DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to the major histocompatibility complex II compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus-1 p55Gag as a chimera with the lysosome-associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long-term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long-lasting B cell- and CD4+ and CD8+ T-cell memory responses induced by DNA encoding non-targeted Gag decay rapidly and elicit very low or undetectable levels of gag DNA is sufficient to generate T-cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B- and T-cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA-encoded vaccine antigens to the MHC II processing compartments for induction of long-term immunological memory.     

含密码子优化的SIV gag基因的DNA疫苗与rAd5疫苗构建及免疫原性评价

目的 构建含有SIVgag基因的DNA疫苗和重组腺病毒疫苗,为后期在SIV感染的猴模型中进行多载体疫苗联合免疫策略的治疗效果评价奠定基础.方法 将SIV gag基因按照哺乳动物偏嗜密码子进行优化并构建至pVR载体,作为DNA疫苗.以Western Blot方法比较优化前后gag基因表达水平.将优化后的gag基因插入重组腺病毒载体,构建rAd5-SIVgag疫苗.在BALB/c小鼠中分别比较DNA疫苗及rAd5-SIV.gag疫苗单独及联合免疫的效果.结果 密码子优化的SIVgag基因的表达水平远高于野生型SIVgag基因.重组腺病毒疫苗免疫一次或两次诱导的细胞免疫反水平分别高于DNA疫苗免疫一次或两次.两种疫苗联合免疫的反应水平与腺病毒疫苗免疫两次的水平相当,高于DNA疫苗单独免疫及腺病毒疫苗单独免疫一次的结果.结论 成功优化了SIV gag基因,使其不依赖Rev高水平表达;成功构建了表达优化后SIV gag基因的DNA疫苗和rAd5疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors

Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination.     

两株登革2型病毒NS1基因真核重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 利用登革2型病毒(dengue type 2 virus,DENV2)M株和NGC株NS1基因部分扇列PcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 分别构建两株DENV2 NS1基因部分序列(1-413 bp)的PcDNA3.1真核重组质粒和pET28a(+)质粒,进行原核蛋白的表达、鉴定、纯化和定量;并用pcDNA3.1重组质粒免疫BALB/c小鼠,初次免疫及第7天、14天分别加强免疫1次,共免疫3次.收集初次免疫后第7、14、28和56天外周血标本,间接ELISA法测定小鼠血清特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异保护性抗体水平.结果 构建了pET28a(+)-NS1m/pET28a(+)-NS1n原核表达重组质粒,SDS-PAGE分析表明,NS1基因部分序列获得表达,其相对分子质量均约22.3×103;Western blot表明该目的 蛋白可与抗His标签单克隆抗体结合;经Ni柱亲和层析法得到纯度达92%的表达蛋白,对C6/36细胞有毒性,并可用于ELASA检测.不同DENV2毒株NS1基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类和中和抗体的产生存在差异,M株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DENV2两毒株NS1基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.

Abstract：

Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.     

CD80-IgG1 Fc段融合蛋白真核表达载体的构建及表达

目的：构建人CD80-IgG1 Fc段融合蛋白的真核表达载体CD80-IgG1 Fc／pcDNA3．1（＋），并住CHO细胞中进行表达。方法：应用T—A克降技术和亚克降技术，构建人CD80膜外段-IgG1 Fc段融合蛋白的真核表达载体CD80-IgG1 Fc／pcDNA3．1（＋），并将其转染人CHO细胞株并筛选，进行稳定表达：通过Western blot及ELISA法，检测融合蛋白的表达。结果：成功地构建了真核表达载体CD80-IgG1 Fc／pcDNA3．1（＋），并建立CHO细胞稳定表达株。Western blot及ELISA法检测到细胞培养上清中有融合蛋白的表达。结论：成功地构建了人CD80膜外段-IgGI Fc段融合蛋白的真核表达载体CD80-IgGI Fc／pcDNA3．1（＋），并在CHO细胞中稳定表达，为下一步抗肿瘤的研究奠定了基础。     

Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis

DNA vaccines whose DNA encodes a variety of antigens from Mycobacterium tuberculosis have been evaluated for immunogenicity and protective efficacy. CD8(+) T-cell responses and protection achieved in other infectious disease models have been optimized by using a DNA immunization to prime the immune system and a recombinant virus encoding the same antigen(s) to boost the response. A DNA vaccine (D) and recombinant modified vaccinia virus Ankara (M) in which the DNA encodes early secreted antigenic target 6 and mycobacterial protein tuberculosis 63 synthesized, and each was found to generate specific gamma interferon (IFN-gamma)-secreting CD4(+) T cells. Enhanced CD4(+) IFN-gamma T-cell responses were produced by both D-M and M-D immunization regimens. Significantly higher levels of IFN-gamma were seen with a D-D-D-M immunization regimen. The most immunogenic regimens were assessed in a challenge study and found to produce protection equivalent to that produced by Mycobacterium bovis BCG. Thus, heterologous prime-boost regimens boost CD4(+) as well as CD8(+) T-cell responses, and the use of heterologous constructs encoding the same antigen(s) may improve the immunogenicity and protective efficacy of DNA vaccines against tuberculosis and other diseases.     

人CD81的克隆及在COS-7细胞中的表达

目的 从人外周血淋巴细胞中克隆出CD81基因，构建真核表达质粒，并在COS－7细胞中进行表达。方法 分离外周血淋巴细胞，提取细胞总RNA，采用RT－PCR扩增CD81基因。将CD81基因克隆至载体pcDNA3.1( )中，进行酶切及测序鉴定。以质粒pcDNA3.1-CD81转染COS－7细胞进行瞬时表达，并用免疫细胞化学染色法和流式细胞术检测蛋白的表达。结果 RT－PCR产物已插入载体pcDNA3.1( )构建成真核表达质粒pcDNA3.1-CD81。经双酶切和测序鉴定表明，克隆出的人CD81全长编码序列同GenBank收录的序列一致，并且真核表达质粒的构建正确。以脂质体转染COS－7细胞后，用免疫细胞化学染色法和流式细胞术检测表明，细胞可表达人CD81。结论 成功地构建真核表达载体pcDNA3.1-CD81，为进一步研究HCV和CD81的相互作用，以及建立可能的HCV细胞感染模型打下了基础。     

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-γ− secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response.     

DNA priming E and NS1 constructs--homologous proteins boosting immunization strategy to improve immune response against dengue in mice

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.     

人巨细胞病毒gB和pp150融合基因真核表达载体的构建及其免疫活性研究

目的 构建人巨细胞病毒（human cytomegalovirus,HCMV）糖蛋白（gB）和胞膜蛋白（pp150）的单基因及融合基因的真核表达载体并比较其免疫学活性,为研制HCMV核酸疫苗奠定实验基础.方法 PCR扩增HCMV gB及pp150并将其连接成融合基因,分别克隆至peDNA3.1（＋）真核表达载体;制备纳米粒DNA疫苗并免疫BALB/e小鼠,检测其体液免疫与细胞免疫应答.结果 成功制备了peDNA3.1（＋）-gB、peDNA3.1（＋）-pp150和peDNA3.1（＋）-gB-pp150纳米核酸疫苗;gB和gB-pp150核酸疫苗均能刺激小鼠产生较强的特异性抗体（P〈0.01）;pp150和gB-pp150核酸疫苗免疫组的小鼠脾细胞能产生较多的IFN-γ和较高的刺激指数.结论 HCMV gB单基因核酸疫苗能诱导较强的体液免疫应答,pp150单基因核酸疫苗则能诱导较强的细胞免疫应答;而gB-pp150的融合基因疫苗能诱导较强的体液免疫和细胞免疫应答.     

Enhanced immunogenicity of SIV Gag DNA vaccines encoding chimeric proteins containing a C-terminal segment of Listeriolysin O

We investigated the potential of the C-terminal 59-amino acid segment of Listeriolysin O (LLO) in enhancing immune responses against the SIV Gag antigen in the context of DNA immunization. Genes with codons optimized for mammalian expression were synthesized for the SIVmac239 Gag, a secreted SIV Gag protein with the tissue plasminogen antigen (tPA) signal fused to its N-terminus (tPA/Gag), as well as their corresponding chimeric proteins Gag/LLO and tPA/Gag/LLO containing the C-terminal 59 amino acids of LLO. Analysis of immune responses to these DNA constructs in a Balb/c mouse model showed that the Gag/LLO construct induced higher levels of both CD4 and CD8 T cell responses against SIV Gag, whereas the tPA/Gag construct induced higher levels of CD4 T cell responses. Moreover, immunization with the tPA/Gag/LLO construct further enhanced both CD4 and CD8 T cell responses. DNA constructs encoding secreted Gag proteins (tPA/Gag and tPA/Gag/LLO) were also more effective in eliciting antibody responses against SIV Gag. Our results demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses in the context of a DNA vaccine.     

免疫途径及载体对乙肝病毒DNA疫苗免疫效果影响的研究

目的探讨ＤＮＡ载体结构及接种途径对ＤＮＡ疫苗免疫效果的影响。方法分别构建插入ＨＢＶ表面抗原编码基因的表达载体ｐｃＤＮＡ１．１／ＳＡ（无抗性基因）和ｐｃＤＮＡＩ／Ａｍｐ／ＳＡ（含氨苄青霉素抗性基因）,肌注小鼠后比较其诱生特异性免疫应答的能力;同时比较不同接种途径（肌内、皮内、皮肤划痕）及ＣｐＧ免疫刺激元件（ＩＳＳ）对ＤＮＡ疫苗诱生免疫效果的影响。结果ｐｃＤＮＡＩ／Ａｍｐ／ＳＡ的免疫效果优于ｐｃＤＮＡ１．１／ＳＡ。ｐｃＤＮＡ１．１／ＳＡ的免疫效果可被ＩＳＳ增强,而ｐｃＤＮＡＩ／Ａｍｐ／ＳＡ诱生特异性免疫应答的能力则可被ＩＳＳ抑制;诱生免疫应答的能力以肌内注射最强,皮内注射免疫其次,皮下划痕法较弱。结论不同ＨＢｓＡｇ表达载体诱生免疫应答的能力不尽相同;ＣｐＧ免疫刺激元件在决定ＤＮＡ疫苗免疫原性中起重要作用,可增强不含相应结构ＤＮＡ疫苗的免疫效果;皮内注射可诱发与肌内接种相似的免疫应答,是一种简便、有效的免疫接种途径     

Comparative immunization in BALB/c mice with recombinant replication-defective adenovirus vector and DNA plasmid expressing a SARS-CoV nucleocapsid protein gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.     

空肠弯曲菌DNA疫苗构建及其疫苗免疫程序研究

目的:构建空肠弯曲菌peb1A基因真核表达载体,探讨DNA疫苗不同免疫程序的免疫效果。方法:以重组pET28a(+)-peb1A质粒为模板,将空肠弯曲菌peb1A基因克隆入真核表达载体pcDNA3.1(-),经酶切和测序鉴定后,将重组质粒转染COS-7细胞中表达,Western blot鉴定表达蛋白;昆明小鼠随机分组:设空白对照组、空载体对照组、pcDNA3.1(-)-peb1A实验组,用ELISA法检测免疫后的昆明小鼠血清IgG和IgM抗体水平。结果:重组质粒pcDNA3.1(-)-peb1A经双酶切和PCR鉴定,构建正确。peb1A基因测序结果与GenBank中peb1A基因序列一致。重组质粒成功转染COS-7细胞,并能正确表达重组蛋白。裸DNA组IgG水平均高于两对照组,有显著性差异(P<0.05)。同一剂量短时间(0、10、20天)免疫程序优于长时间(0、15、25、35天)免疫程序(P<0.05)。结论:成功构建空肠弯曲菌真核表达重组质粒pcDNA3.1(-)-peb1A,其经肌肉注射免疫小鼠,具有较好的免疫原性,短时间免疫程序免疫效果好。     

人CD154基因真核细胞表达载体的构建、鉴定于序列分析

目的 构建人CD154基因真核细胞表达载体。方法 采用RT-PCR方法,从激活的人外周血淋巴细胞的总cDNA中扩增得到820bp的人CD154cDNA片段,再用BamHI和EcoRI双酶切后定向克隆到真核细胞表达载体PcDNA3.1中,限制性内切酶酶切分析重组质粒和DNA序列分析。结果 人CD154cDNA已经正确克隆到真核细胞表达载体pcDNA3.1中。结论本研究为探讨CD154分子与淋巴细胞活化的关系及其信号传导机制。为进一步用于抗移植排斥反应的研究,或对由于CD154遗传缺陷所致的免疫缺陷病的基因治疗奠定了基础。     

Design, construction, and characterization of a dual-promoter multigenic DNA vaccine directed against an HIV-1 subtype C/B' recombinant

An effective vaccine against HIV-1 is generally considered the best hope for controlling the raging AIDS pandemic. As a part of our AIDS vaccine development effort, we constructed a dual-promoter plasmid capable of high-level expression of 2 independent transgenes. HIV-1 gag, pol, env, nef, and tat from a primary subtype C/B' CCR5-tropic HIV-1 were "codon" optimized, modified to eliminate known functional activity, and assembled using an overlapping polymerase chain reaction into 2 plasmids: ADVAX-I (containing env and gag) and ADVAX-II (containing pol and nef-tat). These 2 dual-promoter candidate vaccines showed levels of HIV-1 gene expression comparable to those observed with single-gene plasmids in vitro. Importantly, immunization of mice with these vaccine constructs resulted in dose-dependent multigenic CD4 and CD8 T-cell responses equivalent to those provided by vaccination with single-gene plasmids. With input from the US Food and Drug Administration, ADVAX-I and ADVAX-II have since been combined as a single candidate DNA vaccine, ADVAX. A phase 1 clinical trial of this product has been successfully completed, and its use in prime-boost studies is now underway.     

Evaluation of canarypox-induced CD8(+) responses following immunization by measuring the effector population IFNgamma production

CD8(+) cytolytic activity is traditionally measured by detecting the release of 51Cr after incubation of effector cells with HLA-matched, infected, radiolabeled targets. An alternative method to detect CD8+ activity is to measure the production of intracellular interferon gamma (IFNgamma) after antigen-specific stimulation, either by ELISPOT or by flow cytometry. Studies were performed in 19 volunteers enrolled in a phase 1 trial of candidate canarypox HIV-1 vaccines that encoded multiple HIV-1 genes. The vaccines including vCP205 (Env, Gag, and protease), vCP1433 (Env, Gag, protease, and CTL epitope-rich regions of pol and nef) and vCP1452 (equivalent to vCP1433 with additional immunomodulatory genes of vaccinia). PBMCs were stimulated in vitro with vaccinia constructs encoding env and gag or a lacZ control, and the effectors were cultured for 12-14 days. EBV-transformed B cell lines were infected overnight with the vaccinia vectors, and then incubated with the effector cells for 4 h in the presence of monensin. CD8(+) gene-specific activity was determined as a percentage of IFNgamma cells in the CD3(+)CD8(+)CD45RO(+) gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNgamma production was simultaneously determined in the CD3(+)CD8(-)CD45RO(+) gate. Using these techniques in blinded studies, we found that CD8(+) IFNgamma activity could be measured in the majority of volunteers given four immunizations. Specifically, the responses to the gag gene were control -- 0/2; vCP205 -- 2/4; vCP1433 -- 5/6; vCP1452 -- 4/7. Most of the positive responses were detected after the fourth immunization. Flow cytometric techniques hold promise as a surrogate measure of CTL and for ease of phenotyping of the effector population.     

DNA vaccine expressing repeated carcinoembryonic antigen (CEA)(625-667) induces strong immunity in mice

The efficacy of immunization with DNA plasmids for single truncated carcinoembryonic antigen (CEA) peptide or triple repeated CEA peptides in mice was evaluated. A DNA fragment the truncated CEA gene (nucleotide 625-667) encoding two helper T lymphocyte (HTL) epitopes was amplified by PCR and cloned for generating recombinant plasmids for single CEA(625-667) (pcDNA-CEA(625-667)) or triple CEA(625-667) (pcDNA-triCEA(625-667)), respectively. Subsequently, groups of BALB/c female mice were intramuscularly injected with pcDNA-CEA(625-667,) pcDNA-triCEA(625-667) or control pcDNA3.0 vector, respectively. Ten days after the last immunization, the frequency of splenic CD4(+) and CD8(+) T cells in the mice was determined by flow cytometry. The antigen-specific proliferation of splenic T cells and cytokine production ex vivo were analyzed by (3)H-TdR uptake and cytokine ELISA, respectively. The levels of serum antibodies against CEA in the mice were detected by Western blot and ELISA. Although immunization with plasmid for the CEA(625-667) peptide(s) did not alter the frequency of CD4(+) and CD8(+) T cells in mice, vaccination with plasmid for CEA peptide induced strong antigen-specific T cell proliferation, particularly for the plasmid encoding the triple-repeated CEA peptides, accompanied by significantly elevated levels of IFN-γ secreted by T cells from the mice immunized with triple-repeated peptides. Furthermore, immunization with the plasmid for CEA peptide stimulated higher levels of antigen-specific antibody responses in mice. Vaccination with the plasmid for the triple repeated CEA peptides induced stronger Th1 responses. Our findings may be useful for the development of effective DNA vaccine for the immunotherapy of cancer.