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.     

Immunostimulatory oligodeoxynucleotides are potent enhancers of protective immunity in mice immunized with recombinant ORFF leishmanial antigen

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligonucleotides (ODN) have proved as promising adjuvants for promotion of T helper 1 (Th1) type immune response. The potent Th1 like immune activation by CpG-ODNs suggests a possible utility for vaccination against leishmaniasis. We therefore investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with recombinant ORFF (rORFF) leishmanial antigen. BALB/c mice were vaccinated with the rORFF with or without CpG-ODN as adjuvant and then challenged with Leishmania donovani metacyclic promastigotes. Administration of CpG-ODN alone resulted in partial protection against challenge with L. donovani in BALB/c mice. Combination of rORFF and CpG-ODN showed enhanced reduction in parasite load (84%) when compared to rORFF (56%) vaccinated mice. Immunization with rORFF alone did not induce the typical Th response whereas co-administration of rORFF with CpG-ODN resulted in enhanced production of immunoglobulin G2a and interferon gamma. Our results further demonstrate that CpG-ODN alone or in combination with rORFF resulted in a dose dependent increase of nitric oxide production in activated macrophages. These studies suggest that CpG-ODN are promising immune enhancers for vaccination against visceral leishmaniasis.     

Vaccination of Swiss Albino mice against experimental visceral leishmaniasis with the FML antigen of Leishmania donovani.

The FML antigen of Leishmania donovani in combination with saponin, aluminum hydroxide (Al(OH)3) and Freund's incomplete adjuvant (FIA) was used in vaccines tested in an outbred murine model of visceral leishmaniasis, either through intraperitoneal or subcutaneous routes. The humoral response was significantly higher in the groups treated with FML + saponin or FML + Al(OH)3 than in controls, both before and after the infection. Animals immunized by the i.p. route developed higher antibody titres. A significant and specific reduction of parasitic load in relation to saline (85%, p < 0.01) and saponin (p < 0.025) controls, was seen in animals treated with FML + saponin by the i.p. Coincidentally with this reduction, an increase in antibodies of the IgG2a subtype was detected only in animals treated with FML + saponin i.p. A reduction of 88% in parasitic load was achieved by the combination of FML + Al(OH)3 (s.c.), but the Al(OH)3 treatment itself accounted for 68% of this protection. In our conditions, vaccination with FML + saponin i.p. was superior to other treatments and had no toxic effect due to saponin.     

Vaccination with an alkaline extract of Histoplasma capsulatum packaged in glucan particles confers protective immunity in mice

Infection with the dimorphic fungus, Histoplasma capsulatum, occurs world-wide, but North and South America are regions of high endemicity. Interventions to mitigate exposure and consequent disease are limited to remediating a habitat harboring the fungus. The development of a vaccine to prevent infection or lessen its severity is an important advance in disease prevention. Accordingly, we prepared an alkaline extract from the yeast phase of Histoplasma and encased it in glucan particles that act as an adjuvant and delivery vehicle. Immunization of C57BL/6 mice with this encapsulated extract decreased the number of CFUs in lungs and spleens at days 7 and 14 following intranasal infection. Moreover, this vaccine conferred protection against a lethal challenge with the fungus. Cytokine assessment in lungs at a time when the CFUs were similar between controls and vaccinated groups revealed increased quantities of interferon-γ and interleukin-17 in vaccine recipients. This finding was supported by increased generation of both Th1 and Th17 cells in lungs and draining lymph nodes of vaccinated mice compared to controls. Neutralization of interferon-γ or interleukin-17 blunted the effectiveness of vaccination. To identify the proteins comprising this extract, liquid chromatography tandem mass spectrometry was performed. Thus, an H. capsulatum alkaline extract packaged in glucan particles confers protection in an interferon-γ and interleukin-17-dependent manner. Discovery of a single protein or a few proteins in this admixture that mediate protective immunity would represent significant progress in efforts to prevent histoplasmosis.     

Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis

Leishmania histones are relevant immunogens for the host immune system during both Leishmania infection and disease. In the present paper we have evaluated the prophylactic value of the four Leishmania infantum histones forming the nucleosomal core in the murine model of cutaneous leishmaniasis. In a first stage, the immune response elicited by the intramuscular injection of a mixture of four plasmid DNAs, encoding the L. infantum histones H2A, H2B, H3 and H4, was determined in BALB/c mice. It was found that the immunized animals developed a specific Th1 immune response, which was associated with an antigen-specific production of interferon (IFN-gamma) and a limited humoral response against histones (dominated by antibodies of the IgG2a isotype). According to the pure Th1-type immune response elicited by the DNA vaccination with Leishmania histones, vaccinated mice showed a solid immunity that efficiently controlled the Leishmania major infection. The protection in mice vaccinated with histone-DNAs was associated with a low humoral response against leishmanial antigens, an enhanced IFN-gamma production and little, if any, IL-4 production. The relative contribution of both CD8(+) and CD4(+) T cells to the IFN-gamma production, and the IL-12 dependence were also evaluated. All these data indicated that DNA vaccination with Leishmania histones genes results in a specific Th1-like response during L. major infection, and that both CD4(+) and CD8(+) T cells contribute to the resistance of vaccinated mice to cutaneous leishmaniasis.     

Vaccination of Balb/c mice against experimental visceral leishmaniasis with the GP36 glycoprotein antigen of Leishmania donovani

Leishmania donovani GP36 glycoprotein is the main antigen of the FML Fucose Mannose Ligand (FML) complex specifically recognized by sera of kala-azar human patients. The GP36 was isolated by chemical elution + sonication and used for Balb/c mouse vaccination in combination with saponin, by the s.c. route, inducing a strong and specific protective effect against experimental visceral leishmaniasis shown by the increase of: specific IgG antibodies (82.6%), mainly IgG2a, the delayed type of hypersensitivity to promastigote lysate (37.8%, P < 0.001), the in vitro cellular proliferative response to GP36 of ganglia lymphocytes (53.5%, P < 0.005) and the decrease of liver parasite burden (68.1%, P < 0.025). Saponin treated controls reacted significantly differently from GP36 vaccinated animals at all the assayed variables (P < 0.05). GP36 induced significant protection against murine visceral leishmaniasis at concentrations commonly used for vaccination with recombinant antigens.     

Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice

We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.     

Co-administration of IL-12 DNA with rORFF antigen confers long-term protective immunity against experimental visceral leishmaniaisis

Visceral leishmaniasis, caused by the intracellular parasite Leishmania donovani is a significant public health problem in many regions of the world. Anti-leishmanial immune defences are primarily dependent on the ability of the host to mount an interleukin-12 (IL-12) driven Th1 type of responses. Thus, IL-12 plays a pivotal role in diversification of the immune responses towards Th1 type. In this report, we investigated the effect of IL-12 DNA as an adjuvant with leishmanial recombinant open reading frame F (rORFF) protein. We demonstrate that an expression plasmid encoding both p35 and p40 subunits of IL-12 when co-administered with rORFF induces a significant protection with around 82% protection in both liver and spleen. The protection correlated with increased proliferative response of splenocytes and subsequent release of Th1 cytokine IFN-gamma. The levels of IFN-gamma were sustained 4 and 8 weeks after challenge with L. donovani promastigotes. Interestingly, IL-12 DNA played a key role in modulating the antibody response towards IgG2a isotype suggesting its use as a potential vaccine adjuvant against intracellular infections like leishmaniasis.     

Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.     

DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice

Despite the lack of effective vaccines against parasitic diseases, the prospects of developing a vaccine against leishmaniasis are still high. With this objective, we have tested four DNA based candidate vaccines encoding to immunodominant leishmania antigens (LACKp24, TSA, LmSTI1 and CPa). These candidates have been previously reported as capable of eliciting at least partial protections in the BALB/c mice model of experimental cutaneous leishmaniasis. When tested under similar experimental conditions, all of them were able to induce similar partial protective effects, but none could induce a full protection. In order to improve the level of protection we have explored the approach of DNA based vaccination with different cocktails of plasmids encoding to the different immunodominant Leishmania antigens. A substantial increase of protection was achieved when the cocktail is composed of all of the four antigens; however, no full protection was achieved when mice were challenged with a high dose of parasite in their hind footpad. The full protection was only achieved after a challenge with a low parasitic dose in the dermis of the ear. It was difficult to determine clear protection correlates, other than the mixture of immunogens induced specific Th1 immune responses against each component. Therefore, such an association of antigens increased the number of targeted epitopes by the immune system with the prospects that the responses are at least additive if not synergistic. Even though, any extrapolation of this approach when applied to other animal or human models is rather hazardous, it undoubtedly increases the hopes of developing an effective leishmania vaccine.     

DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase confers protection against Leishmania donovani infection

In this study the potential of using Leishmania donovani gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, gamma-GCS) as a rational target for vaccine development was determined. Mice, immunised with plasmid containing the full gene sequence for gamma-GCS (pVAXgammaGCS) or plasmid alone (pVAX control), were challenged with a high dose of L. donovani amastigotes to give a stringent test of the ability of the vaccine to protect against infection. Vaccination with pVAXgammaGCS resulted in the production of specific IgG1 and IgG2a antibodies and resulted in significantly lower liver parasite burdens compared to controls. Protection was also associated with a significant increase in cell-mediated immunity, demonstrated as an increase in nitrite production by ConA stimulated splenocytes, an increase in the percentage of splenic CD3+CD4+ cells, and enhanced granuloma maturation, compared to control values.     

Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice

A carcinoembryonic antigen (CEA)-based DNA vaccine, adsorbed onto cationic microparticles of poly(DL-lactide-co-glycolide) (PLG) induced tumor-protective immunity against a lethal challenge of MC38-CEA colon carcinoma cells in CEA-transgenic mice that was more potent than that of the corresponding naked DNA vaccine. Boosting with a plasmid encoding murine GM-CSF increased the vaccine's efficacy leading to a complete rejection of tumor cells in 50% of mice. This effect was due to activation of MHC class I-restricted CD8(+) T cells coupled with an increased secretion of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-2. Also, specific activation of dendritic cells was indicated by a two-three-fold upregulation of their costimulatory CD80 and MHC class II molecules. This approach may be a promising new strategy for the rational design of cancer vaccines for future clinical applications.     

Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene

GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-gamma and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.     

Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum

Live attenuated Leishmania donovani parasites such as LdCen^−/− have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen^−/− including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4⁺ and CD8⁺ T cells, IFN-γ production by CD8⁺ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen^−/− parasites as vaccine candidates, we performed a 24-month follow up of LdCen^−/− immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen^−/− (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune^®). The protection was associated with antibody production and CD4⁺ and CD8⁺ proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune^®, with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen^−/− expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune^® or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.     

Vaccination of attenuated EIS-producing Salmonella induces protective immunity against enterohemorrhagic Escherichia coli in mice

There is an urgent need for vaccine against enterohemorrhagic Escherichia coli (EHEC), which causes a wide range of life-threatening diseases in human and animals. E. coli secreted protein A (EspA), intimin and shiga toxin (Stx) are important pathogenic factors and protective antigens of EHEC. In our previous study, we found that recombinant trivalent protein EIS, which is composed of EspA (E), the 300 amino acids of the carboxyl terminus of intimin (I) and the B subunit of Stx2 (S), was able to efficiently elicit protective immunity against EHEC. The application of live attenuated Salmonella as a carrier for vaccine against mucosal pathogens provided unparalleled merits. Therefore, in this study we constructed live attenuated EIS-producing Salmonella vaccine and tested it as vaccine in mice model. We found that the vaccination of EIS-producing recombinant Salmonella was able to induce significant increases of EspA, intimin and Stx2 specific IgG in serum and secretory IgA in feces. Antigen specific T cell proliferation was also observed in the mice immunized with recombinant EIS-producing Salmonella. In addition, this immunity was able to protect mice from a challenge of a lethal dose of EHEC, even after a period of 70 days. Moreover, the EIS-producing Salmonella induced immunity can be boosted by a single subcutaneous injection of purified EIS protein, even after an interval of 70 days. This EIS-producing Salmonella vaccine provides an alternative approach for the prevention of EHEC infection.     

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 recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice

Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis.     

Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A

The potential utility of using DNA vaccination to protect mice from the microbial neurotoxin, botulinum toxin type A, was evaluated. A synthetically derived gene encoding a carboxyl-terminal 50 kDa fragment of the toxin was placed in two sites in the DNA inoculation vehicle pCMVint-BL (Vical), one predicted to lead to MHC I processing (pJT-1 construct) and the other to direct MHC II processing (pJT-2 construct). Mice were then inoculated at 3 week intervals with these two constructs and with the vehicle alone and evaluated for protection from botulinum toxin by i.p. challenges with various toxin doses. Protection was observed at about week 10-11 from toxin doses of 25-100 LD(50). Only animals inoculated with pJT-2 exhibited protection. In dose-response experiments, 50 micrograms of DNA was the minimal dose required to elicit a protective response against serotype A, while protection against serotypes B or E was not obtained. With standard ELISA testing, a relationship was observed between the level of protection and the level of ELISA reactive antibody. Our results support the concept that DNA vaccination is a viable methodology to use in cases where protection from toxins is the goal.     

DNA vaccination with linear minimalistic (MIDGE) vectors confers protection against Leishmania major infection in mice

Immunization protocols based on priming with plasmid DNA and boosting with recombinants of vaccinia virus (rVV) encoding the same antigen offer great promise for the prevention and treatment of many parasitic and viral infections for which conventional vaccination has little or no effect. To overcome some of the potential problems associated to the use of plasmids, we have developed minimalistic, immunogenically defined, gene expression (MIDGE((R))) vectors. These linear vectors contain only the minimum sequence required for gene expression and can be chemically modified to increase the immune response. Here, we demonstrate that MIDGE vectors coding for the LACK antigen confer a highly effective protection against Leishmania infection in susceptible Balb/c mice. Protection is achieved at lower doses of vector compared to conventional plasmids. This efficacy could be greatly improved by the addition of a nuclear localization signal (NLS) peptide to the end of the MIDGE vector. In fact, immunization with two doses of NLS-modified MIDGE conferred similar or even better protection than that achieved by priming with plasmid DNA followed by boosting with rVV. These results demonstrate that MIDGE vectors are a good alternative to plasmid and rVV for immunization.     

Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization did not result in antigen-specific antibody in either feces or sera but induced the release of IFN-gamma on restimulation of primed lymphocytes ex vivo. The results suggested that the protective efficacy provided by flagellin-expressing L. casei is mainly attributable to cell-mediated immune responses. In addition, an adjuvant-type effect of the antigen delivery system with L. casei was also observed.