Induction of systemic immunity through nasal-associated lymphoid tissue (NALT) of mice intranasally immunized with Brucella abortus malate dehydrogenase-loaded chitosan nanoparticles.

Infection with Brucella abortus causes contagious zoonosis, brucellosis, and leads to abortion in animals and chronic illness in humans. Chitosan nanoparticles (CNs), biocompatible and nontoxic polymers, acts as a mucosal adjuvant. In our previous study, B. abortus malate dehydrogenase (Mdh) was loaded in CNs, and it induced high production of pro-inflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In this study, the time-series gene expression analysis of nasal-associated lymphoid tissue (NALT) was performed to identify the mechanism by which Mdh affect the target site of nasal immunization. We showed that intranasal immunization of CNs-Mdh reduced cell viability of epithelial cells and muscle cells at first 1 h, then induced cellular movement of immune cells such as granulocytes, neutrophils and lymphocytes at 6h, and activated IL-6 signaling pathway at 12h within NALT. These activation of immune cells also promoted signaling pathway for high-mobility group box 1 protein (HMGB1), followed by the maturation of DCs required for mucosal immunity. The CNs also triggered the response to other organism and inflammatory response, showing it is immune-enhancing adjuvant. The ELISA showed that significant production of specific IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group after 2 weeks-post immunization. Collectively, these results suggest that B. abortus Mdh-loaded CNs triggers activation of HMGB1, IL-6 and DCs maturation signaling within NALT and induce production of systemic IgG and IgA.

Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase, outer membrane proteins 10 and 19.

Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.

Induction of Th2-related immune responses and production of systemic IgA in mice intranasally immunized with Brucella abortus malate dehydrogenase loaded chitosan nanoparticles.

The aim of this study was to investigate the induction of mucosal immune responses by an important Brucella abortus antigen, malate dehydrogenase (Mdh), loaded in mucoadhesive chitosan nanoparticles (CNs) and immunized intranasally in a BALB/c mouse model. The production of cytokines was investigated in human leukemic monocyte cells (THP-1 cells) after stimulation with the nanoparticles. Mdh-loaded CNs (CNs-Mdh) induced higher interleukin (IL)-6 production than unloaded antigens and TF loaded CNs (CNs-TF). Using ELISpot to quantify cytokines and antibody-secreting cells in the intranasally immunized mice, IL-4 and IgG-secreting cells were found to be significantly increased at 4 weeks and 6 weeks post-immunization in the CNs-Mdh immunized group, respectively. Increases in Mdh-specific IgG, IgG1, and IgG2a antibodies were confirmed at 6 weeks after immunization, indicating a predominant IgG1 response. Analysis of the mucosal immune response in the intranasally immunized mice revealed, Mdh-specific IgA and total IgA in the nasal washes, genital secretions, fecal extracts and sera that were remarkably increased in the CNs-Mdh-immunized group compared to the CNs-TF-immunized group except total IgA of nasal wash. Therefore, the results indicated that the intranasal immunization of CNs-loaded B. abortus Mdh antigen effectively induced antigen-specific mucosal immune responses through the elicitation of Th2-related immune responses.

Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots.

Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Th2-related immune responses by the Brucella abortus cellular antigens, malate dehydrogenase, elongation factor, and arginase.

Brucellosis is an important zoonotic disease caused by Brucella species. The disease is difficult to control due to the intracellular survival of the bacterium and the lack of precise understanding of pathogenesis. Despite of continuous researches on the pathogenesis of Brucella spp. infection, there is still question on the pathogenesis, especially earlier immune response in the bacterial infection. Malate dehydrogenase (MDH), elongation factor (Tsf), and arginase (RocF), which showed serological reactivity, were purified after gene cloning, and their immune modulating activities were then analyzed in a murine model. Cytokine production profiles were investigated by stimulating RAW 264.7 cells and naïve splenocytes with the three recombinant proteins. Also, immune responses were analyzed by ELISA and an ELIspot assay after immunizing mice with the three proteins. Only TNF-α was produced in stimulated RAW 264.7 cells, whereas Th1-related cytokines, IFN-γ and IL-2, were induced in naïve splenocytes. In contrast, Th2-type immune response was more strongly induced in antigen-secreting cells in the splenocytes obtained 28 days after immunizing mice with the three proteins, as were IgM and IgG. The induction of Th2-related antibody, IgG1, was higher than the Th1-related antibody, IgG2a, in immunized mice. These results suggest that the three proteins strongly induce Th2-type immune response in vivo, even though Th1-related cytokines were produced in vitro.

Molecular cloning, purification and immunogenicity of recombinant Brucella abortus 544 malate dehydrogenase protein.

The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.

Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens.

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.

Partial protective immunity against toxoplasmosis in mice elicited by recombinant Toxoplasma gondii malate dehydrogenase.

Toxoplasma gondii can infect humans and wildlife, sometimes causing serious clinical presentations. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii malate dehydrogenase (TgMDH) is a crucial enzyme in cellular redox reactions and has been shown to be an immunogenic compound that could be a potential vaccine candidate. Here, we investigate the protective efficacy of recombinant TgMDH (rTgMDH) against T. gondii infection in BALB/c mice. All mice were vaccinated via the nasal route. We determined the optimal vaccination dose by monitoring systemic and mucosal immune responses. The results showed that mice vaccinated with 30 µg of rTgMDH produced the highest antibody titers in serum, a strong lymphoproliferative response, marked increases in their levels of IL-2 and IFN-γ, and significantly greater levels of specific secretory IgA (sIgA) in mucosal washes. In addition, the vaccinated mice were orally challenged with tachyzoites of the virulent T. gondii RH strain 2 weeks after the final vaccination. Compared to the control group, we found that vaccination with rTgMDH increased the survival rate of infected mice by 47% and also significantly reduced the tachyzoite loads in their liver (by 58%) and brain (by 41%). Therefore, the rTgMDH protein triggers a strong systemic and mucosal immune response and provides partial protection against T. gondii infection.

Characterization of the immunogenicity and pathogenicity of malate dehydrogenase in Brucella abortus.

Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. abortus S2308 was cloned, sequenced and expressed. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein. In addition, recombinant MDH showed sero-reactivity with 30 individual bovine B. abortus-positive sera by enzyme-linked immunosorbent assay, indicates that MDH may be used as a candidate marker for sero-diagnosis of brucellosis. Furthermore, MDH exhibits fibronectin and plasminogen-binding ability in immunoblotting assay. Inhibition assays on HeLa cells demonstrated that rabbit anti-serum against MDH significantly reduced both bacterial adherence and invasion abilities (p < 0.05), suggesting that MDH play a role in B. abortus colonization. Our results indicated that MDH is not only an immunogenic protein, but is also related to bacterial pathogenesis and may act as a new virulent factor, which will benefit for further understanding the MDH's roles in B. abortus metabolism, pathogenesis and immunity.

Evaluation of Mdh1 protein as an antigenic candidate for a vaccine against candidiasis.

Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

[Cloning, expression and immunogenicity analysis of malate dehydrogenase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the malate dehydrogenase (MDH) gene of Toxoplasma gondii, and analyze the immunogenicity. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii (GenBank accession No. AY650028). The coding region of TgMDH was amplified with a pair of specific primers. The product of RT-PCR was digested with double restriction enzyme and ligated into pET30a (+) vector. The recombinant pET30a (+)-TgMDH plasmid was transformed into E. coli DH5alpha. The positive clones were confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity. RESULTS: The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a(+)-TgMDH was confirmed by the double restriction enzyme digestion, PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE, followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T. gondii serum. CONCLUSION: TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.

[Preliminary study on the difference in immune protection between Echinococcus granulosus recombinant ferritin and recombinant mMDH].

OBJECTIVE: To evaluate the immunoprotective activity of the Egrecombinant ferritin and Egrecombinant mMDH proteins in mice. METHODS: Thirty ICR mice were divided into 3 groups and immunized by injection of adjuvant-emulsified rEgferritin, rEgmMDH and PBS, respectively, in multiple spots at back, for 3 times with an interval of 2 wk. Two weeks after the last immunization, the mice of the 3 groups were infected intraperitoneally with 0.1 ml suspension containing about 1 500 Echinococcus granulosus (Eg) protoscoleces. The mice were sacrificed 22 wk after infection and the Eg cysts were collected and measured. Spleens were taken for detecting CD4+ T cells and CD8+ T cells and ratio calculated. RESULTS: Eg cysts were found in 30% (3/10) of the mice in the rEgferritin group with 5 cysts altogether; cysts were received in all the mice in the rEgmMDH group with 118 cysts totally; and cysts were found in 7 of 9 mice in the PBS control with 35 cysts totally. The mice in the rEgferritin group showed an 84.7% protection but revealed no protection in the rEgmMDH group. The CD4+ T cells were significantly higher in the rEgferritin group than the control, but no statistical difference was found in CD8+ T cells and CD4+/CD8+ ratio between the 2 groups. There was no considerable change in the T cells and ratio in the rEgmMDH group compared to the control. CONCLUSION: The Egrecombinant ferritin can inhibit the growth of Eg while the Egrecombinant mMDH seems promoting its growth in mice.

Identification of major allergens in watermelon.

BACKGROUND: Watermelon is a worldwide consumed Cucurbitaceae fruit that can elicit allergic reactions. However, the major allergens of watermelon are not known. The aim of this study is to identify and characterize major allergens in watermelon. METHODS: Twenty-three patients allergic to watermelon took part in the study. The diagnosis was based on a history of symptoms and positive skin prick-prick tests to watermelon, confirmed by positive open oral challenge testing to watermelon pulp. Allergenic components were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by N-terminal amino acid sequencing and mass spectrometry. Allergens were purified combining several chromatographic steps. RESULTS: Several IgE binding bands (8-120 kDa) were detected in watermelon extract. Three major allergens were identified as malate dehydrogenase (36 kDa), triose phosphate isomerase (28 kDa) and profilin (13 kDa). Purified allergens individually inhibited IgE binding to the whole watermelon extract. CONCLUSIONS: All in all these results indicate that malate dehydrogenase, triose phosphate isomerase and profilin are major allergens involved in watermelon allergy.

A proteomic approach for studying the pathogenesis of spontaneous equine recurrent uveitis (ERU).

Equine recurrent uveitis (ERU) is a wide spread disease of the eye, which is the main cause for blindness in horses worldwide. Meanwhile, ERU is also accepted as the only reliable spontaneous model for human autoimmune uveitis. We identified and characterized novel autoantigens by analyzing the autoantibody-binding pattern from ERU cases to the retinal proteome. Cellular retinaldehyde-binding protein (CRALBP) and malate dehydrogenase (MDH) were detected as novel ERU autoantigens by this approach. B- and T-cell autoreactivity was detected to both autoantigens in ERU cases. The evaluation of the pathological relevance of CRALBP and MDH brought surprising results. While CRALBP-induced uveitis with high incidence in rats and horses, MDH was only uveitogenic in Lewis rats, but not in the horse itself.

[Cloning and prokaryotic expression of malate dehydrogenase gene of Taenia saginata asiatica and immunogenicity analysis of the recombinant protein].

OBJECTIVE: To clone and express the lactate dehydrogenase (LDH) gene of Taenia saginata asiatica and analyze the immunogenicity of the recombinant protein. METHODS: By screening the full length cDNA plasmid library, the coding region of LDH was amplified with PCR, and cloned into the prokaryotic expression vector pET-30a (+), then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. RESULTS: PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blotting analysis of LDH recombinant protein testified that the recombinant protein could be recognized by sera of the Taenia saginata asiatica infected swine and the patient. CONCLUSIONS: The LDH gene of Taenia saginata asiatica has been cloned and expressed, and the purified protein has been confirmed with immunogenicity.

Changes in self-reactive IgG antibody repertoire after treatment of experimental autoimmune encephalomyelitis with anti-allergic drugs.

We reduced EAE severity by using two anti-allergic drugs. A control group of mice received i.p. injections of PBS as vehicle while a further two groups were treated either with pyrilamine, a histamine receptor 1 antagonist or with CV6209, a platelet activating factor receptor antagonist. Our results showed that the blockade of the responses to both histamine and PAF leads together to a decline in clinical signs of EAE and significant changes in the serum IgG recognition of some healthy brain antigenic targets. We characterized two discriminant antigens: internexin neuronal intermediate filament protein, and malate dehydrogenase 1, which were able to clearly distinguish untreated mice from treated mice. Their role as potent targets in pathogenic and/or neuroprotective processes is discussed.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.