DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

Bacterial ghosts are an efficient delivery system for DNA vaccines

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52-60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for beta-galactosidase under the control of the CMV immediate early gene promoter (pCMVbeta) stimulates more efficient Ag-specific humoral and cellular (CD4(+) and CD8(+)) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVbeta-containing ghosts also resulted in the elicitation of beta-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.     

Upconversion nanoparticles modified with aminosilanes as carriers of DNA vaccine for foot-and-mouth disease

The potential of the upconversion nanoparticles NaYF(4):Yb/Er@silica(UCPs)/plasmid DNA (pcDNA3.1/VP1-GFP) complex in inducing immune responses was evaluated using the UCPs as carriers of the foot-and-mouth disease virus (FMDV AsiaI/Jiangsu2005) DNA vaccine. The UCPs protection against DNaseI degradation was measured using an in vitro inhibition assay. The expression of the plasmid in vivo was determined via confocal microscopy. Its biocompatibility was evaluated through cytotoxicity assay. Based on the results, the aminosilane-modified UCPs can electrostatically bind, condense, and protect plasmid DNA. Cell viability assays demonstrated that the cytotoxicity of the UCPs/plasmid DNA complex is lower than that of the cationic lipid/plasmid DNA complex, and that the transfection efficiency of UCPs is the same as that of the cationic lipid. Furthermore, the UCP/plasmid DNA complex was intramuscularly administered to guinea pigs. Humoral and cellular immune responses were detected using indirect enzyme-linked immunosorbent assay (ELISA), micro-neutralization assay, and T-lymphocyte proliferation assay. Anti-FMDV specific antibodies, neutralizing antibodies, and T-lymphocyte proliferation responses were induced after vaccination. In the challenge test, all the guinea pigs vaccinated with the UCPs/plasmid DNA complex were fully protected from the FMDV challenge. The current study encourages the use of UCPs as an effective nanosystem for gene delivery to cells for in vitro and in vivo vaccination, and other therapeutic applications.     

Application of the shsp gene,encoding a small heat shock protein,as a food-grade selection marker for lactic acid bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

Suppression of immune response and protective immunity to a Japanese encephalitis virus DNA vaccine by coadministration of an IL-12-expressing plasmid

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-gamma gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-gamma. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

In Vivo Molecular Imaging and Histological Analysis of Changes Induced by Electric Pulses Used for Plasmid DNA Electrotransfer to the Skin: A Study in a Dorsal Window Chamber in Mice

Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a “vascular lock.” Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1?h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24?h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer

Induction of acid resistance (habituation) in Escherichia coli at pH 5·0 took ca 5 min in broth at 37°C and 30–60 min in minimal medium. Induction occurred at a range of pH values from 4·0 to 6·0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5·0 retained most of their resistance after 2 h growth at pH 7·0. Organisms grown at pH 5·0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7·0. DNA repair-deficient strains carrying recA, uvrA or polAl mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5·0. Organisms grown at pH 5·0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7·0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5·0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

DNA Immunization against Herpes Simplex Virus: Enhanced Efficacy Using a Sindbis Virus-Based Vector

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508–519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.     

Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.     

Induction of a cytotoxic T lymphocyte (CTL) response to plasmid DNA delivered via Lipodine liposomes

We have previously shown that liposome-mediated plasmid DNA immunisation may be a preferred alternative to the use of naked DNA. Lipodine DNA formulations consist of liposomes containing entrapped DNA plasmid by the dehydration-rehydration (DRV) method. Such liposome formulations are distinct from liposomes with externally complexed DNA in that the majority of the DNA is "internal" to the liposome structure and hence protected from DNAase degradation. Previous studies on the immune response induced by DNA vaccines entrapped in Lipodine have focused on the humoural response. In the present study, we have expanded the analysis profile in order to include the cytotoxic T lymphocyte (CTL) component of the immune response. We have analysed the immune response induced by DNA entrapped in Lipodine compared to that induced by DNA alone when delivered subcutaneously, a route of administration not normally inducing significant plasmid DNA mediated immune activation. Our results indicate that delivery of a small dose of plasmid DNA in Lipodine results in an improved antibody response to the plasmid encoded antigen and a strong antigen specific CTL response compared to that induced by DNA delivered alone.     

The injection of plasmid DNA in mouse muscle results in lifelong persistence of DNA, gene expression, and humoral response

The duration of the immune response against any vaccine is critical. The present study was performed to determine the stability of injected plasmid deoxyribonucleic acid (DNA), the duration of gene expression in mouse muscle, as well as the duration of the immune response generated in mice after injection of plasmid pSO2C1 harboring the cry11Bb gene of Bacillus thuringiensis serovar. medellin. The localization and the persistence of the inoculated gene were determined by in situ hybridization and polymerase chain reaction (PCR). The results demonstrated that plasmid DNA can persist in mouse muscle for up to 2 yr. Moreover, immunohistochemical analysis showed that Cry11Bb protein was expressed for the lifetime of the mice at a low but significant level. Finally, production of Cry11Bb-specific antibodies in mice injected with pSO2C1 was high and durable as significant antibody titers were observed up to 119 wk after injection of the plasmid. This persistent immune response is likely owing to the existence of a protein and/or DNA depot in the organism, which serves to maintain the immune response, acting as a secondary or booster immunization.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Mechanisms of increased immunogenicity for DNA-based vaccines adsorbed onto cationic microparticles

Investigation into the mechanism of action of vaccine adjuvants provides opportunities to define basic immune principles underlying the induction of strong immune responses and insights useful for the rational development of subunit vaccines. A novel HIV vaccine composed of plasmid DNA-encoding p55 gag formulated with poly-lactide-co-glycolide microparticles (PLG) and cetyl trimethyl ammonium bromide (CTAB) elicits both serum antibody titers and cytotoxic lymphocyte activity in mice at doses two orders of magnitude lower than those required for comparable response to plasmid DNA in saline. Using this model, we demonstrated the increase in potency requires the DNA to be complexed to the PLG-CTAB microparticles. Furthermore, the PLG-CTAB-DNA formulation increased the persistence of DNA at the injection site, recruited mononuclear phagocytes to the site of injection, and activated a population of antigen presenting cells. Intramuscular immunization with the PLG-CTAB-DNA complex induced antigen expression at both the injection site and the draining lymph node. These findings demonstrate that the PLG-CTAB-DNA formulation exhibits multiple mechanisms of immunopotentiation.     

Transplacental genetic immunization after intravenous delivery of plasmid DNA to pregnant mice.

A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.     

INDUCTION OF A CYTOTOXIC T LYMPHOCYTE (CTL) RESPONSE TO PLASMID DNA DELIVERED VIA LIPODINE™ LIPOSOMES*

ABSTRACT

We have previously shown that liposome-mediated plasmid DNA immunisation may be a preferred alternative to the use of naked DNA. Lipodine? DNA formulations consist of liposomes containing entrapped DNA plasmid by the dehydration–rehydration (DRV) method. Such liposome formulations are distinct from liposomes with externally complexed DNA in that the majority of the DNA is “internal” to the liposome structure and hence protected from DNAase degradation. Previous studies on the immune response induced by DNA vaccines entrapped in Lipodine? have focused on the humoural response. In the present study, we have expanded the analysis profile in order to include the cytotoxic T lymphocyte (CTL) component of the immune response. We have analysed the immune response induced by DNA entrapped in Lipodine? compared to that induced by DNA alone when delivered subcutaneously, a route of administration not normally inducing significant plasmid DNA mediated immune activation. Our results indicate that delivery of a small dose of plasmid DNA in Lipodine? results in an improved antibody response to the plasmid encoded antigen and a strong antigen specific CTL response compared to that induced by DNA delivered alone.     

含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

研究了庚型肝炎病毒E2(HGV E2)基因片段作为DNA疫苗的可行性。将来自于质粒pThioHis-E2编码HGV E2的基因片段(559bp)亚克隆到质粒pCMV-S中,使之和HBsAg基因位于同一阅读框,形成重组质粒pCMV-S-E2。用纯化的质粒pCMV-S-E2 DNA注射到昆明小鼠后腿四头肌中来免疫小鼠,同时用pCMV-S作为对照。间隔14天再加强一次免疫。在加强免疫后的第8天眼眶取血。用E2—GST融合蛋白作为固定化抗原,通过ELISA检测受试小鼠的体液免疫应答。结果表明,用质粒pCMV-S-EDNA免疫的小鼠可以产生很强的体液免疫应答。