Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.     

Oral immunogenicity of human papillomavirus-like particles expressed in potato

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.     

Expression of the B Subunit of E. coli Heat-labile Enterotoxin in the Chloroplasts of Plants and its Characterization

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.     

Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice

High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to ～4% and ～40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24. Oral administration of a partially purified preparation of chloroplast-derived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.     

Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system

Vaccine antigens have been successfully produced in transgenic plants for oral immunization. Recently, a fusion strategy has been adopted to produce multicomponent vaccines and to target antigens to mucosal sites for enhanced oral immunogenicity. However, antigen fusions may not be folded correctly due to steric hindrance and may thus lose their potency. Here, we describe an Agrobacterium-mediated transient assay that provides enough antigen-expressing material at 2 days post-transfection to evaluate antigen conformation. Using the hepatitis B surface antigen (HBsAg) as a model antigen and the green fluorescent protein (GFP) as a model fusion partner, we showed that transiently expressed HBsAg and an HBsAg fusion with GFP at the N-terminus (GFP:HBsAg), but not the HBsAg fusion with GFP at the C-terminus (HBsAg:GFP), formed the 'a' determinant and virus-like particles (VLPs), similar to yeast-derived vaccine HBsAg. Thus, it is feasible to modify the HBsAg with an N-terminal fusion of up to 239 amino acids without altering its major antigenic properties. Our results also demonstrate that the Agrobacterium-mediated transient expression system can be used to evaluate the conformation of plant-based vaccines or other pharmaceutical proteins in a high-throughput manner.     

Production of human papillomavirus type 16 L1 virus-like particles by recombinant Lactobacillus casei cells

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.     

Immunization with a pentameric L1 fusion protein protects against papillomavirus infection

The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathione S-transferase (GST) fusion protein in Escherichia coli and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.     

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts,Bio-Encapsulation,Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。     

Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by Recombinant Lactobacillus casei Cells

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.     

Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco

Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production. In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native viral (L1^v gene) or a synthetic sequence optimized for expression in plant plastids (L1^pt gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up to 1.5% total leaf proteins only when vectors included the 5′-UTR and a short N-terminal coding segment (Downstream Box) of a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes and by electron microscopy. Contribution No. 129 from CNR-IGV, Portici.     

Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52–90 μg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection.     

Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T₀, T₁ and T₂ generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Enhancement of vaccine potency by fusing modified LTK63 into human papillomavirus type 16 chimeric virus-like particles

To enhance the immunogenicity of human papillomavirus 16 (HPV 16) virus-like particles (VLPs), the modified adjuvant, mLTK63, was fused to the C-terminus of HPV 16 L2 protein. Coexpression of HPV 16 L1 and L2-mLTK63 proteins in insect cells led to the efficient assembly of HPV 16 L1/L2-mLTK63 chimeric VLPs (cVLPs), which combined the antigen and adjuvant as a unit. Compared with HPV 16 L1/L2 VLPs, the HPV 16 L1/L2-mLTK63 cVLPs had similar structural biology characteristics and binding activities with the cell surface receptors and HPV 16-specific neutralizing monoclonal antibodies. Intramuscular immunization of BALB/c mice with the HPV 16 L1/L2-mLTK63 cVLPs could induce higher titers of HPV 16-specific long-lasting neutralizing serum antibodies and stronger splenocyte proliferation, Th1- and Th2-type cytokines and CD4(+) Th responses than HPV 16 L1/L2 VLPs. The results suggested that it is possible to enhance the immunogenicity of HPV VLP vaccines via a strategy of fusing effective adjuvant protein into cVLPs.     

Synthesis and assembly of dengue virus envelope protein fused to cholera toxin B subunit into biologically active oligomers in transgenic tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

Dengue is the fastest growing mosquito-borne disease worldwide, causing nearly 400 million infections annually. A universally applicable dengue virus vaccine is required to arrest its spread. Here, we generated an edible dengue vaccine by expressing the dengue fusion protein in tomatoes, which is a desirable expression system owing to the inherent adjuvanticity of alpha tomatine and immunogenicity of the tomato lectin/microbial antigen complex. The B subunit of Vibrio cholera toxin (CTB) was genetically fused to dengue envelope antigen for improved delivery to antigen-presenting cells and enhanced immunogenicity, while avoiding immunological tolerance. We utilized domain III of the dengue envelope protein (EDIII), as it has been shown to induce serotype-specific neutralizing antibodies. The CTB–EDIII fusion gene construct containing an endoplasmic reticulum target sequence was introduced into tomato plants by Agrobacterium tumefaciens-mediated gene transformation, and the expression of CTB–EDIII in transgenic plants was confirmed by DNA, RNA and protein analyses. Accumulated fusion protein accounted for up to 0.015 % of total soluble protein, and it assembled into fully functional pentamers as demonstrated by binding to GM1 ganglioside. Future work will involve testing of transgenic tomatoes for immunogenicity in mice following oral delivery.     

A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response

Capsomeres are considered to be an alternative to viruslike particle (VLP)-based vaccines as they can be produced in prokaryotic expression systems. So far, no detailed side-by-side comparison of VLPs and capsomeres has been performed. In the present study, we immunized mice with insect cell-derived human papillomavirus type 16 VLPs and capsomeres. VLPs induced consistently higher antibody titers than capsomeres but the two forms induced similar CD8 T-cell responses after subcutaneous, intranasal, and oral immunization, and at least 20 to 40 times more L1 in the form of capsomeres than in the form of VLPs was needed to achieve comparable antibody responses. These results were confirmed by DNA immunization. The lower immunogenicity of capsomeres was independent of the isotype switch, as it was also observed for the early immunoglobulin M responses. Although there were differences in the display of surface epitopes between the L1 particles, these did not contribute significantly to the differences in the immune responses. capsomeres were less immunogenic than VLPs in Toll-like receptor 4 (TLR4)-deficient mice, suggesting that the lower immunogenicity is not due to a failure of capsomeres to trigger TLR4. We observed better correlation between antibody results from enzyme-linked immunosorbent assays and neutralization assays for sera from VLP-immunized mice than for sera from capsomere-immunized mice, suggesting qualitative differences between VLPs and capsomeres. We also showed that the lower immunogenicity of capsomeres could be compensated by the use of an adjuvant system containing MPL. Taken together, these results suggest that, presumably because of the lower degree of complexity of the antigen organization, capsomeres are significantly less immunogenic than VLPs with respect to the humoral immune response and that this characteristic should be considered in the design of putative capsomere-based prophylactic vaccines.     

Contribution of virus-like particles to the immunogenicity of human immunodeficiency virus type 1 Gag-derived vaccines in mice

The human immunodeficiency virus type 1 (HIV-1) Gag protein is a major target antigen for cytotoxic-T-lymphocyte-based vaccine strategies because of its high level of conservation. The murine model has been used extensively to evaluate potential HIV-1 vaccines. However, the biology of HIV-1 Gag is somewhat different in human and murine tissues. The ability of HIV-1 Gag to form virus-like particles (VLPs) in human cells is severely curtailed in murine cells. Hence, it is not known whether immunizing mice with expression vectors encoding HIV-1 Gag provides an accurate assessment of the immunogenicity of these candidate vaccines in primates. In this report, we made use of a chimeric Moloney murine leukemia virus (MMLV)-HIV-1 Gag in which the p17 matrix domain of HIV-1 was replaced with the p15 matrix and p12 domains from MMLV. Murine cells expressing this construct released significant amounts of VLPs. The construct preserved H-2d-restricted antigenic determinants in the remaining portion of HIV-1 Gag, allowing immunogenicity studies to be performed with mice. We demonstrated that immunizing mice with plasmid DNA or adenoviral vectors encoding this chimeric Gag did not significantly increase the HIV-1 Gag-specific cellular or humoral immune response when compared to immunization with a myristoylation-incompetent version of the construct. Thus, the release of VLPs formed in vivo may not play a major role in the immunogenicity of vectors expressing HIV-1 Gag constructs.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.