Escheriosomes entrapped DNA vaccine co-expressing Cu-Zn superoxide dismutase and IL-18 confers protection against Brucella abortus

In the present study, we evaluated prophylactic prospective of liposome based DNA vaccine co-expressing Cu-Zn superoxide dismutase (SOD) along with interleukin-18 (IL-18) against experimental murine brucellosis. The immunization schedule involves liposome-mediated delivery of pVsod (encoding SOD of Brucella abortus) and pVIL18-sod (encoding IL-18 of mouse and SOD of B. abortus) DNA constructs. The data highlight potential of Escherichia coli lipid liposome (escheriosome) based DNA delivery vehicle to induce SOD specific humoral and cellular immune responses in the immunized mice. The co-expression of SOD along with IL-18 ensued in higher lymphoproliferative response and IFN-gamma production in comparison to the group of animals that were immunized with free form of SOD-DNA. Antibody response developed upon immunization with both DNA vaccines was of IgG2a type mainly. The results of the present study show that co-expression of IL-18 along with SOD polarized the antigen specific immune responses toward Th-1 direction, a desirable feature to control intracellular pathogens.     

Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development.

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.     

CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%.     

Rapid clearance of a recombinant Salmonella vaccine carrier prevents enhanced antigen-specific CD8 T-cell responses after oral boost immunizations

The type III secretion system of Salmonella enterica serovar Typhimurium can be used to target heterologous antigens directly into the cytosol of antigen-presenting cells. Our laboratory has previously reported that the single oral immunization of mice with a recombinant Salmonella strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection. In the present study, we investigated whether these antigen-specific cytotoxic T lymphocytes induced by the prime immunization contribute to a more rapid clearance of the vaccine carrier after subsequent boost immunizations and whether oral boost immunizations lead to an augmented p60-specific CD8 T-cell response. We found that the ability of recombinant Salmonella strains to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after boost immunizations but that this effect was independent of existing CD8 T cells reactive with p60(217-225). A significant elevation of antigen-specific CD8 T cells could not be detected by enzyme-linked immunospot assay after the second or the third oral immunization, possibly due to the rapid clearance of the bacterial vaccine carrier from lymphatic organs.     

携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

丙型肝炎病毒(HCV)核心蛋白是丙肝疫苗的重要候选抗原,然而,该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒,一种是体内激活型原核表达质粒pZW-C,另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207,得到重组菌SL7207/pZW-C和SL7207/pCI-C,分别将重组菌口服接种小鼠,检测小鼠的免疫应答,结果发现:①SL7207/pCI-C免疫鼠的CD3 CD4 T细胞持续降低,而SL7207/pZW-C免疫鼠的CD3 CD4 T细胞无明显改变;②SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体,加强免疫对抗体阳转率及抗体水平无明显影响,而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示:携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式(结构以及磷酸化修饰)的HCV核心蛋白,可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题,并具有接种方便,成本低廉等优点,从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

Fusogenic potential of prokaryotic membrane lipids. Implication in vaccine development.

Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral- and cell mediated-immunity. The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses. In contrast, the activation of a cell-mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells. The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids. These lipids were shown to induce strong membrane-membrane fusion as evident from resonance energy transfer and content mixing assays. Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells. Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes. Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses.     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Prophylactic anti-tumor immunity against a murine fibrosarcoma triggered by the Salmonella type III secretion system

The potential of an attenuated Salmonella enterica serovar Typhimurium strain as a prophylactic anti-tumor vaccine against the murine fibrosarcoma WEHI 164 was evaluated. Tumor cells were transfected with the DNA sequence encoding the MHC class I-restricted peptide p60(217-225) from Listeria monocytogenes. BALB/c mice received a single orogastric immunization with Salmonella that translocates a chimeric p60 protein via its type III secretion system. Mice were subsequently challenged subcutaneously with p60(217-225)-expressing WEHI cells. In vivo protection studies revealed that 80% of these mice remained free of the fibrosarcoma after challenge, whereas all animals of the non-vaccinated control group did develop tumor growth. In further experiments, the distribution of tetramer-positive p60(217-225)-specific effector and memory CD8 T cells after Salmonella-based immunization and tumor application was analyzed. Costaining with CD62L and CD127 revealed a predominance of p60-specific central memory and effector memory CD8 T cells in spleens, whereas in blood samples the majority of p60-specific lymphocytes belonged to effector and effector memory CD8 T cell subsets. This is the first report demonstrating that a bacterial type III secretion system can be used for heterologous antigen delivery to induce cytotoxic effector and memory CD8 T cell responses resulting in an efficient prevention of tumor growth.     

鼠伤寒杆菌主要外膜蛋白作为保护性抗原的研究

采用超声破碎,ＴｒｉｔｏｎＸ─１００处理和Ｓｅｐｈａｃｒａｌ超细Ｓ─３００凝胶过滤技术提取了鼠伤寒杆菌的主要外膜蛋白（ＭＯＭＰｓ）。ＭＯＭＰｓ的脂多糖（ＬＰＳ）含量约为０．２％。经ＳＤＳ─ＰＡＧＥ图谱显示蛋白在３６─４１ＫＤ之间。ＭＯＭＰｓ能使小鼠产生典型的足垫肿胀（ＤＴＨ）及高水平的ＩＬ─２;可保护５００ＬＤ５０鼠伤寒杆菌及伤寒杆菌的攻击,其免疫保护率分别为９０％和３３．３％,用５０ｕｇＭＯＭＰｓ免疫的小鼠的Ｔ淋巴细胞经尾静脉注射给非免疫小鼠,可使后者得到被动免疫保护,其保护率为４２．９％。基于上述实验结果,本文认为鼠伤寒杆菌的ＭＯＭＰｓ是一良好的保护性抗原。     

Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21days. In contrast, the period of colonization was significantly shortened to 6days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis.     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.     

Expressed Salmonella antigens within macrophages enhance the proliferation of CD4+ and CD8+ T lymphocytes by means of bystander dendritic cells

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.     

Critical role of CD28 in protective immunity against Salmonella typhimurium

Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.     

Internal initiation sites of de novo RNA synthesis within the hepatitis C virus polypyrimidine tract

Using fluorescein isothiocyanate (FITC)-conjugated H-2K(b) CTL epitope (SIINFEKL) as a model system, we investigated the antigen delivery route by pH-sensitive liposomes in vivo. Fluorescence was initially detected in lymph nodes at 3 h after immunization, and its intensity reached a peak value in superticial inguinal lymph node at 9 h. No trace could be detected in spleen even with prolonged monitoring for up to 24 h. These results strongly suggest that the presentation of CTL-peptide antigen vehicled by pH-sensitive liposomes exclusively occurs in lymph nodes. In mice immunized with the H-2K(b) CTL epitope encapsulated pH-sensitive liposomes, peptide-specific CTL response was detected at day 3. The response was strongly augmented by the second immunization and persisted up to at least 45 days. These results suggest that pH-sensitive liposome formula functions as a potential adjuvant of peptide antigens and is useful for the induction of antigen specific CTLsv in vivo.     

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.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Mechanism of the suppressive effect of interferon on antibody synthesis in vivo.

Mouse interferon preparations significantly suppress the in vivo antibody response to sheep red blood cells (SRBC), a thymus-dependent antigen, and to Salmonella typhimurium lipopolysaccharide (LPS), a thymus-independent antigen. It is also possible to effect the late responses of antigen sensitive "memory" cells observed during secondary immunization by administration of interferon prior to primary immunization. The immunosuppressive activity of interferon was time- and dose-dependent. Maximum suppression was produced when animals were given 1.5 times 10-5 units of interferon between 4 and 48 hr before antigenic stimulation. These findings suggested that interferon affects some early event(s) in the process of antibody synthesis which might be related to the general inhibitory effect of interferon on rapidly dividing cells and viral m-RNA translation. In addition, the use of nonadherent spleen cell cultures from interferon-treated mice, immunized in vitro with a thymus-independent antigen, indicated that in this situation the inhibitory effect of interferon was due to an action on B lymphocytes. A variety of soluble "suppressive" factors are secreted by T cells as a consequence of activation by mitogens or specific antigens in vitro. Since T cells are recognized as one of the sources of interferon, it is suggested that interferon should be investigated as a suppressor T cell-produced lymphokine which can regulate B cell expression.     

Oral Salmonella typhimurium (AroA-) vaccine expressing a major leishmanial surface protein (gp63) preferentially induces T helper 1 cells and protective immunity against leishmaniasis

The gp63 gene of Leishmania major was transformed into the AroA- vaccine strain of Salmonella typhimurium (SL3261). The construct (SL3261-gp63), which stably expresses the gp63 Ag in vitro, was used to immunize CBA mice by the oral route. Spleen cells from mice inoculated with SL3261-gp63 developed antibody and proliferative T cell response to L. major. They did not express detectable delayed-type hypersensitivity reactivity. The activated T cells are mainly CD4+ and secrete IL-2 and IFN-gamma but no IL-4. The orally immunized mice developed significant resistance against a challenge L. major infection. We have, therefore, demonstrated the feasibility of oral vaccination against leishmaniasis and that the oral route of antigen delivery via the heterologous carrier may preferentially induce Th1 subsets of CD4+ cells.