鼠透明带3(ZP3)融合蛋白表达以及抗血清制备

鼠透明带3（mZP3)作为精子的初级受体,是鼠透明带中的一种主要糖蛋白,抗鼠透明带3抗体能够阻断精卵的结合,达到不育的效果,因此mZP3是免疫避孕研究的重要候选抗原。从小鼠卵巢中提取总RNA,分离mRNA,反转录获得cDNA,将cDNA连接到测序载体pUCm-T质粒上,通过序列测定和分析得到正确的mZP3 cDNA。经内切酶EcoR I和XhoI处理,将mZP3 cDNA克隆至融合蛋白表达载体pGEX-4T-1中,在T4 DNA连接酶的作用下构建融合表达载体pGEX-mZP3,转化大肠杆菌BL－21菌株,利用IPTG诱导后获得可溶性的蛋白质产物,经过SDS－PAGE鉴定,表达的融合蛋白GST－mZP3分子质量约为72kD左右,纯化的融合蛋白免疫兔子,获得效价为1：1000的抗血清,Western blot检测抗血清具有针对mZP3融合蛋白的专一性,为进一步开展mZP3的免疫功能检测的研究奠定 了基础。     

Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro

Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-huIgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.     

Transgenic mice with reduced numbers of functional sperm receptors on their eggs reproduce normally.

To initiate fertilization in mice, free-swimming sperm bind to mZP3, an approximately 83-kDa glycoprotein present in the ovulated egg zona pellucida (ZP). mZP3 is located periodically along the filaments that constitute the ZP. Sperm recognize and bind to specific oligosaccharides linked to one or more of five Ser residues clustered in the carboxy-terminal one-third of the mZP3 polypeptide. When all five Ser residues are converted to nonhydroxy amino acids by site-directed mutagenesis of the mZP3 gene, an inactive form of mZP3, called mZP3[ser], is secreted by embryonal carcinoma cells stably transfected with the mutated gene. Here, seven independent transgenic mouse lines were established that harbor the mutated mZP3 gene. In all lines, the mutant gene is expressed by growing oocytes and mZP3[ser] is synthesized, secreted, and incorporated into the ZP. Purified mZP3[ser] prepared from ovaries of transgenic mice, like mZP3[ser] from transfected embryonal carcinoma cells, is inactive in sperm binding assays in vitro. On the other hand, the presence of mZP3[ser] in the ZP does not significantly affect either the binding of sperm to ovulated eggs in vitro or the reproduction of the mice, i.e., the transgenic mice are fertile, breed at normal intervals, and produce litters of normal sizes. These results indicate that the number of functional sperm receptors in the ZP can be reduced by more than 50% without adversely affecting fertilization of eggs in vivo.     

Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.     

重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

摘要：【目的】为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响。【方法】我们将HBV DNA疫苗（pcDS2）和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTT和流式细胞仪检测T细胞增殖反应,及流式细胞仪检测细胞因子表达水平。【结果】pcDS2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后, 联合免疫组刺激指数(SI)明显降低;经流式细胞仪检测进一步证实联合免疫组T细胞增殖反应被显著抑制;联合免疫组T细胞表达IL-10和Foxp3水平显著升高。【结论】pcDS2和HBsAg联合免疫能诱导产生特异性体液免疫应答,但不能诱导产生抗原特异性T细胞增殖反应;T细胞增殖反应被显著抑制可能与T细胞表达IL-10和Foxp3上调有关;本研究为急性乙型肝炎和爆发性乙型肝炎治疗及HBV疫苗的研究奠定了基础。     

Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3+/+ and mZP3−/− mice

The mouse egg extracellular coat, or zona pellucida (ZP), is composed of three glycoproteins, called mZP1–3, which are synthesized and secreted concomitantly by growing oocytes. Disruption of the mZP3 gene by targeted mutagenesis yields mice that are homozygous nulls (mZP3^−/−). Growing oocytes from mZP3^−/− mice do not synthesize mZP3 mRNA or protein and, as a result, do not assemble a ZP. Here, we examined secretion of mZP2 by growing oocytes and eggs from mZP3^−/− mice, as well as incorporation of mZP2 into the ZP of oocytes from mZP3^+/+ mice. Laser scanning confocal microscopy (LSCM) of antibody‐labeled samples showed that, indeed, mZP2 was synthesized and secreted by oocytes isolated from mZP3^−/− mice and cultured in vitro. Nascent mZP2 was found in the culture medium, associated with the surface of the plasma membrane of growing oocytes, and in the oocyte cytoplasm. By contrast, mZP2 was barely detectable at any of these sites when ovulated eggs from mZP3^−/− mice were examined. Examination of oocytes from wild‐type (mZP3^+/+) mice showed that, while a portion of nascent mZP2 was assembled into the ZP (approximately 40%), here too a significant fraction was secreted into the culture medium (approximately 60%). Similar results also were obtained when intact pre‐antral follicles were isolated from mZP3^+/+ mice and cultured in vitro. Several of these observations are consistent with previous results obtained with oocytes from heterozygous null mice (mZP3^+/−). Furthermore, the results suggest that ZP assembly from nascent glycoproteins may be a stochastic process that requires the presence of both mZP2 and mZP3 and occurs completely outside the growing oocyte. Dev. Genet. 25:95–102, 1999. © 1999 Wiley‐Liss, Inc.     

Zona pellucida glycoproteins based immunocontraceptive vaccines: strategies for development and their applications

The mammalian oocyte is surrounded by an extra-cellular matrix, the zona pellucida (ZP), composed of three major glycoproteins (ZP1, ZP2 and ZP3). The ZP glycoproteins, by virtue of their tissue specificity and critical role during mammalian fertilization, have emerged as potential candidate antigens for the development of an immunocontraceptive vaccine. Molecular characterization of ZP glycoproteins from several species, reveals a variable degree of homology among the deduced primary amino acid sequences, which provided an opportunity to undertake active immunization studies in heterologous animal models. Active immunization of various animal species with either native ZP glycoproteins or those obtained by recombinant DNA technology led to the inhibition of fertility. Thus ZP glycoproteins based immunocontraceptive vaccines offer an attractive proposition for controlling wild life population. To make it a practical proposition, additional research inputs are required to optimize and devise novel strategies for vaccine delivery. Observed ovarian dysfunction, often associated with immunization by ZP glycoproteins is one of the major stumbling blocks for their use in humans. Ongoing studies to delineate appropriate B cell epitopes of ZP glycoproteins that are devoid of oophoritogenic T-cell epitopes, which will inhibit fertility without concomitant oophoritis, will be critical to determine their feasibility for human use.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3 glycoproteins from mouse zona pellucida

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian egg plasma membrane, is a relatively simple structure consisting of three to four glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) binding sites for spermatozoa. The two zona components are highly glycosylated containing N-linked and O-linked glycan units. In an attempt to characterize N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate chains were released by exhaustive digestion with N-glycanase. The released oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks indicating the presence of a variety of N-linked glycans. Interestingly, the radioactive peaks resolved from mZP2 and mZP3 were quite different, a result suggesting qualitative and quantitative differences in the glycans. The [SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and fractionated by serial lectin chromatography. Experimental evidence included in this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans eluted from the immobilized lectin columns were further characterized by lectin and sizing column chromatography before or after digestion with endo-/ exo-glycohydrolases. Data revealed the presence of a variety of OSs, including poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and high-mannose-type glycans. Taken together, these results provide additional evidence on the complex nature of the glycan chains present on mZP glycoconjugates.     

Differential O-glycosylation of a conserved domain expressed in murine and human ZP3

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.     

Autoimmune ovarian inflammation triggered by proinflammatory (Th1) T cells is compatible with normal ovarian function in mice.

The detection of noninfectious ovarian inflammation (oophoritis) and serum ovarian autoantibodies in a patient with premature ovarian failure is indicative of an autoimmune etiology. The mechanisms of autoimmune ovarian injury leading to loss of function are currently unknown. In this study we investigated the impact of oophoritis on ovarian function based on two murine autoimmune ovarian disease (AOD) models. AOD can be induced by thymectomy at Day 3 after birth (d3tx). D3tx mice develop ovarian inflammation and atrophy with loss of oocytes. In these mice, ovarian atrophy and not oophoritis correlated with abnormal estrous cyclicity. The second AOD model is induced by active immunization of adult mice with a murine ZP3 peptide (pZP3) in adjuvant. After active immunization, the zona pellucida antibody titer, not oophoritis, correlated with reduced fertility. To investigate the effect of oophoritis in the absence of antibody response or ovarian atrophy, pZP3-specific T cells were passively transferred into naive syngeneic mice. This recruited cytokine-producing cells into the ovaries so that elevated cytokine production and its effect on ovarian function could be examined. Recipients of pZP3-specific T cells developed severe granulomatous oophoritis, and the diseased ovaries had elevated ovarian mRNA levels of interferon-gamma, interleukin-1beta, and tumor necrosis factor alpha. Despite these changes, fertility rates and gonadotropin-induced follicular development remained essentially normal. Therefore, normal ovarian function is compatible with severe ovarian inflammation mediated by autoreactive T cells.     

Contribution of mouse egg zona pellucida glycoproteins to gamete recognition during fertilization

For sperm to fertilize eggs, they must first bind to the thick zona pellucida (ZP) that surrounds the plasma membrane of all unfertilized mammalian eggs. An extensive literature suggests that mouse sperm recognize and bind to a specific ZP glycoprotein called mZP3. However, the role of individual ZP glycoproteins in binding of mouse sperm to eggs has been called into question by recent transgenic experiments with null mice. Results of such experiments have been interpreted to mean that binding of sperm depends on the supramolecular structure of the ZP, not on an individual ZP glycoprotein. Here, it is argued that results of these transgenic experiments actually are consistent with the prevailing view of gamete recognition that implicates a specific ZP glycoprotein in both binding of mouse sperm to eggs and induction of the acrosome reaction.     

The Parkes Lecture. Zona pellucida glycoprotein mZP3: a versatile player during mammalian fertilization

All mammalian eggs are surrounded by a relatively thick extracellular coat, the zona pellucida (ZP), which facilitates fertilization of eggs by a single spermatozoon. The mouse egg ZP is constructed of only three glycoproteins, termed mZP1-3. Each of these glycoproteins consists of a unique polypeptide that is heterogeneously glycosylated with both asparagine-(N-)linked and serine/threonine-(O-)linked oligosaccharides. Polypeptides of ZP glycoproteins are highly conserved among mammalian species and are similar to polypeptides of egg vitelline envelope glycoproteins of fish, birds and amphibians. One of the mouse ZP glycoproteins, mZP3, serves as both a receptor for spermatozoa and an inducer of the acrosome reaction during fertilization. Free-swimming acrosome-intact spermatozoa recognize and bind to certain serine-(O-)linked oligosaccharides located close to the carboxy terminus of mZP3 polypeptide and, after binding, undergo the acrosome reaction (cellular exocytosis). In this review, in addition to the background information presented, results of recent experiments using homologous recombination to produce mZP3 null mice and site-directed mutagenesis to inactivate mZP3 as a sperm receptor and inducer of the acrosome reaction are presented and discussed.     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Identification of a region of mouse zona pellucida glycoprotein mZP3 that possesses sperm receptor activity.

The ability of mouse zona pellucida glycoprotein ZP3 (mZP3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 108, 1-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, approximately 55,000 apparent M(r), that exhibited sperm receptor activity in vitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function.     

小鼠卵ZP3蛋白的可溶性表达、纯化及免疫活性鉴定

哺乳动物卵透明带3(Zona pellucida 3,ZP3)在诱导获能精子发生顶体反应中发挥着重要作用,其在大肠杆菌中主要以包涵体形式表达。本研究在大肠杆菌中诱导表达可溶性的mZP3融合蛋白,并鉴定该蛋白的免疫活性。将小鼠卵透明带3克隆至pMAL-p2x质粒,转化至大肠杆菌BL21表达菌中。用不同温度、不同IPTG浓度、不同诱导时间及不同浓度的添加剂诱导目的蛋白表达,以筛选出mZP3融合蛋白可溶性表达的最佳条件。大量表达纯化后以Western blotting和ELISA检测蛋白的免疫活性。酶切鉴定及DNA测序表明mZP3已克隆入pMAL-p2x质粒。经优化诱导表达条件,筛选出mZP3融合蛋白可溶性表达的最佳条件为:当A600为0.6时添加0.02 mol/L葡萄糖,以0.6 mmol/L IPTG于25oC条件下诱导表达4 h。ELISA及Western blotting检测结果表明诱导表达的蛋白具有免疫活性。在大肠杆菌中表达并纯化获得可溶性mZP3蛋白,为mZP3免疫不育疫苗研制及其免疫效果检测提供了可溶性抗原。     

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.