Browsing by Author "Schurig, Gerhardt G."

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    Antigenic Characterization of Haemophilus somnus Lipooligosaccharide
    Howard, Michael D. (Virginia Tech, 1998-10-23)
    Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of Haemophilus and Neisseria. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of H. somnus, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to H. somnus LOS were produced and used with cross-reacting MAbs to H. aegyptius LOS (MAb 5F5) and Neisseria gonorrhoeae LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of H. somnus. Using three MAbs, thirty-nine of these H. somnus strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of H. somnus strains. The anti-H. somnus LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the H. somnus strains. Six strains (13%) were not recognized by any of these MAbs. Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in H. somnus LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed.
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    Applicability of vaccinia virus as cloning and expression vector for bacterial genes: mice immune responses to vaccinia virus expressing Brucella abortus and Listeria monocytogenes antigens
    Baloglu, Simge (Virginia Tech, 2001-07-27)
    Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant.
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    Approaches towards vaccine development against Neospora caninum
    Ramamoorthy, Sheela (Virginia Tech, 2006-06-05)
    Neospora caninum is an apicomplexan parasite that causes neuromuscular paralysis in dogs and abortions in cattle. N. caninum is responsible for losses of several million dollars to the dairy and beef industries in several parts of the world. The key players in the host immune response to N. caninum include CD4+ T cells, the Th1 cytokines IL-12, Interferon gamma and IgG2a isotype antibodies. There are currently no chemotherapeutic agents that are effective against adult cattle neosporosis. A commercially available, inactivated vaccine induces the undesirable Th2 type of immunity against N. caninum. Therefore, two approaches towards vaccine development against N. caninum that were designed to induce potent cell mediated immunity have been explored in this dissertation. The first approach consisted of the development of a bivalent recombinant vaccine for both brucellosis and neosporosis, while the second approach involved gamma irradiation of N. caninum tachyzoites for use as an attenuated vaccine against N. caninum. Since N. caninum research has been conducted with several strains of mice and the different strains of mice vary in their susceptibility to infection with N. caninum, there is a need to develop a standard lab animal model for N. caninum. A gerbil and a C57BL/6 mouse model for N. caninum vaccine testing have been developed. It was found that the LD50 of N. caninum tachyzoites in gerbils was 9.3 x105 tachyzoites per gerbil delivered intra-peritoneally, (i.p) while for C57BL/6 mice the LD50 was 1.5 x107 tachyzoites per mouse delivered i.p. Vertical transmission rates in C57BL/6 mice infected with N. caninum tachyzoites during mid-gestation were determined and found to be in the range of 96-100%. Putative protective antigens of N. caninum that included MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in B. abortus strain RB51 to create recombinant vaccine strains. C57BL/6 mice were vaccinated with either the recombinant strains or the irradiated tachyzoites. Antigen specific IgG2a and IgG1 responses and high levels of interferon gamma and IL-10 were induced by vaccination. Mice vaccinated with irradiated tachyzoites, RB51-MIC1 and RB51-GRA6 were completely protected against lethal challenge, while the mice vaccinated with RB51-SRS2, RB51-GRA2 and RB51-MIC3 were partially protected. To determine the efficacy of the vaccines in preventing vertical transmission of N. caninum, mice were vaccinated and bred after administration of a booster dose four weeks after the primary vaccination. Antigen specific IgG1 and IgG2a and significant levels of IFN-ã and IL-10 were detected in vaccinated, pregnant mice. Pregnant mice were challenged with 5 x 106 N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups three weeks after birth and examined for the presence of N. caninum by a semi-nested PCR. Protection against vertical transmission elicited by the RB51-GRA6, RB51-MIC3, irradiated tachyzoite, RB51-GRA2, RB51-MIC1 and RB51-SRS2 vaccinated groups were 43%, 38%, 34%, 34%, 18%, and 7% respectively. Since not all the antigens that were highly protective against acute disease were not very effective in preventing vertical transmission, the role of the selected antigens in preventing acute disease and vertical transmission appear to differ. Only GRA6 was found to be effective in protecting against an acute lethal challenge as well as preventing vertical transmission 43% of the time. In summary, two animal models for the testing of N. caninum vaccines were developed. N. caninum protective antigens were successfully expressed in B. abortus strain RB51. The irradiated tachyzoite and recombinant RB51-Neospora vaccines were highly effective in protecting against acute neosporosis and partially protective against vertical transmission. Therefore, both these approaches show great promise as practical and effective means to achieve the goal of successful prophylaxis against N. caninum induced abortions and reduce the chances of vertical transmission.
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    Assessment of the Expression of Brucella Abortus Heat Shock Protein, Groel, in Vaccinia Virus to Induce Protection Against a Brucella Challenge in Balb/C Mice
    Baloglu, Simge (Virginia Tech, 1997-07-08)
    B. abortus is an intracellular facultative bacterial pathogen which causes abortion in cattle and undulant fever in humans. Cattle vaccines such as B. abortus strains 19 and RB51 are live vaccine strains which protect approximately 75% of the vaccinated animals. No effective vaccines are available for the prevention of brucellosis in humans. We are developing vaccinia virus recombinants expressing various B. abortus proteins to prevent brucellosis in susceptible mammalian species. In this work the B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination. Expression of the GroEL protein in vaccinia infected cells in-vivo was confirmed by immunoblotting. Groups of 5 female BALB/C mice were injected with the vaccinia recombinant or appropriate positive and negative control vaccines. Mice were bled and their humoral immune responses assessed. In addition, mice were challenged with virulent B. abortus strain 2308 and protection measured by the rate of splenic clearance of live Brucella. In spite of demonstrating specific GroEL antibodies in recombinant vaccinia injected mice, no significant level of protection was demonstrable. Preliminary lymphocyte transformation assays were carried out to establish if a cell mediated immune response to GroEL was induced in the vaccinated animals.
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    The behavior and effects of Brucella abortus rough strain RB51 in mice and cattle
    Buhrman, Dianne L. (Virginia Tech, 1989-07-05)
    Brucella abortus st. RB51 is a rough mutant of smooth st. 2308 devoid of O-side chain and resistant to rifampin. The purpose of this investigation was to study the behavior and effects of viable st. RB51 organisms in inoculated mice and cattle and to further substantiate the lack of O-side chain antigens in this strain. A single injection of live st. RB51 persisted in BALB/C mice up to 28 days. A secondary exposure was cleared in 7-21 days. One or 2 injections of st. RB51 did not induce detectable titers of anti-O-side chain antibodies, although antibody titers to st. RB51 whole cell and cytoplasmic antigens were detected. Mice infected with st. RB51 alone or followed by infection with st. 2308, demonstrated a very strong reaction to a 14-18 Kd antigen which was believed to be the core of the LPS complex. When st. RB51 was administered after injection of st. 2308 the response to the core determinants were inhibited. One vaccination with st. RB51 was able to significantly protect mice against challenge with st. 2308 at one and four weeks post challenge. Two st. RB51 vaccinations were able to protect mice as well as one vaccination at one week post challenge but protection increased by four weeks post challenge. Strain RB5l was able to survive in cattle a for at least twenty-two days. The organism remained stable, rifampin resistant, and may have induced minor amounts of transient anti-O-side chain antibodies in some cows late in the experiments.
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    Brucella abortus RB51 vaccine: Testing its Spectrum of Protective and Curative Characteristics
    Contreras Rojas, Andrea Paz (Virginia Tech, 2004-07-30)
    Brucella abortus (BA) are gram-negative, facultative intracellular bacteria that cause abortions in cattle and debilitating illness in humans. The US is now virtually free of bovine brucellosis, but the disease is endemic in wildlife. The official brucellosis vaccine in the US is strain RB51 (RB51). It elicits protective cell-mediated immunity (CMI) against BA infections. Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis in ruminants. It is a slow growing intracellular parasite that requires CMI for its control, belongs to the genus Mycobacterium, and is closely related to M. avium avium (MA). Using RB51 as a vector that induces strong protective CMI may be useful to protect against MAP if it expresses MAP protective antigens. Therefore, MAP 85A and 35kDa proteins were expressed at low levels in RB51, and the immune responses elicited by these vaccines in BALB/c mice were evaluated. Strong anti-Brucella immunity was generated, but the anti-mycobacterial response was low. To evaluate protective efficacy, a BALB/c model using MA was developed. When mice were challenged with MA, protection was obtained in some experiments but was inconsistent. This may be due to the low expression of MAP antigens in RB51. Another objective was to evaluate the effect of an ongoing Brucella-infection on the efficacy of RB51 vaccination, and whether vaccination of already infected animals could have a curative effect. Mice acutely or chronically infected with virulent BA, rapidly cleared the RB51 vaccine organisms, but there was no significant decrease in the number of virulent BA. Brucella spp. have been developed as biological weapons, but there are no vaccines to protect humans. The development of a very attenuated protective vaccine is necessary to prevent human infections, as well as to protect wildlife. To generate such a vaccine, RB51 based vaccines were irradiated to render them non-replicative, but metabolically active. We demonstrated that in general, irradiated and non-irradiated RB51 vaccines remain protective at levels similar to those elicited by the live vaccines. Therefore, irradiation of strain RB51 is an effective means of attenuating the strain without affecting its protective characteristics, and could eventually be used as a vaccine for wildlife and humans.
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    Changes in macrophage functions and gene expression during tumor growth
    Askew, David (Virginia Tech, 1993-01-25)
    Functions and phenotypes change in macrophages (Mφ) during tumor growth. Although analyzing functional and phenotypic changes are important in understanding the mechanism of tumor-induced immunosuppression, it is necessary to look beneath the surface and expose the mechanisms behind these changes. Flow cytometrically isolated Mac-1⁺, -2⁺, or -3⁺ Mφ showed that although both normal host and tumor-bearing host (TBH) Mac-2⁺ Mφ were the primary source of prostaglandin E2, no specific TBH suppressor Mφ could be identified. To determine if normal host and TBH Mφ respond to in vitro activating agents differently, normal host and TBH Mø, were treated with lipopolysaccharide. Functional, phenotypic, and molecular changes were observed in the lipopolysaccharide-treated M4J. Three- and 24-h lipopolysaccharide treatment reduced TBH Mφ-mediated suppression, while only 24-h lipopolysaccharide treatment reduced it in normal host Mφ. Prolonged adherence, which induces Mφ differentiation, increases the number of Mac-2⁺ TBH Mφ. Tumor growth causes an increase of Mφ in the S and G₂/M phases of the cell cycle. Lipopolysaccharide and adherence increase the number of normal host Mφ in Sand G₂/M; however, these same treatments reduce the number of TBH Mφ in these same phases. Earlier work showed an increase in the number of TBH Mφ that did not express class II major histocompatibility complex molecules and that there was an increase in the suppression mediated by these Mφ. TBH Mφ have a decreased response to interferon-γ-induced class II mRNA expression and a decrease in its mRNA stability. TBH Mφ have an increase in lipopolysaccharide- and prostaglandin E₂-mediated suppression of class II mRNA. Tumor necrosis factor-α can induce class II mRNA expression in TBH Mφ, but suppresses it in normal host Mφ. The effects of tumor necrosis factor-α on class II mRNA is due, in part, to the maturation stage of the Md. To examine further mechanisms that regulate Mφ maturation, intracellular Mac-2 expression was examined. The expression of nuclear Mac-2 increases during tumor growth and after 24-h adherence. This increase in Mac-2 protein parallels the increase in Mac-2 mRNA expression. Because there is a change in TBH Mφ cell-cycle kinetics and maturation, proto-oncogene expression was examined in normal host and TBH Mφ. The proto-oncogenes c-myb, c-myc, c-fos, and c-fms are constitutively expressed in TBH Mφ, while normal host M@ express c-fos and c-fms but at lower levels. Adherence suppresses the expression of the proto-oncogenes c-myb, c-myc, and c-fms in TBH Mφ, while inducing c-fos and c-fms in the normal host. Lipopolysaccharide induces c-myc, c-fos and c-fms in both normal and TBH Mφ, but suppresses c-myb expression in TBH Mφ. The results suggest that tumor growth causes a shift in Mφ maturity, and that this shift is responsible for alterations in Mφ function and phenotype. It is possible, however, to activate Mφ by lipopolysaccharide in the absence of triggering differentiation, and to trigger differentiation by adhesion without activating Mφ. TBH Mφ are more suppressive than normal host Mφ, but TBH Mφ can respond to activating signals.
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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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    Conjugated Linoleic Acid in the treatment of murine autoimmune glomerulonephritis
    Hammond, Sarah Elizabeth (Virginia Tech, 2015-10-15)
    Conjugated linoleic acid (CLA) has been shown to reduce inflammation via Peroxisome Proliferator-Activated Receptor (PPAR)-γ in inflammatory disorders such as Crohn's Disease and Inflammatory Bowel Disease. We sought to determine whether CLA isomers would reduce inflammation via PPAR-γ in cultured mesangial cells, and in murine models of anti-glomerular basement membrane (anti-GBM) glomerulonephritis and Systemic Lupus Erythematosus (SLE). SV40-transformed mouse mesangial cells (MES13) were cultured with pure CLA isomers (c9,t11 or t10,c12-CLA or a 50:50 mixture prior to immune stimulation with lipopolysaccharide and interferon-γ. Next, cultured mesangial cells were transfected with small interfering RNA (siRNA) targeting PPAR-γ and treated with CLA isomers prior to immune stimulation. ELISA, qPCR, Western blot, and Griess reaction were performed to measure cytokine production, mRNA expression, induced nitric oxide synthase (iNOS) and nitrite production, respectively. Next, myeloid-specific (LysM creR2+) PPAR-γ knockout mice were treated with CLA prior to the induction of anti-GBM glomerulonephritis and evaluated for disease. Finally, NZM2410/J mice (a natural model of SLE) were treated with c9,t11-CLA and evaluated for disease progression. Treatment with CLA reduced IL-6 production in cultured mesangial cells, but not in siRNA-treated mesangial cells, supporting a PPAR-γ-mediated mechanism. CLA treatment increased both Transforming Growth Factor (TGF-β) and Interleukin-1 Receptor Antagonist (IL-1RA) mRNA expression independent of PPAR--γ. While CLA treatment reduced nitrite production and iNOS production to some degree, this was an inconsistent finding. Conversely, in the induced anti-GBM mouse model, CLA treatment increased mesangial cell IL-6 mRNA expression, reduced TGF-β expression, and had no effect on IL-1RA. Moreover, NZM2410/J mice that were fed a c9,t11-CLA-supplemented diet had reduced survival times, increased renal inflammation and increased serum IgG2a relative to controls. Taken together, these studies indicate that the in vitro MES13 cell line does not translate to the in vivo mouse model of anti-GBM induced glomerulonephritis. Furthermore, while CLA may have beneficial effects in other mouse models, it worsens disease in NZM2410/J mice. Findings from these models should be interpreted with caution.
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    The Development and Application of a Hemolytic Plaque Forming Cell Assay (PFC) and a Cytotoxic T-Lymphocyte Assay (CTL) in Tilapia (Oreochromis niloticus) for Immunotoxicity Risk Assessment of Environmental Contaminants
    Smith, Dorinda Ann (Virginia Tech, 1998-07-24)
    The prospect of utilizing the cichlid teleost tilapia (Oreochromis niloticus) as an alternative experimental model to mammals for immunotoxicity risk assessment is currently being proposed. As such, the National Toxicology Program's (NTP) standard battery of rodent immunotoxicity assays is being developed for use in this fish species. Included in the testing series are the hemolytic plaque forming cell (PFC) and the cytotoxic T-lymphocyte (CTL) assays, quantitative indicators of antibody production and cell-mediated activity, respectively. The assays were modified in consideration of specific tilapian immune parameters, then tested using fourteen environmental contaminants or drugs, ten of which are classified by the NTP as immunotoxic in rodents. Reduced antibody production via a decrease in plaque number was observed in response to exposure of tilapia to eight of the nine humoral immunotoxicants, and five of the five non-immunotoxicants. Under specific immunization circumstances, immunostimulation (also a response to immunotoxicity) was noted via an increase in plaque number in benzo[a]pyrene (B[a]P) exposed fish using the PFC assay, a result noted in rodents as well. Reduced T-cell recognition and lysis of allogeneic tilapian lymphocytes via a decrease in the percentage of specific 51Chromium (51Cr) release was observed in response to exposure of tilapia to the nine of the ten cell-mediated immunotoxicants, and four of the four non-immunotoxicants. Although the normal teleost immune responsiveness was slightly weaker than seen with mice under comparable conditions (presumably due to differences in antibody structure and decreased cells counts), tilapia were found to exhibit well-defined humoral and cell-mediated immune responses, and responses to immunotoxic and non-immunotoxic chemicals comparable to the rodent model.
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    Development of a cloning system for gene expression in Pasteurella multocida
    Jablonski, Lynn McGonagle (Virginia Tech, 1993-01-14)
    To identify antigens unique to live Pasteurella multocida P1059, 10 week old specific pathogen-free (SPF) chickens were vaccinated three times with one of the following: viable cells from P. multocida P1059, 3865, 3866, or cells from formalin-killed strain PI059 or formalin-killed strain P1059 that were opsonized with antiserum directed against killed strain PI059 prior to immunization. Vaccinated birds were challenged with 1.5 x 10⁷ CFU of live strain P1059. Eight, 71, 86, and 50% of the birds that received live strains P1059, 3865, 3866 and killed strain P1059 (respectively), exhibited clinical signs of fowl cholera. Antisera directed against live strain PI059 recognized 23 proteins ranging from 14- to 92-kilodaltons (kDa); 20 of which were adsorbed by strain 3865. The molecular masses of the three remaining proteins were 25-, 30- and 43-kDa. A genomic library of strain P1059 was constructed using the plasmid vector pUC-19 and screened with antisera against live strain P1059; 12 out of 4,100 clones were recognized. The inserts of the plasmids from these clones ranged from 0.48- to 6.S-kilobases (kb) in length. Five of the 12 clones expressed proteins with molecular masses of 34-, 37-, 42-, 46- and 55-kDa. Escherichia coli CSR603(pOP43- 2G) and CSR603(pOP33-SF) expressed proteins recognized by antisera directed against live strain P1059. E. coli CSR603(pOP43-2G) expressed an epitope(s) which was recognized by antisera directed against strains 3865 and 3866. Conditions for transformation were optimized and attempts were made to create a shuttle vector in order to establish a cloning system for gene expression in P. multocida. The highest efficiency of transformation (1.25 x 10⁷ CFU/μg DNA) was obtained when 7.6 x 10¹⁰ cells of P. multocida R473 were electroporated at 12.5 kV cm⁻¹ for 10 ms with 5 ng of the plasmid, p VM109. Of the six strains tested, representing serogroups A, B, D and E, all were transformed successfully. Vectors including pBR322, pUC19, pJFF224-NX and pSP329 were unable to transform P. multocida. To create a shuttle vector for gene expression in P. multocida, a Pasteurella plasmid (pLAR-1) was cloned in both orientations into the BamH I site of pBR322. These plasmids, pLRBR-21 and pLRBR-67, had a transformation efficiency of 4.5 to 8 x 10⁴ CFU/μg of DNA in strain R473. Chromosomal DNA containing the Brucella abortus copper-zinc superoxide dismutase gene was cloned into the Cla I site of pLRBR-21. The 1.8-kb fragment encoding a 42-kDa Pasteurella protein was cloned into an additional unique site (Nru 1) of pLRBR-21 to determine if this plasmid was a viable shuttle vector for gene expression in P. multocida.
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    Development of a live, attenuated, recombinant vaccine for Brucellosis
    (United States Patent and Trademark Office, 2008-04-29)
    A recombinant, attenuated strain of Brucella suis or Brucella melitensis with a deficiency in carboxyl-terminal protease activity or tail-specific protease activity can be used as a vaccine for the prevention or treatment of Brucellosis. Prior exposure to the Brucella species is identified by detecting a genetic sequence for carboxyl-terminal (i.e. tail-specific) protease activity in a biological sample.
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    Development of an Antibiotic Resistance Free Bivalent Vaccine Against Swine Brucellosis and Swine Influenza
    Rajasekaran, Parthiban (Virginia Tech, 2009-12-09)
    Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform.
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    Development of an antigen-specific ELISPOT to detect intestinal antibody responses to the swine whipworm, Trichuris suis
    Kellman, Maxine Franchestcê (Virginia Tech, 1997-06-05)
    The swine whipworm, Trichuris suis, is a parasite present throughout the United States and is of concern to the swine industry worldwide because it is very pathogenic to growing pigs. The economic threat posed by T. suis and other intestinal parasite infections has created a strong interest in the development of parasite vaccines for the swine industry. Use of a vaccine either alone or with anthelmintics should reduce the economic losses. However, before effective parasite vaccines can be created, the swine gastrointestinal immune response to parasite antigens must be understood. In this study, an enzyme-linked immunospot (ELISPOT) assay was developed to measure total and antigen-specific IgG and IgA antibody secreting cells (ASC) from gut-associated lymphoid tissues (GALT) [mesenteric lymph node explants from jejunal region of small intestine (SI-MLN) and cecum in large intestine (C-MLN); and ileocecal Peyer's patches (IC-PP)] and lamina propria from the proximal colon removed from T. suis infected pigs. Tbe local antibody responses were compared to peripheral antibody responses found in the spleen and submandibular lymph nodes. The hypotheses to be tested was that parasite antigen-specific antibody secreting cells would be greatest in lymphoid tissue draining the site of infection compared to peripheral lymphoid tissues and that 19A ASC would predominate over IgG ASC in the lamina propria of T. suis infected pigs. The total IgG and IgA ASC frequencies for the spleen, SI-MLN, and ICPP did not significantly change (P> 0.05) over time. For C-MLN, there was a significant increase (p< 0.05) of total IgG ASC during a primary infection with T. suis. Antigen-specific IgG ASC were greatest at the GALT site closest to the infection, CMLN, whereas, antigen-specific IgA ASC predominated in the proximal colonic: lamina propria. Host protection to T. suis develops after anthelmintic: treatment of a primary exposure to parasite. The ELISPOT assay provided valuable information on the localization and compartmentalization of the swine gastrointestinal immune response to T. suis which resides in the cecum and proximal colon. In the future, this technique may be useful for monitoring gastrointestinal immune parameters of pigs exposed to a T. sllis vaccine.
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    Development of Brucella abortus RB51 as a Vaccine to Protect Against Brucellosis and Anthrax
    Poff-Reichow, Sherry Ann (Virginia Tech, 1999-01-14)
    Bacillus anthracis is a facultative extracellular bacterial pathogen that causes cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are known to provide protection of limited duration and may not protect against the respiratory form of the disease. Commercially available live vaccines for animals have been shown to cause disease in certain species. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective CMI and antibody responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce antibodies, the pag gene encoding PA was expressed in B. abortus RB51, producing a dual vaccine to protect against both brucellosis and anthrax. In a previous study, the entire pag gene was expressed in strain RB51 and following immunization the vaccine induced antibodies against PA in A/J mice. However, PA stability and protective efficacy were less than desirable as only 1/6 were protected. The studies in this dissertation involved synthesizing a gene corresponding to domain 4 (PA4) of the pag gene utilizing the native codon usage of Brucella. The PA4 domain was fused to Brucella signal sequences of Brucella 18kDa protein, superoxide dismutase or no signal sequence to localize the PA4 to the outside cell envelope, periplasmic space or cytosol respectively. Comparisons of the expression level and stability of the native and synthetic PA4 in B. abortus strain RB51 were assessed by immunoblot. The protective efficacy of PA4 expressed in Brucella was assessed by immunization and protection studies in A/J mice against a live challenge with either B. abortus or B. anthracis Sterne spores. ELISA and western blot indicate the induction of PA specific antibodies by these recombinant strain RB51 vaccine constructs. Results based on subisotype antibody ELISA (IgG, IgG1, IgG2a and IgM) and CMI assays (cytokine ELISA of IL-4 and INF-g, and LPA) suggest a Th1 based immune response to strain RB51 and PA. B. abortus strain RB51 expressing PA4 fused to the signal sequence of Brucella 18kDa protein was able to induce 50% protection, while strain RB51 expressing PA4 with no signal sequence gave 17% protection against B. anthracis Stern spore challenge. Mice were boosted with an intraperitoneal injection of purified PA after an initial immunization with Brucella vaccine candidates, sterile saline or pure PA. Protection assessed by live challenge with B. anthracis Sterne spores increased following boosting with PA in 4 cases. Immunization with purified PA, and 3 strain RB51/PA vaccines and a PA boost gave protection against a spore challenge ranging from partial to full. This study suggests that additional work is needed to define the antigens of B. anthracis involved in the induction of specific CMI.
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    Effect of homozygous lpr and gld mutations on the immune functions and induction of autoimmunity
    Hammond-McKibben, Denise M. (Virginia Tech, 1995-04-05)
    The murine lpr gene encodes for an aberrant form of Fas (CD95), a molecule involved in apoptosis. The mouse gld gene leads to the expression of a defective Fasligand. Mice homozygous for lpr or gld mutations develop severe lymphoproliferative and autoimmune disease characterized by the accumulation of unique CD4⁻CD8⁻ (double-negative, DN) T cells. Because of these poor functions in vitro, the nature and significance of DN T cells in the autoimmune disease process is not clear. In the current study we found that lpr DN T cells could mediate spontaneous lysis of certain tumor cells as well as mediate redirected lysis of various tumor targets when stimulated through the CD3/αβTCR complex and certain adhesion molecules, such as, CD44 and gp90MEL-14. The DN T cells constitutively transcribed perform, TNF-α and IFN-γ genes. Unlike the DN T cells from lpr mice, similar cells from gld mice failed to exhibit spontaneous cytotoxicity despite expression of similar levels of cytokines and adhesion molecules. Furthermore, lpr DN T cells could mediate redirected lysis of Fas⁺ but not Fas⁻ target cells. Together, these studies suggested that lysis of target cells by DN T cells was dependent on the interaction between Fas and Fas-ligand. The fact that lpr DN T cells can be activated via CD44 and gp-90MEL-14 suggested that these T cells may be able to mediate lysis of endothelial cells which bear the ligand for these adhesion molecules. Further studies revealed that the lpr DN T cells could mediate spontaneous lysis of endothelial cells and that CD44-hyaluronate interactions were important for endothelial cell lysis. Thus, interactions between DN T cells and endothelial cells in vivo may trigger an inflammatory response and contribute to the vasculitis seen in lpr and gld mice. We also addressed the hypothesis that acquired immunodeficiency syndrome (AIDS) may be a consequence of destabilization of the idiotypic network. These studies demonstrated that auto- or allo-immunizations involving recognition of class II MHC antigens can trigger an anti-HIV response and such possibilities should be taken into consideration while delineating the pathogenesis of AIDS.
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    Estrogen Regulates Interferon-gamma (IFN-g) and IFN-g-Inducible iNOS Gene Expression: Implications to Immunity and Autoimmunity
    Sahin, Ebru Karpuzoglu (Virginia Tech, 2005-04-07)
    It is now clear that estrogen not only modulates the differentiation and function of reproductive systems, but it also profoundly regulates the immune system of normal and autoimmune individuals. An important mechanism by which estrogen regulates the immune system is by altering the secretion and/or response to cytokines. We hypothesized that estrogen may alter the levels and/or response to IFN-g, a prototype Th1 cytokine, that plays a pivotal role in immunity against intracellular infections and in many autoimmune and inflammatory disorders. We found that estrogen treatment tended to upregulate the secretion of IFN-g protein and mRNA expression from Concanavalin-A (Con-A)-activated splenic lymphocytes. Impressively, we found that splenocytes from estrogen-treated mice when activated with Con-A also resulted in increased release of nitric oxide compared to placebo-treated mice. Furthermore, Con-A-activated splenocytes from estrogen-treated mice also had upregulated iNOS mRNA, iNOS protein, and nitric oxide-regulated COX-2 protein when compared to control mice. Blocking co-stimulatory signals mediated through interactions of CD28 and B7 molecules by using CTLA-4Ig markedly decreased not only IFN-g, but also nitric oxide, thereby implying an important role for CD28/B7 interactions in IFN-g/nitric oxide. Estrogen-induced upregulation of iNOS/nitric oxide is mediated through IFN-g since: (i) Estrogen alone did not upregulate iNOS/nitric oxide in IFN-g knockout mice; (ii) addition of rIFN-g to activated splenocytes from estrogen-treated mice further upregulated nitric oxide levels. We next investigated whether estrogen also upregulated IFN-g-inducing cytokines and select IFN-g-inducing transcription factors. Estrogen treatment resulted in increased mRNA and/or protein expression of IFN-g inducing cytokines and their receptors, including: IL-18, IL-15, IL-27, IL-12Rb2, and IL-18Rb. We also found that T-bet, a critical Th1 transcription factor, and STAT-4 phosphorylation, a key molecule in IL-12 signaling were both increased, while IRF-4, an important player in Th2 differentiation, was diminished in Con-A-activated splenocytes from mice treated with estrogen. Altogether, these studies are the first to demonstrate that estrogen regulates IFN-g-dependent iNOS and describes the potential mechanisms of how estrogen alters IFN-g-inducible genes, IFN-g inducing cytokines, and transcription factors in normal C57BL/6 mice. These studies may have profound implications to many autoimmune and inflammatory disorders, where estrogen is known to regulate the course of these diseases. Since estrogen may promote inflammatory disorders by upregulating pro-inflammatory biomolecules including IFN-g, nitric oxide, and COX-2, these studies may help in the design of therapeutic agents that regulate or block secretion and/or response to these inflammatory molecules.
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    Evaluation of immunological techniques for host fish identification, and cryopreservation of embryos for conserving rare freshwater mussels
    Chang, Yunsheng (Virginia Tech, 1993-09-16)
    Glochidia (larvae) of freshwater mussels are obligate parasites which attach to and become encysted in the gills or fins of host fish species. The immune responses of the host fish to the parasite affects the susceptibility of the fish to glochidia of different mussels. The immune response provides an opportunity to identify which fish species are hosts. The number and variety of mussels in rivers and lakes has sharply declined since the last century due to various anthropogenic factors, and some mussels species are facing extinction. It is an urgent task to preserve these vanishing mussels, or extinction will be inevitable. An attempt was made to develop an assay, using the immunological response to glochidia, to screen fish species for appropriate hosts. This would facilitate the production and rearing of juveniles. In order to design these assays, reagents such as anti-immunoglobulins which can react with antibodies from many different fish species have to be developed. This work was carried out to develop such reagents. Host and non-host fish were immunized with killed bacteria (Brucella abortus) to study their humoral immune response to an antigen. All fish were able to respond well, as measured by agglutination and Western Blot assays. Antibodies produced by the Brucella injections were used to stimulate anti-fish immunoglobulins in goats, and the antisera were tested for their ability to recognize immunoglobulins from different host fish species. The specificities of these reactions were compared to the reactivity of Protein A. Goat antisera were able to cross-react with different fish antibodies, but it was found that Protein A was a more suitable reagent. Protein A is seemingly suitable to identify the host-fish species and could be used as a reagent for the serological diagnosis of various fish diseases.
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    Expression and Localization of Green Fluorescent Protein in B. abortus strain RB51
    Liu, Hailan (Virginia Tech, 2003-05-06)
    Brucella abortus is a facultative intracellular bacterial pathogen, which causes abortion in cattle and undulant fever in human. B. abortus strain RB51 (Strain RB51) is the official vaccine for bovine brucellosis in the USA. B. abortus strain RB51 can be used as a vector for the over-expression of its own (homologous) as well as heterologous protective antigens. The immune system can detect these heterologous antigens and produce a response. Expressing a protein in different bacterial compartments has been shown to affect its accessibility to the immune system and the way the antigen is processed by antigen presenting cells. In order to determine if the immune response is affected by the localization of the antigen, green fluorescent protein (GFP) was expressed at three different locations in B. abortus strain RB51, outer-membrane (OM), periplasmic space (PS) and in the cytoplasmic region (CR) of B. abortus strain RB51. This localization was obtained by transforming strain RB51 with plasmids pBBg18sGFP and pBBgSsGFP, in which the 18 kDa Brucella lipoprotein and the Brucella Cu/Zn SOD protein signal sequences were added to the GFP sequence to cause OM and PS expression respectively. No signal sequences were added to the plasmid pBBgGFP for CR only expression. Expression and localization of GFP in the different compartments in recombinant B. abortus strain RB51 were confirmed by electron microscopy and antibody absorption experiments. Groups of 5 female BALB/c mice each were injected and boosted with three recombinant strains and appropriate controls. Mice were bled and their anti-GFP antibody production was assessed. None of the immunized mice produced specific antibodies against GFP, probably due to the low expression of the heterologous antigen observed in this study by strain RB51 observed in this study. It will be necessary to produce new recombinants which are able to express higher amounts of GFP to answer if localization of heterologous antigen within the recombinant RB51 affects the level of a specific immune response.
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    Expression of Bacillus Anthracis Protective Antigen in Vaccine Strain Brucella Abortus Rb51
    Poff, Sherry Ann (Virginia Tech, 1997-12-15)
    Bacillus anthracis is a facultative intracellular bacterial pathogen that can cause cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are of limited duration and do not protect against the respiratory form of the disease. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective cell mediated immunity and humoral responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce protective antibodies, it was decided that the objective of this research was to test whether the gene encoding PA could be expressed in Brucella producing a bivalent vaccine to protect against both brucellosis and anthrax. The pag gene was transcriptionally fused to promoters of genes encoding superoxide dismutase or heat shock protein groE, subcloned into a broad host range plasmid (pBBR1MCS) and shown to express in E. coli by immunoblotting using antiserum specific for PA. The immunoblot results revealed that E. coli produced a PA protein of the expected size. In addition, the culture medium was shown to contain the same PA protein using immunoblotting. These results show that E. coli is capable of expressing B. anthracis PA in both the cellular and extracellular forms. The pBB/PA plasmid was used to transform B. abortus RB51 and CmR clones screened for the expression of PA by immunoblotting. Twenty clones of strain RB51/pBBSOD were show to express a 30kDa PA protein. Three clones of strain RB51/pBBGroE-PA were shown to express a 63-83kDa protein as detected by antiserum specific for PA. Using the A/J mouse, an immunocompromised vertebrate model, immunization and challenge studies were performed. Preliminary results demonstrate that the bivalent vaccine is capable of producing protection against a live challenge with B. abortus and some protection against live non-disease producing spores of B. anthracis.