A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.     

Immunization with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I (CPB) and Type II (CPA), partially protects against leishmaniasis

Cysteine proteinases (CPs) are enzymes that belong to the papain superfamily, which are found in a number of organisms from prokaryotes to mammals. On the parasitic protozoan Leishmania, extensive studies have shown that CPs are involved in parasite survival, replication and the onset of disease, and have, therefore, been considered as attractive drugs and/or vaccine targets for the control of leishmaniasis. We have previously shown that cysteine proteinases, Type I (CPB) and Type II (CPA), in Leishmania major (L. major), delivered as recombinant proteins or in plasmid DNA, induce partial protection against infection with the parasite in BALB/c mice. We had shown that the level of protection was greater if a cocktail of cpa and cpb containing DNA constructs was used. Therefore, to reduce the costs associated with the production of these vaccine candidates, a construct was developed, whereby the cpa and cpb genes were fused together to give rise to a single hybrid protein. The genes were fused in tandem where the C-terminal extension (CTE), encoding region of CPB, was located at the 3' of the fused genes, and ultimately expressed in the bacterial expression construct pET-23a. The expression of the CPA/B hybrid protein (60 kDa) was verified using rabbit anti-CPA and anti-CPB antibodies by SDS-PAGE and immunoblotting. The protective potential of the CPA/B hybrid protein against the infection with Leishmania was then assessed in BALB/c mice. The animals were vaccinated with CPA/B, challenged with live L. major promastigotes, and the degree of protection was examined by measuring footpad lesion sizes. It was found that there was a delay in the expansion of lesions size compared to control groups. Furthermore, an immunological analysis of antibody isotypes, before and after infection, showed high levels of IgG2a compared to IgG1 (more than five-fold) in the CPA/B hybrid protein vaccinated group. In addition, a predominant Th1 immune response characterized by in vitro IFN-gamma production was observed, along with little, if any, IL-5 production. This finding indicates that the hybrid CPA/B is able to elicit a protective immune response against L. major in the mice model. In addition, 54% of individuals tested, who had recovered from cutaneous leishmaniasis, produced more than 50 pg/ml IFN-gamma, in response to the CPA/B hybrid protein in an in vitro assay, demonstrating the importance of cysteine proteinases as targets of immune response in humans.     

Protective effect of a naked DNA vaccine cocktail against lethal toxoplasmosis in mice

BALB/c mice were intramuscularly immunized with low doses (25-50microg) of DNA cocktail containing plasmids encoding the full-length SAG1/P30 and the 196-561 terminal sequence of ROP2 genes. This immunization resulted in a Th1-type response with predominance of IgG2a and a specific T-cell proliferation with high levels of interferon-gamma (IFN-gamma) secretion, whereas no IL-4 was detected. Moreover, DNA cocktail immunization induced a long-lasting protection against a lethal challenge with the highly virulent Toxoplasma gondii RH strain, whereas low doses of single genes were not protective. These results support further investigations to achieve a multigene anti-T. gondii DNA vaccine.     

Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice

Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.     

Recombinant cysteine proteinases-based vaccines against Leishmania major in BALB/c mice: the partial protection relies on interferon gamma producing CD8(+) T lymphocyte activation

Together with poloxamer 407 as adjuvant the recombinant type I (rCPB) or type II (rCPA) cysteine proteinases of Leishmania major were screened as potential vaccines against L. major in a mouse model. The vaccines were delivered subcutaneously twice at 3 weeks intervals. Three weeks after booster injection, 5x10(5) stationary phase L. major promastigotes were inoculated subcutaneously in one footpad. Using the footpad thickness increase to monitor the clinical outcome/cutaneous lesion at site of L. major delivery, it was possible to document that rCPB but not rCPA allowed BALB/c mice to mount a partial protective response: indeed over the period under study (weeks 1-12) a clear delay was noticed after the immunization with rCPB. This partial protective effect was no more detectable if CD8 depleting antibody was given intravenously to rCPB-immunized mice, at the time of parasite challenge. Seven weeks after challenge, the draining lymph nodes were monitored for their frequencies of IFN-gamma positive CD4(+) and CD8(+) T lymphocytes using PMA and ionomycin as re-activating signals: interestingly the partial protection achieved in BALB/c mice immunized with rCPB together with poloxamer was correlated only to one immunological parameter, namely the higher frequency of IFN-gamma producing CD8(+) T lymphocytes. Of note also, in the lymph node draining the L. major-loaded footpad of C57BL/6 mice otherwise known to develop a transient lesion, the frequency of IFN-gamma producing CD8(+) T lymphocytes reach similar value 7 weeks after challenge and in absence of any prior immunization. Taken together, it was shown that the induced partial protection was mainly dependent on IFN-gamma producing CD8(+) T cells.     

Protective vaccination against experimental canine visceral leishmaniasis using a combination of DNA and protein immunization with cysteine proteinases type I and II of L. infantum

Leishmania infantum is known to be associated with visceral leishmaniasis in Iran and canids are natural reservoirs. Control of disease in dogs appears to be one of the most effective approaches for interrupting the domestic cycle of the disease. In search for successful vaccine strategies, we evaluated the cysteine proteinases (CPs) type I and II using a heterologous prime-boost regime for vaccination against experimental visceral leishmaniasis in dogs. Following vaccination and challenge, dogs were followed for 12 months. Ten dogs vaccinated by prime/boost with DNA/recombinant CPs (in combination with CpG ODN and Montanide 720) remained free of infection in their bone morrow. In contrast, three out of four dogs in the control groups had infection in their bone marrow. The peripheral lymphocytes from protected animals had generally higher proliferation responses to F/T antigen, recombinant CPA (rCPA) and recombinant CPB (rCPB) than controls. During post-challenge period, the difference in stimulation index is significant (p<0.05) on months 11 and 12 to F/T antigens, all months for rCPA and 5, 7, 9, 11 and 12 months for rCPB. Analysis of cytokine mRNA level suggested that vaccinated dogs had elevated IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC), whereas there was a consistent increase in the level of IL-10 in the control groups and some vaccinated dogs. The level of total IgG and IgG2, but not IgG1, to rCPA and rCPB was significantly higher in the vaccinated group (p<0.05) than the control groups. We also showed that with the exception of one dog, all dogs in the vaccinated group in comparison to control dogs had strong DTH responses. We propose that the combination of DNA and recombinant protein vaccination using CPs could be instrumental to control (VL) in dogs.     

Immunization with the cysteine proteinase Ldccys1 gene from Leishmania (Leishmania) chagasi and the recombinant Ldccys1 protein elicits protective immune responses in a murine model of visceral leishmaniasis

The gene Ldccys1 encoding a cysteine proteinase of 30 kDa from Leishmania (Leishmania) chagasi, as well as the recombinant cysteine proteinase rLdccys1, obtained by cloning and expression of the Ldccys1 gene in the pHIS vector, were used to evaluate their ability to induce immune protective responses in BALB/c mice against L. (L.) chagasi infection. Mice were immunized subcutaneously with rLdccys1 plus Bacille Calmette Guerin (BCG) or Propionibacterium acnes as adjuvants or intramuscularly with a plasmid carrying the Ldccys1 gene (Ldccys1/pcDNA3) and CpG ODN as the adjuvant, followed by a booster with rLdccys1 plus CpG ODN. Two weeks after immunization the animals were challenged with 1 x 10(7) amastigotes of L. (L.) chagasi. Both immunization protocols induced significant protection against L. (L.) chagasi infection as shown by a very low parasite load in the spleen of immunized mice compared to the non-immunized controls. However, DNA immunization was 10-fold more protective than immunization with the recombinant protein. Whereas rLdccys1 induced a significant secretion of IFN-gamma and nitric oxide (NO), animals immunized with the Ldccys1 gene increased the production of IgG2a antibodies, IFN-gamma and NO. These results indicated that protection triggered by the two immunization protocols was correlated to a predominant Th1 response.     

Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep

In this work we tested the ability of plasmid DNA constructs encoding structural Rift Valley fever virus (RVFV) antigens to induce specific immune responses in sheep. The sole immunization of DNA constructs encoding the glycoprotein precursor NSm/G2/G1 did not suffice to induce a detectable antibody response. In contrast, immunization of sheep with a plasmid vector encoding the viral nucleocapsid protein N elicited a potent and long lasting induction of antibodies but with low neutralizing titers. After DNA immunization, no antigen-specific proliferating cells were detected in sheep PBLs. Boosting with the attenuated vaccine strain MP12 was able to increase the levels of proliferating memory cell pools and induction of IFN-gamma in response to purified virus or recombinant proteins, particularly in sheep vaccinated with a combination of both plasmid constructs. These results open the possibility to exploit this strategy to improve the induction of immune responses against RVFV in sheep.     

Evaluation of protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital toxoplasmosis in mice

To develop a multiantigenic vaccine against toxoplasmosis, two Toxoplasma gondii antigens, SAG1 and GRA4 selected on the basis of previous immunological and immunization studies, were chosen. We showed that DNA-based immunization with plasmids expressing GRA4 (pGRA4) or SAG1 (pSAG1mut) reduced mortality of susceptible C57BL/6 mice upon oral challenge with cysts of the 76K type II strain (62% survival). Immunization with pGRA4 and pSAG1mut, enhanced the protection (75% survival). This protection was further increased by co-inoculation with a plasmid encoding the granulocyte-macrophage colony-stimulating factor (GM-CSF) (87% survival). This latter DNA cocktail provided significant protection of less susceptible outbred Swiss OF1 mice against the development of cerebral cysts. A significantly higher survival of newborns from immunized outbred mice exposed to infection during gestation was observed (4.25+/-3.77 live pups/litter) in comparison to non-immunized mice (1.08+/-2.15 live pups/litter) without preventing parasite vertical transmission. Analysis of the immune response showed that protected animals developed a specific humoral and cellular Th1 response to native T. gondii SAG1 and GRA4 antigens. Our data demonstrated that protection was improved by associating antigens (SAG1 and GRA4) and cytokine (GM-CSF) for further development of a multiantigenic vaccine against toxoplasmosis.     

Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus

Vaccination with a membrane-bound thiol Sepharose-binding fraction (TSBP) of adult Haemonchus contortus has been shown to confer significant levels of protection against homologous challenge in sheep. This fraction is greatly enriched for cysteine proteinase activity. Following fractionation of TSBP by anion-exchange chromatography on MonoQ, protection was found to partition with those fractions further enriched for cysteine proteinase activity. In this study, the cysteine proteinases of adult H. contortus TSBP were specifically purified by affinity chromatography using recombinant H. contortus cystatin, a potent cysteine proteinase inhibitor. Although only 1-1.5% of total TSBP bound to cystatin-Sepharose, this fraction contained 100% of the cysteine proteinase activity, as determined by gelatin substrate gel analysis. When used to immunise sheep, less than 3microg per dose of this cysteine proteinase fraction was found to confer a substantial and repeatable level of protection against homologous challenge infection, reducing faecal egg counts by 48 and 28% and worm burdens by 44 and 46% over two trials. Host serum immunoglobulin levels and abomasal mast cell and eosinophil numbers were evaluated, although no correlation with protection was observed. Three cathepsin B-like cysteine proteinases present in TSBP (hmcp1, 4 and 6) have been identified previously by cDNA library immunoscreening. The predicted mature forms of these three cysteine proteinases were expressed in bacteria as insoluble, GST-fusion proteins. Following solubilisation in urea/DTT, the protective capacity of a cocktail of recombinant proteins was evaluated in sheep. Although no reduction in faecal egg counts was observed, sheep vaccinated with recombinant cysteine proteinases showed a highly significant 38% reduction (P <0.01) in worm burdens.     

Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis

Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.     

Immune responses in mice to intranasal and intracutaneous administration of a DNA vaccine encoding Helicobacter pylori-catalase

We previously reported that the intracutaneous injection of DNA vaccines encoding Helicobacter pylori heat shock proteins elicited specific immune responses, and led to reduced infection in mice. In this study, we constructed DNA vaccine encoding H. pylori-catalase (pcDNA3.1-kat) and investigated the immune responses to intranasal and intracutaneous administration of pcDNA3.1-kat. C57/BL6 mice were immunized intracutaneously with 10 microg of pcDNA3.1-kat or intranasally with 50 microg of pcDNA3.1-kat. Catalase-specific IgG antibody was detected in the sera of intranasal and intracutaneous immunized mice. Both intranasal and intracutaneous immunized mice were significantly protected from colonization by H. pylori and had significantly reduced degrees of gastritis. These results demonstrate that DNA vaccine encoding H. pylori-catalase can induce an immune response against H. pylori, and that intranasal immunization works as well as intracutaneous immunization.     

Vaccination with DNA encoding cysteine proteinase confers protective immune response to rats infected with Clonorchis sinensis

Cysteine proteinases of C. sinensis are important virulence factors that induce pathological changes associated with larval migration and localized biliary epithelial destruction. This study investigated the immunogenicity and protective efficacy of a DNA vaccine encoding Clonorchis sinensis cysteine proteinase (CsCP). The CsCP cDNA sequence displays significant homology to the mammalian or trematode cathepsin L. Plasmid DNA carrying the CsCP gene (pcDNA3.1-CsCP) was injected into Sprague-Dawley (SD) rats intradermally. Animals injected with pcDNA3.1-CsCP developed CsCP-specific antibodies, which exhibited an IgG2a dominance in sera. In addition, the DNA vaccine elicited the production of IFN-gamma, but not IL-4 in splenocytes, suggesting the induction of a typical Th-1 dominated immune response in rats. The pcDNA3.1-CsCP induced a significant level of protection (31.5%, p<0.05) in SD rats challenged with C. sinensis metacercariae. These results indicate that pcDNA3.1-CsCP induces both humoral and cellular immune responses. The CsCP gene may be a good candidate for use in future studies of vaccination against clonorchiasis.     

Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting

The study evaluated the immune response elicited by a DNA vaccine encoding ESAT6 protein of Mycobacterium tuberculosis by DNA prime-protein boost protocol. BALB/c mice were respectively vaccinated with plasmid DNA encoding ESAT6 protein, with ESAT6 protein in IFA adjuvant, or a combined DNA prime-protein boost regimen. While DNA immunization induced Th1-polarized immune response, protein-in-adjuvant vaccination elicited a Th2-dominant response. When animals were primed with DNA and boost with protein, both antibodies and Th-cell proliferative response were significantly enhanced. Moreover, production of Th1-type cytokine (IFN-gamma) was increased significantly by DNA priming-protein boosting. This protocol also resulted in an increased relative ratio of IgG2a to IgG1 and the cytotoxicity of T cells. Thus, this study demonstrated that the formation of ESAT6 DNA prime-protein boost inoculation could improved antigen-specific cellular immune responses, which are important for protection against TB infection.     

Evaluation of Immunogenicity of Cocktail DNA Vaccine Containing Plasmids Encoding Complete GRA5, SAG1, and ROP2 Antigens of Toxoplasma gondii in BALB/C Mice

Background:

Severe and fatal complications of toxoplasmosis urge development of effective vaccines against the disease. The current study was performed to evaluate cocktail DNA vaccine containing plasmids encoding GRA5, SAG1, and ROP2 genes of Toxoplasma gondii in BALB/c mice in Tarbiat Modares University in 2012.

Methods:

The plasmids containing complete GRA5, SAG1, and ROP2 genes were mass extracted and then the recombinant plasmids were administered via intramuscular injections according to immunized mice three times with three-week intervals. Then splenocytes were cultured, and proliferation as well as cytokine assays were carried out. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis.

Results:

The results of cytokine assay for INF-γ were higher in the mice that received the cocktail DNA containing recombinant plasmids. Evaluation of proliferation of splenocytes using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay indicated induction of cellular response. Measurement of total IgG and the isotypes of IgG1 and IgG2a showed that the cocktail DNA stimulated IgG and IgG2a production in comparison with the control groups (P<0.05). Furthermore, the survival rate of mice in the groups that received the cocktail DNA was significantly higher than that in the control groups (P<0.05).

Conclusion:

Administration of the cocktail DNA vaccine led to production of higher levels of IFN-γ, confirmed by secretion of IgG2a, and the immune response was shifted toward Th1. Thus, the cocktail DNA containing the recombinant plasmids can be an appropriate candidate for immunization against toxoplasmosis.     

Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection.

Several defined vaccine candidate antigens of Schistosoma japonicum have shown promise in large animal vaccination experiments. However, vaccination of mice in the laboratory with either single recombinant antigens or DNA encoding forms of the individual antigens has so far failed to induce significant protection against S. japonicum cercarial challenge infection as judged by worm reduction, although specific antibodies were generated. This is in contrast to the results achieved using radiation-attenuated vaccines which are highly protective. Even in large animal vaccination experiments, the protection levels obtained with single defined antigens were far below those achieved using the attenuated vaccines. One possible interpretation is that the immune responses induced by single antigen vaccination may not be strong enough to combat the challenging infection. We, therefore, carried out mouse vaccination experiments using a cocktail DNA vaccine comprising four DNA plasmids encoding four different S. japonicum antigens, Sj62, Sj28, Sj23 and Sj14-3-3, respectively. We, also investigated whether co-injection of the mouse IL-12 encoding plasmid with the cocktail DNA vaccine was able to enhance the Th1 responses and hence the protective immunity. Three intramuscular injections of the cocktail DNA vaccine induced a significant Th1-type cellular response with high level of IFN-gamma production by splenocytes upon in vitro stimulation with recombinant antigens. Importantly, significant IgG antibody responses were also induced against crude worm antigens. In two out of three experiments, significant resistance (34-37 and 44-45%, respectively) was demonstrated while another experiment did not show any protection against S. japonicum cercarial challenge infection. Co-injection of the IL-12 encoding DNA did not further enhance these responses, nor the level of resistance, compared with the cocktail DNA alone.     

Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine

We investigated the DNA immunization approach in order to induce a protective immune response against hepatitis delta virus (HDV) superinfection of chronically woodchuck hepatitis virus (WHV) infected woodchucks. The animals were immunized with an expression vector encoding HDAg by gene gun. T cell and humoral immune responses induced by this protocol were determined and compared with those induced by HDAg immunization using a CpG oligonucleotide as an adjuvant. After immunization the woodchucks were challenged with 10⁶ genome equivalents of HDV. The protein immunization with HDAg induced good humoral and T helper cell responses in the woodchucks, but did not protect them from HDV superinfection. The DNA immunized woodchucks were also not protected from HDV superinfection, however, the course of infection was modified: HDV viremia occurred later, the typical fluctuation of the HDV RNA titer with several peaks was absent, and antibodies to HDV were not detectable.     

Vaccination of mice with DNA vaccine induces the immune response and partial protection against T. spiralis infection

Trichinellosis is a serious parasitic zoonosis which is widely distributed in the world. Pork still is the predominant source of outbreaks of human trichinellosis in many countries. Vaccines are urgently needed to prevent swine from Trichinella infection. The gene (TspE1) encoding a 31 kDa antigen of T. spiralis was cloned to a eukaryotic expression plasmid pcDNA3 as DNA vaccine. The ability of the DNA vaccine to express antigen in mammalian CHO cells was previously confirmed by indirect fluorescencent antibody test (IFAT) and Western blotting. To evaluate its immunogenicity and its host protective potential, BALB/c mice were immunized with the DNA vaccine by intramuscular injection and gene-gun delivery. The serum antibody response was assayed by IFAT, enzyme linked immunosorbant assay (ELISA) and Western blotting. The cellular immune response was investigated by splenocyte proliferation assay. Vaccine administration by either route induced both humoral and cellular immune response against TspE1, which provided the partial protection against challenge infection with T. spiralis, as shown by significant reduction of larval burden in muscles. Thus, DNA immunization may offer an attractive alternative strategy against swine trichinellosis.     

Poly(propyleneimine) dendrimer and dendrosome mediated genetic immunization against hepatitis B

The purpose of the present research work is to explore the potential of dendrosomes in genetic immunization against hepatitis B. Plasmid DNA encoding pRc/CMV-HBs[S] (5.6 kb), encoding the small region of the hepatitis B surface antigen, was complexed with 5th generation poly(propyleneimine) dendrimer (PPI) in different ratios. Transfection of CHO cells revealed that a ratio of 1:50 for pDNA:PPI was optimum for transfection. Results of cytotoxicity studies showed that the toxicity of PPI-DNA complex was significantly (p<0.05) higher for PPI 75 and PPI 100 as compared to the other PPI-DNA complexes. PPI 50 was employed for preparation of dendrosomes by reverse phase evaporation method. The dendrosomal formulation DF3 was found to possess optimum vesicle size, zeta potential and entrapment efficiency. In vitro production of HBsAg in CHO cells showed that DF3 possess maximum transfection efficiency. In vivo immunization studies were carried out by giving a single intramuscular injection of 10 microg of plasmid DNA (pDNA) or its dendrimeric or dendrosomal formulation to female Balb/c mice, followed by estimation of total IgG, IgG(1), IgG(2a), IgG(2b), biweekly. DF3 was found to elicit maximum immune response in terms of total IgG and its subclasses under study as compared to PPI 50 and pDNA at all time points. Animals immunized with DF3 developed very high cytokine level. Higher level of IFN-gamma suggests that the immune response was strictly Th1 mediated. Our observations clearly prove the superiority of dendrosomes over PPI-DNA complex and pDNA for genetic immunization against hepatitis B.