Browsing by Author "He, Yongqun"

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    Characterization of recombinant B. abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51
    Zhu, Jianguo; Larson, Charles B.; Ramaker, Megan Ann; Quandt, Kimberly; Wendte, Jered M.; Ku, Kimberly P.; Chen, Fang; Jourdian, George W.; Vemulapalli, Ramesh; Schurig, Gerhardt G.; He, Yongqun (Frontiers, 2011-11-25)
    Brucella abortus is a Gram-negative, facultative intracellular pathogen for several mammals, including humans. Live attenuated B. abortus strain RB51 is currently the official vaccine used against bovine brucellosis in the United States and several other countries. Overexpression of protective B. abortus antigen Cu/Zn superoxide dismutase (SOD) in a recombinant strain of RB51 (strain RB51SOD) significantly increases its vaccine efficacy against virulent B. abortus challenge in a mouse model. An attempt has been made to better understand the mechanism of the enhanced protective immunity of RB51SOD compared to its parent strain RB51. We previously reported that RB51SOD stimulated enhanced Th1 immune response. In this study, we further found that T effector cells derived from RB51SOD-immunized mice exhibited significantly higher cytotoxic T lymphocyte activity than T effector cells derived from RB51-immunized mice against virulent B. abortus-infected target cells. Meanwhile, the macrophage responses to these two strains were also studied. Compared to RB51, RB51SOD cells had a lower survival rate in macrophages and induced lower levels of macrophage apoptosis and necrosis. The decreased survival of RB51SOD cells correlates with the higher sensitivity of RB51SOD, compared to RB51, to the bactericidal action of either Polymyxin B or sodium dodecyl sulfate (SDS). Furthermore, a physical damage to the outer membrane of RB51SOD was observed by electron microscopy. Possibly due to the physical damage, overexpressed Cu/Zn SOD in RB51SOD was found to be released into the bacterial cell culture medium. Therefore, the stronger adaptive immunity induced by RB51SOD did not correlate with the low level of innate immunity induced by RB51SOD compared to RB51. This unique and apparently contradictory profile is likely associated with the differences in outer membrane integrity and Cu/Zn SOD release.
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    Induction of Protection, Antibodies and Cell Mediated Immune Responses by Brucella Abortus Strain RB51, Ochrobactrum Anthropi and Recombinants Thereof
    He, Yongqun (Virginia Tech, 1999-04-08)
    Although it is known that cell-mediated immunity (CMI) plays a key role in protection against brucellosis, the exact immune mechanisms leading to protection are still not fully understood. Better understanding of the mechanisms would help in the development of a human Brucella vaccine and help in improving animal vaccines. In this research, B. abortus strain RB51 and a closely-related, nonpathogenic Ochrobactrum anthropi (strain 49237) bacterium were used to study the immune response against brucellosis in mice. Both O. anthropi strain 49237 and recombinant strain 49237 expressing Brucella protective antigen copper-zinc superoxide dismutase (Cu/Zn SOD) induced a mix of Th1 and Th2 type immune responses but failed to provide protection against virulent Brucella challenge. After changing the immune response to a predominantly Th1 type of response using CpG oligonucleotides as an adjuvant, both strains provided protection with the recombinant strain inducing significantly higher protection. It was also demonstrated that vaccination with strain RB51 induced Th1 immune responses characterized by high interferon-gamma (IFN-g) production with no interleukin-4 (IL-4) secretion as well as high IgG2a and minimal IgG1 production. A colorimetric cytotoxic T lymphocytes (CTL) assay was developed to demonstrate that strain RB51 induced an antigen-specific CTL reaction that probably plays an important role in protection. The results suggest that optimal protection against brucellosis requires IFN-g-secreting T cells and antigen-specific CTLs. Recombinant strain RB51 overexpressing Brucella Cu/Zn SOD and simultaneously expressing mycobacterial 85A antigen induced higher IFN-g production and CTL activity than the parent RB51 strain. The combined results suggest that the recombinant O. anthropi strain could be used as a human vaccine against brucellosis and that the recombinant RB51 strain could be used as an effective vaccine against both brucellosis and tuberculosis in animals.
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    Molecular targets for rapid identification of Brucellaspp
    Ratushna, Vladyslava G.; Sturgill, David M.; Ramamoorthy, Sheela; Reichow, Sherry A.; He, Yongqun; Lathigra, Raju; Sriranganathan, Nammalwar; Halling, Shirley M.; Boyle, Stephen M.; Gibas, Cynthia J. (2006-02-22)
    Background Brucella is an intracellular pathogen capable of infecting animals and humans. There are six recognized species of Brucella that differ in their host preference. The genomes of the three Brucella species have been recently sequenced. Comparison of the three revealed over 98% sequence similarity at the protein level and enabled computational identification of common and differentiating genes. We validated these computational predictions and examined the expression patterns of the putative unique and differentiating genes, using genomic and reverse transcription PCR. We then screened a set of differentiating genes against classical Brucella biovars and showed the applicability of these regions in the design of diagnostic tests. Results We have identified and tested set of molecular targets that are associated in unique patterns with each of the sequenced Brucella spp. A comprehensive comparison was made among the published genome sequences of B. abortus, B. melitensis and B. suis. The comparison confirmed published differences between the three Brucella genomes, and identified subsets of features that were predicted to be of interest in a functional comparison of B. melitensis and B. suis to B. abortus. Differentiating sequence regions from B. abortus, B. melitensis and B. suis were used to develop PCR primers to test for the existence and in vitro transcription of these genes in these species. Only B. suis is found to have a significant number of unique genes, but combinations of genes and regions that exist in only two out of three genomes and are therefore useful for diagnostics were identified and confirmed. Conclusion Although not all of the differentiating genes identified were transcribed under steady state conditions, a group of genes sufficient to discriminate unambiguously between B. suis, B. melitensis, and B. abortus was identified. We present an overview of these genomic differences and the use of these features to discriminate among a number of Brucella biovars.
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    Plant-derived Murine IL-12 and Ricin B-Murine IL-12 Fusions
    Liu, Jianyun (Virginia Tech, 2006-12-06)
    Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12.