High-affinity antibody induced by immunization with a synthetic peptide is associated with protection of cattle against foot-and-mouth disease.

Previous work has shown that the synthetic peptide C-C-(200-213)-P-P-S-(141-158)-P-C-G, in which residues 200-213 and 141-158 correspond to immunogenic regions of the VP1 protein of foot-and-mouth disease virus (FMDV), is capable of inducing high levels of neutralizing antibody but only inconsistent protection of cattle against virulent FMDV challenge. The possibility exists that differences in affinity may well underlie the observed variations in biological effectiveness of the anti-peptide antibodies in immunized animals. This has been investigated by assessing the affinity for peptide and whole virus of the anti-peptide antibodies in sera from protected and non-protected cattle using both fluid-phase and solid-phase assays. The results obtained show that the affinities of serum antibodies for peptide and virus in protected cattle were significantly higher than those in non-protected animals. Thus in order to assess vaccine efficacy, particularly in the case where synthetic immunogens are employed, consideration should be given to the determination of antibody affinity in addition to other parameters of the antibody response.     

AsiaI口蹄疫病毒VP1蛋白单克隆抗体的制备与鉴定

目的：制备AsiaI口蹄疫病毒VP1蛋白单克隆抗体（mAb）并进行鉴定。方法：用纯化的AsiaI型口蹄疫病毒VP1表位重组蛋白为抗原免疫6～8周龄的雌性BALB／c小鼠，经过3次免疫后，取其脾细胞与sp2／0骨髓瘤细胞融合。采用有限稀释法和间接ELISA克隆和筛选阳性杂交瘤细胞株，用SDS-PAGE电泳、间接ELISA以及微量细胞中和试验对所获得的mAb的特异性进行鉴定。结果：成功获得3株能稳定传代并分泌抗AsiaImAb的杂交瘤细胞株，分别命名为：188、5E1、5E2，其分泌的mAb为IgG1（188）和IgG2a（5E1、5E2）亚类，他们均能特异性的识别VP1重组蛋白和AsiaI型全病毒，其腹水效价在1：10^5～1：10^6。微量细胞中和试验表明该mAb能很好地识别灭活的FMDV，中和效价达1：1024以上。交叉试验表明该mAb具有高度特异性，型间无交叉反应，证明所获得的mAb均完全针对AsiaI FMDV抗原决定簇。结论：在口蹄疫病毒mAb的研究中，VP1重组蛋白有望代替活病毒来制备mAb。AsiaI口蹄疫病毒VP1 mAb的成功制备，为进一步研究和开发新型口蹄疫的检测方法和抗原表位奠定了基础。     

AsiaⅠ口蹄疫病毒VP1蛋白单克隆抗体的制备与鉴定

目的:制备AsiaI口蹄疫病毒VP1蛋白单克隆抗体(mAb)并进行鉴定。方法:用纯化的AsiaI型口蹄疫病毒VP1表位重组蛋白为抗原免疫6～8周龄的雌性BALB/c小鼠,经过3次免疫后,取其脾细胞与Sp2/0骨髓瘤细胞融合。采用有限稀释法和间接ELISA克隆和筛选阳性杂交瘤细胞株,用SDS-PAGE电泳、间接ELISA以及微量细胞中和试验对所获得的mAb的特异性进行鉴定。结果:成功获得3株能稳定传代并分泌抗AsiaImAb的杂交瘤细胞株,分别命名为:1B8、5E1、5E2,其分泌的mAb为IgG1(1B8)和IgG2a(5E1、5E2)亚类,他们均能特异性的识别VP1重组蛋白和AsiaI型全病毒,其腹水效价在1:105～1:106。微量细胞中和试验表明该mAb能很好地识别灭活的FMDV,中和效价达1:1024以上。交叉试验表明该mAb具有高度特异性,型间无交叉反应,证明所获得的mAb均完全针对AsiaI FMDV抗原决定簇。结论:在口蹄疫病毒mAb的研究中,VP1重组蛋白有望代替活病毒来制备mAb。AsiaI口蹄疫病毒VP1mAb的成功制备,为进一步研究和开发新型口蹄疫的检测方法和抗原表位奠定了基础。     

Protective immune responses against foot-and-mouth disease virus by vaccination with a DNA vaccine expressing virus-like particles

To display antigenic protein on the surface of virus-like particles (VLPs) presents a potentially powerful strategy for vaccine development. We genetically engineered the major capsid protein VP1 of foot-and-mouth disease virus (FMDV) into the predominant epitope C of HBV core gene to yield a chimeric core-VP1 VLP. The VLP was successfully expressed in HeLa cells transfected with core-VP1 DNA construct. Compared with a regular VP1 DNA construct, immunization with core-VP1 DNA induced significantly higher levels of antigen-specific IgG production, T cell proliferation, cytotoxic T lymphocyte response, and cytokine production in mice. Most importantly, the level of neutralizing antibody elicited by core-VP1 immunization was significantly higher than that with VP1 DNA immunization, which correlated well with animal protection level from subsequent live FMDV challenge. Thus, immunization with chimeric VLP induces higher efficacy and provides an attractive DNA vaccine strategy for controlling FMDV infection in future.     

Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein

Summary  Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E. coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection. Received June 17, 1996 Accepted September 11, 1996     

Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs

In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.     

Recombinant adenovirus co-expressing capsid proteins of two serotypes of foot-and-mouth disease virus (FMDV): in vitro characterization and induction of neutralizing antibodies against FMDV in swine

Human adenovirus type 5 (Ad5) has been evaluated as a novel gene delivery vector for the development of live-viral vaccines for foot-and-mouth disease (FMD). In this study, we constructed an Ad5 vector co-expressing the capsid precursor proteins, P1, of FMD virus (FMDV) field strains A24 Cruzeiro and O1 Campos and examined the neutralizing antibody responses in swine after inoculation with the vector. To construct the Ad5 vector, a bicistronic expression cassette containing a cytomegalovirus promoter, the P1 coding sequence of FMDV A24, the internal ribosomal entry site (IRES) of FMDV A12, the P1 coding sequence of FMDV O1 Campos and the coding region of A12 3C protease was inserted into the E1 region of an E1/E3-deleted Ad5. The recombinant adenovirus, Ad5A24+O1, was generated by transfection of 293 cells with full-length pAd5A24+O1 recombinant plasmid DNA. The recombinant Ad5 co-expressed P1 of both A24 and O1 in infected 293 cells and P1 of both serotypes was processed to produce VP0, VP3, and VP1. We further demonstrated the formation of capsid protein complexes by co-precipitation of VP0, VP3, and VP1 with monoclonal antibodies against viral capsid proteins. Swine inoculated with Ad5A24+O1 generated neutralizing antibodies against both A24 and O1. However, the overall neutralizing antibody response was considerably lower than that induced by a commercial FMD vaccine or a monovalent Ad5-A24 vaccine.     

Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a?bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A?and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a?challenge with the virus of serotype A?as well as O. This is the first report on the development of a?bivalent FMD vaccine using a?forage crop. Keywords: foot-and-mouth disease; sunnhemp; Agrobacterium tumefaciens; FMDV-VP1 gene; serotype O?and A; in planta transformation; transgenic plants; bivalent vaccine.     

O型口蹄疫病毒VP1表位重组蛋白疫苗的构建、表达和纯化

目的 为了克服灭活口蹄疫病毒疫苗可能存在的传播病毒的潜在危险,构建一种能预防O型口蹄疫病毒感染的VPI表位重组蛋白疫苗。方法采用O型口蹄疫病毒（FMDV）表面VP1蛋白上的B细胞表位肽和T细胞表位肽,以PCR扩增和克隆连接等方法构建VP1表位六聚体重组蛋白（vP1epi）基因,并在大肠杆菌中进行诱导表达,镍亲和层析纯化。用ELISA法检测VPlepi免疫豚鼠血清中抗FMDV抗体的水平。结果构建了一种由FMDV表面vP1中B细胞表位肽重复六次的蛋白质多肽,采用pET28原核表达系统,在大肠杆菌BL21（DE3）中获得了高效表达,表达产量约占菌体蛋白的30％左右,经镍亲和层析后获得纯度高于90％的VP1表位重组蛋白。VP1epi免疫的豚鼠血清中含有一定量的抗FMDV抗体。结论VP1epi重组蛋白能诱导机体产生抗O型FMDV的抗体,说明这种重组蛋白可能成为预防O型FMDV感染的基因工程蛋白质疫苗。     

Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle

Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin α_vβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4⁺ and CD8⁺ gamma interferon (IFN-γ)⁺ cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle.     

Enhanced immunogenicity and antitumour effects with heterologous prime-boost regime using vaccines based on MG7-Ag mimotope of gastric cancer

MG7-Ag, gastric cancer-associated antigen, has been shown to be immunogenic and has been used as marker molecule for prognosis. In a previous study, we developed an oral DNA vaccine based on MG7-Ag mimotope. However, we failed to detect cellular immune response using the oral MG7-Ag mimotope DNA vaccine. To induce significant T cell response, we developed a recombinant adenovirus vaccine based on MG7-Ag mimotope and evaluated the efficacy and protective effects of heterologous prime-boost immunization protocol with an oral DNA vaccine previously developed. We found that both vaccines were able to elicit a significant humoral response against MG7-Ag, while the highest serum titre MG7 antibody was detected in mice immunized with the heterologous prime-boost immunization protocol. Enzyme-linked immunospot (ELISPOT) assay demonstrated that the heterologous prime-boost immunization strategy was more efficient in inducing T cell response than the homologous prime-boost strategy. In the tumour challenge assay, 2 of 5 mice immunized with the heterologous prime-boost protocol were tumour free, while none of the mice in homologous prime-boost groups or control groups was tumour free. Those tumour-bearing mice in the heterologous prime-boost regime had smaller tumour masses than their counterparts in the homologous prime-boost groups or control groups. Therefore, our study suggests that vaccines against MG7-Ag induce significant immune response against gastric cancer, and that the heterologous prime-boost protocol using different types of vaccines could achieve better protective effect than the homologous prime-boost protocol.     

High-titred neutralizing antibodies to human enterovirus 71 preferentially bind to the N-terminal portion of the capsid protein VP1

Summary Human enterovirus 71 has emerged as an important pathogen of children in the Asia Pacific region, and it may be important to consider the development of a vaccine against this virus. Human cord serum was used as a source of neutralizing antibodies to determine whether the N- or C-terminal half of the VP1 capsid protein was more likely to harbour neutralizing determinants. Cord sera from 205 individuals were tested for neutralizing antibodies against human enterovirus 71 in an indirect ELISA against recombinant VP1 antigen as well as the N- and C-terminal portions of VP1 antigen. High-titred human neutralizing antibodies were significantly more reactive with the N-terminal half of VP1 than weak or negative sera. The N-terminal half of human enterovirus 71 is likely to have important neutralizing antibody determinants and should be investigated further in vaccine development efforts.     

Immunization of DNA vaccine encoding C3d-VP1 fusion enhanced protective immune response against foot-and-mouth disease virus

Because foot-and-mouth disease virus (FMDV) remains a great problem to many livestock of agricultural importance, safe, effective vaccines are in great need. DNA vaccine would be a promising candidate but the design remains to be optimized. VP1 gene of FMDV strain O/ES/2001 was linked to three copies of either porcine or murine C3d or four copies of a 28-aa fragment of murine C3d containing the CR2 receptor binding domain (M28). The resultant plasmids encoding C3d/M28-VP1 fusion or only VP1 as control were immunized guinea pigs. Both cellular and humoral immune responses were evaluated and protection was observed after virus challenge. As a result, although the plasmid encoding only VP1 could elicit virus-binding antibody detected by ELISA, splenocyte proliferation, IL-4 and IFN-γ production, the levels were significantly less than C3d/M28-VP1 fusion. Furthermore, VP1 failed to induce neutralization antibody and protect animals against virus challenge, while murine C3d-VP1 fusion efficiently induced neutralization antibody response and provided 87.50% of the animals with complete protection and 12.50% with partial protection. Among murine C3d, M28, and porcine C3d, the adjuvant effect of murine C3d is strongest, followed by porcine C3d, and last murine M28. In conclusion, the fact that C3d genes, when coupled to VP1 gene, are able to greatly enhance the protective immune response of VP1 DNA in guinea pigs suggests that C3d-VP1 DNA chimera has a significant potential for use as a novel DNA vaccine against FMDV.     

Enhanced immunogenicity to food-and-mouth disease virus in mice vaccination with alphaviral replicon-based DNA vaccine expressing the capsid precursor polypeptide (P1)

Recently, alphavirus replicon-based DNA vaccines, also known as suicidal DNA vaccines, have emerged as an important strategy to enhance the potency of DNA vaccines. In this study, two different types of DNA vaccines encoding the capsid precursor polypeptide (P1) of foot-and-mouth disease virus (FMDV) were constructed and the immunogenicity were investigated and compared in mouse model. The first DNA vaccine, pcDP1, is a conventional plasmid DNA vaccine in which P1 was driven directly by a cytomegalovirus promoter. The second DNA vaccine, pSCAP1, is a Semliki Forest virus (SFV) replicon-based DNA vaccine encoding the same antigen. In vitro expression and characterization indicated that two vaccine vectors could correctly produce the P1 antigen. However, pSCAP1 could induce obvious apoptosis of the transfected cells. After immunization in BALB/c mice, the P1-specific ELISA antibodies, neutralizing antibodies, as well as lymphocyte proliferative responses induced by pSCAP1 were significantly higher than those obtained in mice immunized with pcDP1. Notably, mice immunized with the pSCAP1 had the determined ability of clearing virus in their sera after FMDV challenge. These results indicate that the SFV replicon-based DNA vaccine pSCAP1 are more effective than conventional DNA vaccine and it can be considered a promising approach for the development of a safety and efficacious vaccine against FMDV.     

Comparison of immune responses against foot-and-mouth disease virus induced by fusion proteins using the swine IgG heavy chain constant region or beta-galactosidase as a carrier of immunogenic epitopes

Previously, we demonstrated that a fusion protein (Gal-FMDV) consisting of beta-galactosidase and an immunogenic peptide, amino acids (141-160)-(21-40)-(141-160), of foot-and-mouth disease virus (FMDV) VP1 protein induced protective immune responses in guinea pigs and swine. We now designed a new potential recombinant protein vaccine against FMDV in swine. The immunogenic peptide, amino acids (141-160)-(21-40)-(141-160) from the VP1 protein of serotype O FMDV, was fused to the carboxy terminus of a swine immunoglobulin G single heavy chain constant region and expressed in Escherichia coli. The expressed fusion protein (IgG-FMDV) was purified and emulsified with oil adjuvant. Vaccination twice at an interval of 3 weeks with the emulsified IgG-FMDV fusion protein induced an FMDV-specific spleen proliferative T-cell response in guinea pigs and elicited high levels of neutralizing antibody in guinea pigs and swine. All of the immunized animals were efficiently protected against FMDV challenge. There was no significant difference between IgG-FMDV and Gal-FMDV in eliciting immunity after vaccination twice in swine. However, when evaluating the efficacy of a single inoculation of the fusion proteins, we found that IgG-FMDV could elicit a protective immune response in swine, while Gal-FMDV only elicited a weak neutralizing activity and could not protect the swine against FMDV challenge. Our results suggest that the IgG-FMDV fusion protein is a promising vaccine candidate for FMD in swine.     

Proliferative lymphocyte responses to foot-and-mouth disease virus and three FMDV peptides after vaccination or immunization with these peptides in cattle.

We studied proliferative responses of bovine T lymphocytes to foot-and-mouth disease virus (FMDV) serotypes A, O and C as well as to three peptides including the two major B-cell epitopes of FMDV (VP1[141-156] and VP1[200-213]). Peripheral blood mononuclear cells (PBMC) from cattle previously vaccinated with monovalent vaccine responded to both homotypic and heterotypic virus strains. Of 14 FMDV-specific bovine T-cell clones, which were prepared from PBMC of an animal vaccinated with the trivalent vaccine, 11 reacted to each of the three serotypes A, O and C. This indicates that several T-cell epitopes might be conserved among these serotypes. PBMC from one of two cattle immunized with VP1[141-156]KLH, one of two cattle immunized with VP1[200-213]KLH and two of three cattle immunized with CC-VP1[200-213]-PPS-VP1[141-156]-PCG responded to the homotypic virus strain. After immunizations with VP1[200-213]KLH also heterotypic responses were found. Thus, it appears that these two B-cell sites include T-cell determinants that are recognized by some cattle. However, when proliferative responses of PBMC from an animal vaccinated with the trivalent vaccine were tested, no responses were found to VP1[141-156] and VP1[200-213], whereas the response was very poor to CC-VP1[200-213]-PPS-VP1[141-156]-PCG. These results suggest that these sequences do not represent dominant T-cell epitopes and/or that T-cell reactivity towards these synthetic peptides does not completely cover the T-cell reactivity towards the fragments present after processing of the whole virus.     

Antibody responses against epitopes on the F protein of bovine respiratory syncytial virus differ in infected or vaccinated cattle

Summary.  The fusion protein F of bovine respiratory syncytial virus (BRSV) is an important target for humoral and cellular immune responses, and antibodies against the F protein have been associated with protection. However, the F protein can induce antibodies with different biological activity, possibly related to distinct antigenic regions on the protein. Therefore, epitopes were mapped on the F protein using monoclonal antibodies. Two epitopes (A and B) were identified that induced neutralizing antibodies, and one epitope (C) that did not elicit neutralizing antibodies. Subsequently, antibody responses were analysed against these epitopes in cattle sera after natural infection, experimental infection or vaccination. After natural infection or reinfection, the antibody titres against epitope A were significantly higher than those against epitope B or C. After experimental infection and after vaccination with an inactivated vaccine, antibody titres against epitope B and C were significantly higher than after natural infection. Conversely, virus neutralizing antibody titres were significantly lower in these animals with higher antibody titres against epitopes B and C than in naturally infected cattle. Because after natural infection the epitope-specific-antibody titres against epitope A, B or C differed markedly between the cattle, the magnitude of the antibody titres against epitope A, B or C in relation to the major histocompatibility complex (MHC) genes of cattle (BoLA) was studied. The magnitude of the antibody responses against epitope A of the F protein, but not against the G protein, appeared to be associated with the bovine lymphocyte antigen (BoLA) haplotype. Received February 4, 1997 Accepted July 4, 1997     

A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis

The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.     

探讨BCG初次免疫结核杆菌DNA疫苗加强免疫方案的效应

目的:探讨BCG初次免疫(BCG-prime),结核杆菌共表达DNA疫苗加强免疫(DNA疫苗-boost)的策略对小鼠的免疫效果。方法:将BCG及结核杆菌重组DNA疫苗依次免疫小鼠,通过检测CTL和NK细胞的杀伤活性和特异性淋巴细胞增殖,以及小鼠血清抗体及细胞因子的水平,观测BCG-prime、共表达结核杆菌Ag85A/GM-CSFDNA疫苗boost策略对小鼠的免疫效果。结果:采用prime-boost免疫策略组的小鼠CTL的杀伤活性明显增强、特异性淋巴细胞明显增殖、IFN-γ的水平明显增高,NK细胞杀伤活性与对照组相比也有一定提高,但未超过BCG单独免疫效果。免疫小鼠血清特异性抗体的滴度超过单独DNA疫苗免疫组。结论:在采用BCG-prime-结核杆菌DNA疫苗boost免疫策略后,能增强对小鼠的免疫效应,尤其是Th1型细胞免疫反应增强明显,为进一步在动物体内进行保护性效应试验的研究提供了实验依据。