltaF-L2 elicited the highest L2 response. This study provides support for the use of PV particles as vectors to deliver various epitopes in a number of locations internal to the L1 protein and for the potential of using chimeric PV particles as multivalent vaccines. Moreover, it contributes to knowledge of the structure of HPV-16 L1 VLPs and their derivatives.     

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.     

Crystal structures of four types of human papillomavirus L1 capsid proteins: understanding the specificity of neutralizing monoclonal antibodies

Human papillomaviruses (HPVs) are known etiologic agents of cervical cancer. Vaccines that contain virus-like particles (VLPs) made of L1 capsid protein from several high risk HPV types have proven to be effective against HPV infections. Raising high levels of neutralizing antibodies against each HPV type is believed to be the primary mechanism of protection, gained by vaccination. Antibodies elicited by a particular HPV type are highly specific to that particular HPV type and show little or no cross-reactivity between HPV types. With an intention to understand the interplay between the L1 structure of different HPV types and the type specificity of neutralizing antibodies, we have prepared the L1 pentamers of four different HPV types, HPV11, HPV16, HPV18, and HPV35. The pentamers only bind the type-specific neutralizing monoclonal antibodies (NmAbs) that are raised against the VLP of the corresponding HPV type, implying that the surface loop structures of the pentamers from each type are distinctive and functionally active as VLPs in terms of antibody binding. We have determined the crystal structures of all four L1 pentamers, and their comparisons revealed characteristic conformational differences of the surface loops that contain the known epitopes for the NmAbs. On the basis of these distinct surface loop structures, we have provided a molecular explanation for the type specificity of NmAbs against HPV infection.     

Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco

Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1^v gene) or a synthetic sequence optimized for expression in plant plastids (L1^pt gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5′-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy. Contribution No. 129 from CNR-IGV, Portici.     

大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

摘要：【目的】 利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒（HPV11 VLPs）,并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。 【方法】 在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11 VLPs的免疫原性及型交叉反应性。 【结果】 HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉     

Production of human papillomavirus 6b L1 virus-like particles incorporated with enhanced green fluorescent whole protein in silkworm larvae

Using human papillomavirus (HPV) as a subunit vaccine and its manipulation of surface loops is current trending research. Since the atomic model of L1 protein conformations were deciphered, their manipulations of epitopes bring multivalent vaccines. Here, in the present study, we have manipulated antigenic loops of HPV 6b L1 capsid proteins in the amino acid regions 174 ～ 175 (L1:174EGFP) and 348 ～ 349 (L1:348EGFP) with whole enhanced green fluorescent protein(EGFP), expressed in the silkworm larva using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid technology. The expressed proteins were partially purified using sucrose density-gradient centrifugation and size-exclusion chromatography (SEC). The display of EGFP in virus-like particles (VLPs) was confirmed by immuno-fluorescence microscopy, Western blots and immune-transmission electron microscopy (immuno-TEM). There was higher expression of EGFP incorporated L1:174EGFP than L1:348EGFP. Hydrodynamic diameter of VLPs was corroborated by dynamic light scattering, confirming the size of expected range of around 160 nm and substantiating the incorporation of EGFP. From immuno-TEM, each L1:EGFP VLP formed small particles, suggesting that small particles of L1:EGFP fusion protein were aggregated. Our study illustrates that incorporation of whole protein can efficiently form chimeric VLPs, without hindering the conformation. HPV L1 protein accommodated a whole protein on its antigenic loop as a small particle, but an inserted whole protein was unstable.     

HPV18晚期蛋白L1在毕赤酵母菌中的表达及鉴定

目的:用毕赤酵母胞内表达载体构建含人乳头瘤病毒18型(HPV18)L1基因质粒,诱导表达并进行鉴定。方法:按照毕赤酵母密码子偏爱性原则,合成全长L1基因,然后克隆到pAO819表达载体上,在体外分别构建含一个拷贝和二个拷贝的L1基因载体。线形化后转化到GS115酵母细胞,经G418抗性筛选,获高拷贝重组子并经甲醇诱导表达,表达产物采用化学发光Western blot鉴定,一抗为抗HPV18L1蛋白鼠抗血清。结果:在55kDa处有诱导蛋白免疫印迹出现,并在电镜下观察到HPV18的病毒样颗粒(VLPs),证明该表达系统能表达出HPV18 L1蛋白。结论:本实验构建的毕赤酵母表达菌株,可经甲醇诱导表达HPV18L1晚期蛋白,为进一步研制人乳头瘤病毒18型基因工程疫苗打下基础。     

Identification of neutralizing conformational epitopes on the human papillomavirus type 31 major capsid protein and functional implications

The aim of this study was to characterize the conformational neutralizing epitopes of the major capsid protein of human papillomavirus type 31. Analysis of the epitopes was performed by competitive epitope mapping using 15 anti‐HPV31 and by reactivity analysis using a HPV31 mutant with an insertion of a seven‐amino acid motif within the FG loop of the capsid protein. Fine mapping of neutralizing conformational epitopes on HPV L1 was analyzed by a new approach using a system displaying a combinatorial library of constrained peptides exposed on E. coli flagella. The findings demonstrate that the HPV31 FG loop is dense in neutralizing epitopes and suggest that HPV31 MAbs bind to overlapping but distinct epitopes on the central part of the FG loop, in agreement with the exposure of the FG loop on the surface of HPV VLPs, and thus confirming that neutralizing antibodies are mainly located on the tip of capsomeres. In addition, we identified a crossreacting and partially crossneutralizing conformational epitope on the relatively well conserved N‐terminal part of the FG loop. Moreover, our findings support the hypothesis that there is no correlation between neutralization and the ability of MAbs to inhibit VLP binding to heparan sulfate, and confirm that the blocking of virus attachment to the extracellular matrix is an important mechanism of neutralization.     

HPV 58 L1改造基因的表达及其VLP产物抗血清制备

高危型人乳头瘤病毒（human papillomavirus, HPV）慢性持续性感染是诱发宫颈癌的主要病因．体外表达的HPV主要衣壳蛋白（L1）可自组装成病毒样颗粒（virus-like particle, VLP）,免疫后可诱导产生型别特异性中和抗体,有效保护机体免受同型病毒的感染,因此可望预防病毒感染及感染相关的宫颈癌等病变．HPV 58是诱发我国妇女宫颈癌的主要高危型病毒之一,目前尚无针对HPV 58的疫苗问世．本研究联合采用多种策略对HPV 58 L1野生型基因进行改造,获得HPV 58 L1改造基因,命名为HPV 58mL1,用杆状病毒 昆虫细胞表达系统进行HPV 58 mL1的表达,CsCl密度梯度离心法纯化获得HPV 58 mL1重组蛋白,电镜分析结果显示,重组蛋白形成直径约55 nm的VLP．皮下免疫新西兰兔和豚鼠,ELISA检测显示,免疫动物产生高滴度针对HPV 58 mL1 VLP的抗血清,免疫斑点印迹检测显示,抗血清是针对VLP表面表位的．本研究表达了均一性好的HPV 58 mL1 VLP,并获得两个种属的HPV 58 mL1 VLP抗血清,为进一步研究有效预防HPV 58感染的疫苗打下基础．     

Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.     

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2

Background

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.

Methodology/Principal Findings

L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.

Conclusion/Significance

VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.     

Characterization of HPV16 L1 loop domains in the formation of a type-specific,conformational epitope

Background  

Virus-like particles (VLPs) formed by the human papillomavirus (HPV) L1 capsid protein are currently being tested in clinical trials as prophylactic vaccines against genital warts and cervical cancer. The efficacy of these vaccines is critically dependent upon L1 type-specific conformational epitopes. To investigate the molecular determinants of the HPV16 L1 conformational epitope recognized by monoclonal antibody 16A, we utilized a domain-swapping approach to generate a series of L1 proteins composed of a canine oral papillomavirus (COPV) L1 backbone containing different regions of HPV16 L1.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Background

Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.

Methodology/Principal Findings

Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.

Conclusion/Significance

We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.     

Humoral immune response recognizes a complex set of epitopes on human papillomavirus type 6 l1 capsomers

Although epitope mapping has identified residues on the human papillomavirus (HPV) major capsid protein (L1) that are important for binding mouse monoclonal antibodies, epitopes recognized by human antibodies are not known. To map epitopes on HPV type 6 (HPV6) L1, surface-exposed loops were mutated to the corresponding sequence of HPV11 L1. HPV6 L1 capsomers had one to six regions mutated, including the BC, DE, EF, FG, and HI loops and the 139 C-terminal residues. After verifying proper conformation, hybrid capsomers were used in enzyme-linked immunosorbent assays with 36 HPV6-seropositive sera from women enrolled in a study of incident HPV infection. Twelve sera were HPV6 specific, while the remainder reacted with both HPV6 and HPV11 L1. By preadsorption studies, 6/11 of these sera were shown to be cross-reactive. Among the HPV6-specific sera there was no immunodominant epitope recognized by all sera. Six of the 12 sera recognized epitopes that contained residues from combinations of the BC, DE, and FG loops, one serum recognized an epitope that consisted partially of the C-terminal arm, and three sera recognized complex epitopes to which reactivity was eliminated by switching all five loops. Reactivity in two sera was not eliminated even with all six regions swapped. The patterns of epitope recognition did not change over time in women whose sera were examined 9 years after their first-seropositive visit.     

Identification of two cross-neutralizing linear epitopes within the L1 major capsid protein of human papillomaviruses

The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.     

The choice of resin-bound ligand affects the structure and immunogenicity of column-purified human papillomavirus type 16 virus-like particles

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.     

密码子优化型HPV16L1基因在两种昆虫细胞中的表达

目的:提高16型人乳头瘤病毒（HPV16）L1基因在杆状病毒昆虫细胞中的表达水平,为研制预防性HPV疫苗奠定基础。方法:根据昆虫细胞密码子偏性对野生型HPV16L1基因进行改造,利用Bac-to-Bac表达系统获得重组杆状病毒,感染昆虫细胞Sf9和High Five。Western blot鉴定表达产物;电镜下观察病毒样颗粒形成。利用ELISA法评价HPV16L1基因的优化效果,探讨L1蛋白表达的最佳条件。结果:在相对分子质量56kDa处出现HPV16L1的特异性条带;电镜下可见病毒样颗粒在昆虫细胞的核内形成;优化型HPV16L1基因的表达水平显著高于野生型。High Five细胞表达的最佳条件为MOI=10,表达时相72h,其L1蛋白表达量至少比Sf9细胞高3倍。结论:密码子优化技术确实能够促进HPV16L1蛋白的高效表达,而High Five细胞表现出的显著优势尤其值得关注。     

人乳头瘤病毒11型L2NE7E6融合蛋白的原核表达及其诱发的T细胞免疫反应

目的:在原核表达系统中表达人乳头瘤病毒11型(HPV11)L2NE7E6融合蛋白,纯化蛋白后免疫小鼠,检测其诱发的T细胞免疫水平,并筛选HPV11 E6、E7特异的T细胞表位肽。方法:用重叠PCR方法构建HPV11L2NE7E6融合基因并插入原核表达质粒,在大肠杆菌中经IPTG诱导表达融合蛋白L2NE7E6,用SDS-PAGE和West-ern印迹鉴定融合蛋白的表达。Q柱纯化蛋白后免疫C57BL/6小鼠,分别用覆盖HPV11 E6和E7蛋白序列的肽库,采用酶联免疫斑点法(ELISPOT)检测其诱发的E7、E6特异的T细胞免疫反应,并筛选E7、E6特异的T细胞表位肽。结果:在原核表达系统中有效表达了HPV11 L2NE7E6融合蛋白,蛋白纯化后免疫C57BL/6小鼠,分别能检测到针对HPV11 E6、E7肽库刺激产生的特异性T细胞免疫反应。经肽池筛选到1条强的E6 T细胞表位肽E6aa41-55(AEI-YAYAYKNLKVVW);而E7只筛选到2条弱的T细胞表位肽,分别为E7aa53-67(QILTCCCGCDSNVRL)和E7aa73-87(DGDIRQLQDLLLGTL)。结论:HPV11 L2NE7E6融合蛋白能诱发小鼠产生E6、E7特异性细胞免疫反应,可作为尖锐湿疣免疫治疗候选疫苗。