Construction of CpG motif-enriched DNA vaccine plasmids for enhanced early immune response

A DNA vaccine methodology using eukaryote expression vectors to produce immunizing proteins in the vaccinated hosts is a novel approach to the development of vaccine and immuno-therapeutics, and it has achieved considerable success over several infectious diseases and various cancers. To further enhance its efficiency, attempts were made to develop novel plasmid vectors containing multiple immunostimulatory CpG motifs, for rapid and strong immune response. First, a 2.9 kb compact plasmid vector (pVAC), containing CMV promoter, polycloning site, BGH poly(A) terminator, ampicillin resistance gene and pBR322 origin was constructed. A pVAC-hEPO was also constructed, which contained a human erythropoietin gene, for evaluating the transfection efficiency of naked plasmid DNA bothin vitro andin vivo. To examine the adjuvant effect of multi-CpG motifs on naked plasmid DNA, 22 and 44 enriched and unmethylated CpG motifs were introduced into pVAC to generate pVAC-ISS1 and pVAC-ISS2, respectively. 100 μg of pSecTagB, pVAC, pVAC-ISS1 or pVAC-ISS2 were each injected intramuscularly into the tibilias anterior muscle of Balb/c mice. The level of interleukin-6 induced in the mice injected with pVAC-ISS1 and pVAC-ISS2 were significantly elevated, after 12 hours, which were almost 2 and 2.5 times higher than that in the mice injected with pSecTagB, respectively. These results suggest that DNA vaccine plasmids with enriched CpG motifs can induce rapid secretion of interleukin-6 by lymphocytes. In conclusion, these vectors can contribute to the development of adjuvant-free DNA vaccinations against infectious diseases and various cancers.     

分枝杆菌Ag85A DNA疫苗对膀胱癌小鼠细胞免疫功能的影响

为探讨分枝杆菌Ag85A DNA疫苗能否提高荷膀胱癌小鼠保护性免疫应答水平,将1×10^6个MBT-2细胞注射于C3H/HeN小鼠的右侧背部皮内,待肿瘤长至直径约5～8 mm时,将动物随机分成实验组、空质粒组和空白对照组3组,分别于小鼠双侧股四头肌内注射Ag85A-V1 Jns.tPA质粒,V1 Jns.tPA质粒总量0.1 mL（100μg）,或生理盐水0.1 mL,每14 d 1次,共3次,末次免疫后第14天分别检测T细胞亚群数量、NK细胞活性、FasL mRNA表达情况、淋巴细胞增殖水平及肿瘤重量。结果显示,实验组较空白质粒组和空白对照组免疫指标均无明显增高（P〉0.05）,提示肌肉注射Ag85A DNA疫苗不能明显提高荷瘤小鼠免疫功能。     

Early events of electroporation-mediated intramuscular DNA vaccination potentiate Th1-directed immune responses

BACKGROUND: Application of electrical pulses after DNA injection into muscle increases expression of the encoded genes, and is shown to improve antigen-specific immune responses when used for DNA vaccination. In addition, electroporation causes tissue injury and inflammatory reactions. Together with immune stimulatory motifs in the injected DNA these factors may potentiate the immune response by acting as adjuvants for the antigen. Here, we have examined the role of these factors in promoting the efficiency of DNA vaccination. METHODS: We injected a plasmid DNA vector containing the gene Ag85B from M. tuberculosis into mouse quadriceps muscles followed by electroporation. Ag85B was under control of a Tet-responsive promoter, and was expressed either immediately or up to 28 days later by administrating doxycycline to the mice. Delayed expression was combined with injection of non-coding DNA or saline with or without electroporation to examine the ability of these factors to enhance the Ag85B-specific antibody response in the blood and cellular responses in the spleen. Blood samples were analysed with ELISA, while the number of Ag85B-specific IFN-gamma- and IL-4-producing spleenocytes was analysed with ELISpot. RESULTS: Delaying Ag85B expression by 5 or 28 days caused lower anti-Ag85B-specific IgG2a levels. In contrast, the IgG1 antibody response was not significantly affected. Injection of non-coding DNA followed by electroporation moderately increased the IgG2a response. Delaying the Ag85B expression by 28 days reduced the average number of Ag85B-specific IFN-gamma-producing spleenocytes by over 60%. No significant change in the number of IL-4-producing Ag85B-specific spleenocytes was observed. CONCLUSIONS: These results suggest that DNA and electroporation per se may act as good adjuvants in promoting efficient Th1-directed responses during DNA vaccination.     

乙肝病毒DNA疫苗的构建及其诱导小鼠的免疫应答

构建含adr亚型HBV表面抗原基因的核酸疫苗 ,考察人白细胞介素II基因及重组白细胞介素II的免疫佐剂作用。用含有人白细胞介素II基因的真核表达质粒及基因重组白细胞介素II蛋白作为佐剂 ,将编码乙型肝炎病毒表面抗原的重组真核表达质粒 pVAX/HBS免疫BALB/C小鼠 (试验组 ) ,同时设置注射质粒pVAX的阴性对照组 ,并分别于第 2 ,4周后加强免疫各 1次。试验组在第 4周时开始有HBsAb产生 ,阴性对照组未测到HbsAb ,试验组和对照组均未检测到HBsAg。乙肝病毒DNA疫苗能引起小鼠特异性体液免疫应答 ,白细胞介素II的真核表达质粒的佐剂作用不明显 ,基因重组白细胞介素II蛋白具有提高小鼠对乙肝病毒核酸疫苗免疫应答水平的佐剂活性。     

High-level expression of naked DNA delivered to rat liver via tail vein injection

Background

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Methods

We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS‐Epo.

Results

We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose‐response relationship between serum Epo levels and the amount of injected DNA up to 800 µg. Using quantitative real‐time PCR, the vector‐derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, β‐galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose‐dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis.

Conclusions

These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats. Copyright © 2002 John Wiley & Sons, Ltd.

     

Approaches towards DNA Vaccination against a Skin Ciliate Parasite in Fish

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.     

Kidney-targeted naked DNA transfer by retrograde injection into the renal vein in mice

We recently developed a novel kidney-targeted gene transfer technique in rats, using the retrograde renal vein injection of naked plasmid DNA. Many animal disease models are created in mice by transgenic or knockout technologies. However, it is much harder to perform renal vein injection in mice than in rats because they have a thin and short vein. Here we transferred the mouse interleukin (IL)-10 gene into mice by retrograde renal vein injection, using an IL-10 and immunoglobulin fusion protein (IL-10/Fc) (96-kDa) expression plasmid, pCAGGS-IL10/Fc. We observed a dose-response relationship between serum IL-10 levels and the amount of injected DNA. The serum IL-10 levels peaked at day 1 and then were sustained for at least 2 weeks. These results demonstrate that the kidney-targeted naked plasmid DNA transfer of mice by retrograde renal vein injection can be achieved, and the kidney serves as a depot organ for the production of large proteins.     

Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge.

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.     

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.     

A DNA vaccine candidate for <Emphasis Type="Italic">B. anthracis</Emphasis> immunization,pcDNA3.1+PA plasmid,induce Th1/Th2 mixed responses and protection in mice

The protective antigen (PA) of Bacillus anthracis (B. anthracis) is a potent immunogen and a candidate subunit vaccine. To address the question whether antibodies raised against PA following injection of pcDNA3.1+PA plasmid, encoding PA, can protect against virulent B. anthracis two different regimens of PA based vaccines (DNA and live spore) were used. The groups of BALB/c mice that received live spores of the Sterne strain, naked pcDNA3.1 and naked pcDNA3.1+PA were compared to control groups. All groups were injected three times with 30-day intervals. Two weeks after the last immunization, all mice were subjected to challenge with a pathogenic strain of B. anthracis (C2). Blood samples were taken before each injection and challenge. Evaluation of the sera by ELISA method showed that DNA immunization using pcDNA3.1+PA plasmid resulted in an antibody profile representative of a mixed Th1 and Th2 response, with a skewing to a Th1 response. The group which received the naked pcDNA3.1+PA had a survival rate of >80%. This challenge assay revealed that antibodies raised following DNA vaccination against PA can confer strong protection, and resistance against virulent species of B. anthracis.     

In vivo DNA electrotransfer into muscle

Naked plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed. Among the non-viral techniques for gene transfer in vivo , this method is especially simple, inexpensive, and safe. However, the relatively low expression levels attained by this method have limited its applications for uses other than as a DNA vaccine. We and other groups investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA vector. The results demonstrated that gene transfer into muscle by in vivo electroporation is far more efficient than simple intramuscular DNA injection and provides a potential approach to systemically delivering cytokines, growth factors, and other serum proteins for basic research and human gene therapy.     

Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor γ (TCR Vγ) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (Vγ^LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted Vγ locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (Vγ^Δ) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vγ^Δ locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined Vγ^LNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the Vγ^LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. Mol Reprod Dev 46:109–113, 1997. © 1997 Wiley-Liss, Inc.     

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Modulation of ATR-mediated DNA damage checkpoint response by cryptochrome 1

Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent manner. The DDC capacity in response to UV irradiation showed a circadian rhythm. Interestingly, clock-deficient Cry1 and Cry2 double knockout (Cry^DKO) cells retained substantial DDC capacity compared with clock-proficient wild-type cells, although the Cry1-modulated oscillation of the DDC capacity was abolished in Cry^DKO cells. We found temporal interaction of Cry1 and Tim in the nucleus. When Cry1 was expressed in the nucleus, it was critical for circadian ATR activity. We regenerated rhythmic DDC responses by ectopically expressing Cry1 in Cry^DKO cells. In addition, we also investigated the DDC capacity in the liver of mice that were intraperitoneally injected with cisplatin at different circadian times (CT). When mice were injected at CT20, about 2-fold higher expression of phosphorylated minichromosome maintenance protein 2 (p-MCM2) was detected compared with mice injected at CT08, which consequently affected the removal rate of cisplatin-DNA adducts from genomic DNA. Taken together, our data demonstrate the intimate interaction between the circadian clock and the DDC system during genotoxic stress in clock-ticking cells.     

Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.     

Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-microl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 microl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-microl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.     

重组恶性疟原虫DNA质粒免疫小鼠及抗原表达的调控

将恶性疟原虫MSP1-31基因序列,引入四环素(Tc)控制的真核表达载体pTRE,获得重组质粒pTRE-31。将MSP1-31的原核表达载体pDS56T1转化大肠杆菌表达MSP1-31,亲和纯化后作检测用抗原。pTRE-31与辅助质粒pTet-off(tTA)肌肉注射4周龄BALB/c小鼠,观察DNA介导免疫情况。结果显示,四环素饲喂的小鼠4周时血清抗体阳转率为7.1%(1/14),而不饲喂四环素组可达100%(14/14),表明用pTRE-31/pTet-off重组质粒组合直接注射小鼠有效地引发了针对疟原虫MSP1-31抗原的体液免疫反应,且可受控于四环素。不饲喂四环素组小鼠在12周后仍能维持抗体阳性,倍比稀释ELISA显示血清抗体滴度在4周、8周和12周内持续上升。饲喂四环素组小鼠4周后停止饲喂Tc,第8周和第12周检测仍有部分(60%)小鼠血清抗体阳转,而继续饲喂Tc的小鼠未有抗体阳转,暗示重组质粒DNA在小鼠体内可持续存在至少4周并仍具备表达功能。