Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: The focus of our studies is to assess the feasibility of attenuated Salmonella vectors as a vaccine delivery vehicle for cows similar to that described for humans and for chickens. We propose to use attenuated Salmonella vectors as a means of immunizing heifers against bovine enterotoxigenic Escherichia coli (ETEC). We hypothesize that protective immunity to ETEC is dependent upon how the fimbriae is expressed, i.e., extracellular versus intracellular expression. These studies examined humoral immunity induced following oral immunization with the bovine ETEC vaccines in heifers for subsequent challenge studies in newborn calves. During this study, we were able to conduct a vaccine trial. Nine heifers were divided into three groups: vector only (three orally immunized with H647), PBS (two given vehicle only), and Salmonella-K99 (four orally immunized with strain AP112). Heifers were given three oral doses of vaccine on days 0, 30, and 60. Thirty days after the last dose, heifers were artificially inseminated. Nine of nine became pregnant, and successfully calved. Newborn calves were allowed to receive colostrum for the first 12 hrs post-parturition, and then challenged with wild-type K99+ ETEC (strain B41). Calves were followed daily and in some instances, supportive fluids were provided. One of two calves from vehicle-treated heifers scoured. This one scouring calf was able to clear after one day the K99+ ETEC, but did later develop a rotavirus infection. Evaluation of fecal CFU revealed that those calves from PBS-treated and H647-vaccinated heifers shed K99+ ETEC for greater lengths of time whereas calves from AP112-vaccinated heifers shed K99+ ETEC for a shorter duration. Thus, these studies suggest that Salmonella-K99 vaccine was effective for stimulating long-lasting immunity in heifers to confer protection for their newborn calves. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Currently, the industry lacks an efficacious method for delivering vaccines orally into cattle. Current studies are evaluating the efficiency of Salmonella vectors and other methods as a means to vaccinate cattle against enteric diseases. A successful outcome from these studies will facilitate development of new livestock vaccines.
Publications
- Rynda, A., Maddaloni, M., Mierzejewska, D., Ochoa-Reparaz, J., Maslanka, T., Crist, K., Riccardi, C., Barszczewska, B., Fujihashi, K., McGhee, J.R., and Pascual, D.W. 2008. Low-dose tolerance is mediated by microfold cell ligand, reovirus protein sigma1. J. Immunol. 180: 5187-5200.
- Suo, Z., Avci, R., Yang, X., and Pascual, D.W. 2008. An efficient immobilization and patterning of live bacterial cells. Langmuir 24:4161-4167.
- Suzuki, H., Sekine, S., Kataoka, K., Pascual, D.W., Maddaloni, M., Kobayashi, R., Fujihashi, K., Kozono, H., McGhee, J.R., and Fujihashi, K. 2008. Ovalbumin-protein sigma1 M cell targeting facilitates oral tolerance with loss of antigen-specific CD4+ T cells. Gastroenterology 135:917-925.
- Ochoa-Reparaz, J., Rynda, A., Ascon, M.A., Yang, X., Kochetkova, I., Riccardi, C., Callis, G., Trunkle, T., and Pascual, D.W. 2008. IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen. J. Immunol. 181:954-968.
- Pascual, D.W., Wang, X., Kochetkova, I., Callis, G., and Riccardi, C. 2008. Absence of CD8+ lymphoid dendritic cell maturation in L-Selectin-/- respiratory compartment attenuates anti-viral immunity. J. Immunol. 181:1345-1356.
- Rynda, A., Maddaloni, M., Callis, G., Riccardi, C., and Pascual, D.W. 2008. Single dose tolerance protects against experimental autoimmune encephalomyelitis (EAE) via immune deviation and regulatory T (Treg) cells. Experimental Biology Meeting 2008, San Diego, CA, 366, #1074.4.
- Kochetkova, I., Trunkle, T., Callis, G., and Pascual, D.W. 2008. Vaccination without auto-antigen protects against collagen II-induced arthritis via immune deviation and regulatory T cells. J. Immunol. 181:2741-2452.
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Progress 07/01/07 to 06/30/08
Outputs
OUTPUTS: Outputs are activities and teaching that reached the scientific community. Activities included conducting and analyzing experiments, teaching, and mentoring. Events include conferences, interviews, workshops, symposia, workshops, and trainings. Services include consulting, counseling, and tutoring. Products included the development of new vaccines, publications, and dissmenation of information via the public press or scientific press, meetings, and symposia. Knowledge was disseminated via publications, symposia, and workshops.
TARGET AUDIENCES: To date, vaccine development for ruminants consists largely of inactivating the pathogen and its subsequent administration without concern to possible side effects or efficacy. Thus, we will test our mucosal vaccine delivery system using attenuated, Salmonella vectors to express our vaccine antigen, K99 fimbriae, in cattle. Our delivery system will provide the basis for future generation ruminant vaccines to learn how cattle respond to vaccine antigens delivered by Salmonella, and whether they are effective in providing protection in ETEC challenge studies.
Impacts
1. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic
derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against ~1000 LD50 Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD50 Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis. 2. Regulatory T (Treg) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on auto-antigen reactive Treg cells, stimulation to myelin-independent antigens (Ags) may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I (CFA/I)
possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain Treg cell-dependency, a kinetic analysis was performed showing increased levels of FoxP3+CD25+CD4+ T cells. Inactivation of these Treg cells resulted in loss of protection. Adoptive transfer of the vaccine-induced Treg cells protected mice against EAE with greater potency than naive or Salmonella vector-induced Treg cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these Treg cells in conjunction with immune deviation by Th2 cells optimally induced protective Treg cells when compared those induced in the absence of Th2 cells. These data show that Treg cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.
Publications
- Yang, X., Hinnebusch, B.J., Trunkle, T., Bosio, C.M., Suo, Z., Tighe, M., Harmsen, A., Becker, T., Crist, K., Walters, N., Avci, R., and Pascual, D.W. (2007) Oral vaccination with Salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague. J. Immunol. 178:1059-1067.
- Ochoa-Reparaz, J., Riccardi, C., Rynda, A., Jun, S., Callis, G., and Pascual, D.W. (2007) Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis. J. Immunol. 178:1791-1799.
- Suo, Z., Yang, X., Avci, R., Kellerman, L., Pascual, D.W., Fries, M., and Steele, A. (2007) HEPES-stabilized encapsulation of Salmonella typhimurium. Langmuir 23:1365-1374.
- Fujihashi, K., Staats, H.F., Kozaki, S., and Pascual, D.W. (2007) Mucosal vaccine development for botulinum intoxication. Expert Rev. Vaccines 6:35-45.
- Yang, X., Walters, N., Robison, A., Trunkle, T., and Pascual, D.W. (2007) Nasal immunization with recombinant Brucella melitensis bp26 and trigger factor with cholera toxin reduces B. melitensis colonization. Vaccine 25:2261-2268.
- Pascual, D.W., Ochoa-Reparaz, J., Rynda, A., and Yang, X. (2007) Tolerance in the absence of auto-antigen. Endocrine, Metabolic and Immune Disorders - Drug Targets 7:203-210.
- Pascual, D.W. 2007. Commentary: Vaccines are for dinner. Proc. Natl. Acad. Sci., USA 104:10757-10758. Hedges J.F., Buckner, D.L., Rask, K.M., Kerns, H.M., Jackiw, L.O., Trunkle, T.C., Pascual, D.W., and Jutila, MA. (2007) Mucosal lymphatic-derived gd T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2Ra. Cell. Immunol. 246:8-16.
- Ochoa-Reparaz, J., Sentissi, J., Trunkle, T., and Pascual, D.W. (2007) Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and toll-like receptor 2 knockout mice and protects against reinfection. Infect. Immun. 75:5845-5858.
- Pascual, D.W., Riccardi, C., and Csencsits, K. (2008) Distal IgA immunity can be sustained by aEb7+ B cells in L-Selectin-/- mice following oral immunization. Mucosal Immunol. 1:68-77.
- Riccardi, C. and Pascual, D.W. 2007. Head and neck lymph node (HNLN) B cells from L-Selectin-/- (L-Sel-/-) mice show alphaE expression independent of cholera toxin (CT) exposure. J. Immunol. 178: #41.9.
- Maslanka, T., Riccardi, C., and Pascual, D.W. 2007. alphaE Integrin compensates for beta7 deficiency in the nasal passages (NP) following nasal immunization with cholera toxin (CT). J. Immunol. 178: #41.10.
- Trunkle, T., Yang, X., Bosio, C.M., and Pascual, D.W. 2007. Co-Expression of Yersinia pestis F1- and V-Ags by the same Salmonella vaccine protects against nasal Y. pestis challenge. J. Immunol. 178: #41.14.
- Kochetkova, I., Trunkle, T., Callis, G., and Pascual, D.W. 2007. Diverse subsets of regulatory T (Treg) cells mediates suppression of arthritis by an oral bacterial-based diarrheal vaccine. J. Immunol. 178:#47.22.
- Hedges, J.F., Buckner, D., Rask, K., Kerns, H., Jackiw, L., Trunkle, T., Pascual, D.W., Dobrinin, E., Radke, M., Holderness, J., and Jutila, M.A. 2007. Priming of gamma/delta T cells. J. Immunol. 178: #52.4.
- Yang, X., Suo, Z., Walters, N., Trunkle, T., Crist, K., Becker, T., Kellerman, L., Avci, R., Kochetkova, I., and Pascual, D.W. 2007. Adjuvant properties of colonization factor antigen I (CFA/I) are dampened when Yersinia pestis F1 or V antigens are co-expressed in Salmonella enterica serovar Typhimurium vaccine. 107th ASM General Meeting, Toronto, Canada.
- Ochoa-Reparaz, J., Ascon, M.A., Riccardi, C., Rynda, A., Callis, G., and Pascual, D.W. 2007. Salmonella vaccines confers protection against experimental autoimmune encephalomyelitis (EAE) devoid of autoantigen by stimulation of regulatory T (Treg) cells. 13th Intern. Cong. Mucosal Immunol., Tokyo, Japan.
- Yamanaka, H., Hoyt, T., Golden, S., Bosio, C.M., Crist, K., and Pascual, D.W. 2007. A nasal IL-12 DNA vaccine co-expressing Yersinia pestis F1-V fusion protein confers protection against pneumonic plague. 13th Intern. Cong. Mucosal Immunol., Tokyo, Japan.
- Rynda, A., Maddaloni, M., Callis, G., Riccardi, C., Crist, K., Ochoa-Reparaz, J., and Pascual, D.W. 2007. Adapter molecule mediates single-dose tolerization to prevent autoimmunity via the induction of antigen-specific IL-10+ and TGF-b+ regulatory T cells. 13th Intern. Cong. Mucosal Immunol., Tokyo, Japan.
- Skyberg, J.A., Jutila, M.A., and Pascual, D.W. 2007. Protective role for gamma/delta T cells in the innate immune response against Brucella abortus. 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil.
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Progress 10/01/05 to 09/30/06
Outputs
1. Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaption of the K99 scaffold to display heterologous B and T cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished IgA and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial
reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane, but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection. 2. An experimental vaccine for ETEC composed of a live, attenuated Salmonella vector expressing ETEC fimbriae, colonization factor antigen I (CFA/I), stimulated a biphasic T cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-alpha, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with
Salmonella-CFA/I construct 1 or 4 wks prior to induction of experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic proteolipid protein (PLP) peptide, PLP139-151. Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-gamma-secreting T cells and replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, EAE, via immune deviation.
Impacts
These results support the contention that variation in the expression of ETEC fimbriae within different compartments of Salmonella vaccine vectors alters the type of Th cell responses elicited.
Publications
- Suzuki, H., Sekine, S., Kobayashi, R., Fukuiwa, T., Kataoka, K., Fujihashi, K., McGhee, J.R., Pascual, D.W., and Fujihashi, K. 2005. Enhanced mucosal tolerance induction by reovirus protein sigma1. 12th International Congress of Mucosal Immunology, #52321.
- Rynda, A., Maddaloni, M., and Pascual, D.W. 2005. A shaft-less protein sigma 1 as a potent immune response modulator. 12th International Congress of Mucosal Immunology, #52560.
- Crist, K., Maddaloni, M., and Pascual, D.W. 2005. Modified reovirus protein sigma1 for in vitro and in vivo tracing studies. 12th International Congress of Mucosal Immunology, #52607.
- Hoyt, T., Becker, T., Haddad, A., and Pascual, D.W. 2005. Co-expression of Bcl-2 or BLC with beta-galactosidase (betagal) elevates mucosal and systemic antibodies (Abs) following protein sigma1-targeted intranasal DNA vaccination. 12th International Congress of Mucosal Immunology, #52849.
- Kochetkova, I., Trunkle, T., Callis, G., and Pascual, D.W. 2005. Oral immunization with a Salmonella vaccine for enterotoxigenic E. coli (ETEC) inhibits autoimmunity via TGF-beta+ CD4+CD25+ T Cells. 12th International Congress of Mucosal Immunology, #52880.
- Barszczewska, B., Riccardi, C., Robison, A. and Pascual, D.W. 2005. L-selectin and beta7 activation on B lymphocytes following intranasal (i.n.) Immunization with cholera toxin (CT). 12th International Congress of Mucosal Immunology, #53566.
- Pascual, D.W., Staats, H., Mierzejewska, D., Hoyt, T., Robinson, A., Fujihashi, K., and Maddaloni, M. 2005. Mucosal vaccine targeting circumvents the use of adjuvant for development of mucosal immunity to botulinum neurotoxin A (BoNT/A). 12th International Congress of Mucosal Immunology, #53587.
- Jun, S.M., Callis, G., and Pascual, D.W. 2005. Antigen-independent suppression of experimental autoimmune encephalomyelitis (EAE) by a Salmonella vaccine expressing enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I). FASEB J. 19:A3, #43.9.
- Yang, X., Trunkle, T., Becker, T., and Pascual, D.W. 2005. Salmonella vaccine expressing Yersinia pestis V Antigen is regulated by promoters. 105 ASM General Meeting, Atlanta, GA, Abstract #4229.
- Maddaloni, M., Riccardi, C., Rynda, A., Mierzejewska, D., Crist, K., and Pascual, D.W. 2005. A viral device for targeted induction of tolerance. 12th International Congress of Mucosal Immunology, #52234.
- Yang, X., Hudson, M., Walters, N., Bargatze, R.F., and Pascual, D.W. 2005. Selection of protective epitopes for Brucella melitensis using DNA vaccination. Infect. Immun. 73:7297-7303.
- Jun, S., Gilmore, W., Callis, G., Rynda, A., Haddad, A., and Pascual, D.W. 2005. A live diarrheal vaccine imprints a Th2 cell bias and acts as an anti-inflammatory vaccine. J. Immunol. 175:6733-6740.
- Bost, K.L., Clemente, T.E., Sato, S., Pascual, D.W., and Piller, K.J. 2005. Expression and immunogenicity of an E. coli K99 fimbrial subunit in transgenic soybeans. FASEB J. 19:A896, #551.17.
- Pascual, D.W., Wang, X., Kochetova, I., Callis, G., and Riccardi, C. 2005. Selective loss of respiratory plasmacytoid CD8+ dendritic cells (DC) in L-Selectin-/- (L-Sel-/-) mice: CD8- DC confer tolerance to viral vector. FASEB J. 19:A947, #561.5.
- Riccardi, C. and Pascual, D.W. 2005. The distal mucosal B cell immunity in L-Selectin-/- (L-Sel-/-) mice after oral cholera toxin (CT) immunization is due to sustained B cell activation. FASEB J. 19:A929, #557.14.
- Pascual, D. W. and Bost, K. L. 2005. Neuropeptides for mucosal immunity. Mucosal Immunology, 3rd edition, Section B: Inductive and Effector Tissues and Cells of the Mucosal Immune System, J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock, J. R. McGhee, and L. Mayer eds., Academic Press, Inc., San Diego, CA., Chap. 38, pp. 737-748.
- Piller, K.J., Clemente, T.E., Jun, S.M., Petty, C.C., Sato, S., Pascual, D.W., and Bost, K.L. 2005. Expression and immunogenicity of an E. coli K99 fimbriae subunit antigen in soybean. Planta 222:6-18.
- Wang, X., Kochetkova, I., Haddad, A., Hoyt, H., Hone, D.M., and Pascual, D.W. 2005. Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope. Vaccine 23:3836-3842.
- Ascon, M.A., Ochoa-Reparaz, J., Walters, N., and Pascual, D.W. 2005. Partially assembled K99 fimbriae required for protection. Infect. Immun. 73:7274-7280.
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Progress 07/01/05 to 06/30/06
Outputs
1. Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaption of the K99 scaffold to display heterologous B and T cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished IgA and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial
reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane, but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection. 2. An experimental vaccine for ETEC composed of a live, attenuated Salmonella vector expressing ETEC fimbriae, colonization factor antigen I (CFA/I), stimulated a biphasic T cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-alpha, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with
Salmonella-CFA/I construct 1 or 4 wks prior to induction of experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic proteolipid protein (PLP) peptide, PLP139-151. Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-gamma-secreting T cells and replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, EAE, via immune deviation.
Impacts
These results support the contention that variation in the expression of ETEC fimbriae within different compartments of Salmonella vaccine vectors alters the type of Th cell responses elicited.
Publications
- Yang, X., Becker, T., Jun, S.M., Walters, N., and Pascual, D.W. 2006. Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge. Infect. Immun. 74:3874.
- Maddaloni, M., Staats, H.F., Mierzejewska, D., Hoyt, T., Robinson, A., Callis, G., Kozaki, S., Kiyono, H., McGhee, J.R., Fujihashi, K., and Pascual, D.W. 2006. Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A (BoNT/A). J. Immunol. 177:5524.
- Ochoa-Reparaz, J., Riccardi, C., Rynda, A., Jun, S.M., Callis, G., and Pascual, D.W. 2006. An oral bacterial-based diarrheal vaccine is therapeutic for experimental autoimmune encephalomyelitis (EAE) due to induced regulatory T (Treg) cells. J. Immunol. 176:S144, #95.1.
- Rynda, A., Maddaloni, M., Mierzejewska, D., and Pascual, D.W. 2006. M cell ligand induces tolerance to ovalbumin (OVA) via TGF-b and IL-4. J. Immunol. 176:S244, #115.6.
- Kochetkova, I., Trunkle, T., Callis, G., and Pascual, D.W. 2006. Suppression of arthritis by an oral bacterial-based diarrheal vaccine is mediated via enhanced function by Th2 and regulatory T (Treg) cells. J. Immunol. 176:S245, #115.8.
- Pascual, D.W., Kochetkova, I., Trunkle, T., and Ochoa-Repraz, J. 2006. Anti-inflammatory Salmonella vaccine therapy elicits regulatory T cells and protects sgainst sutoimmunity independent of autoantigen. 2nd ASM Conference on Salmonella: Pathogenesis to Therapeutics.
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Progress 10/01/04 to 09/30/05
Outputs
1. The development of the neurokinin-1 receptor-deficient (NK1R-/-) mouse permitted inquiry into the regulation of secretory IgA (S-IgA) responses by substance P (SP) subsequent to oral immunization with a Salmonella vector expressing colonization factor antigen I (CFA/I) from enterotoxigenic Escherichia coli (ETEC). In NK1R-/- mice, mucosal and serum IgA anti-CFA/I fimbrial responses were augmented while secreted IgG anti-CFA/I fimbrial responses remained unaffected when compared to BALB/c (NK1R+/+) mice. Supportive antibody-forming cells (AFC) were present in the small intestinal lamina propria (iLP) and spleen. To gain insight as to why the augmented S-IgA responses occurred, minimally, it was not attributed to differences in vaccine colonization of PP and spleen or in their respective tissue weights. However, these S-IgA responses were supported by increased numbers of Peyer's patch (PP) CD4+ Th cells secreting IL-5 and IL-6 and splenic CD4+ Th cells secreting
IL-6 when compared to NK1R+/+ mice. Challenge of naive NK1R-/- mice with wild-type Salmonella showed improved median survival when compared to naive NK1R+/+ mice. Data from peritoneal macrophage infection studies suggest that this survival is in part contributed by increased IL-10 production. Oral vaccination with Salmonella-CFA/I or Salmonella vector showed no significant differences in conferred protection against wild-type challenge for either NK1R-/- or NK1R+/+ mice. Thus, these studies suggest that SP mediation contributes to proinflammatory responses to Salmonella infections. 2. Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies have shown that murine dams immunized with Salmonella vaccine vectors stably expressing the K99 operon confer protection to ETEC-challenged neonatal pups. To address adaption of the K99 scaffold to display heterologous epitopes, studies were conducted to determine how much of the assembled
K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 operon were made, resulting in the K99 fimbrial subunit localized to the periplasmic or cytoplasmic compartments. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished IgA and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Retention of the K99 fimbrial subunit in the periplasmic compartment showed only partial reduction in protective immunity, unlike when expressed in the cytoplasm, protective immunity was dramatically reduced.
Impacts
These results support the contention that variation in the expression of K99 fimbrial subunit within different compartments of Salmonella vaccine vectors alters the type of Th cell responses elicited.
Publications
- Mierzejewska, D., Maddaloni, M., and Pascual, D.W. 2004. Targeting protein vaccines to M cells enhances mucosal IgA responses. FASEB J. 18:A821, #558.5.
- Riccardi, C., Walters, N., and Pascual, D.W. 2004. Cervical lymph nodes (CLN) are phenotypically and functionally heterogenous as evidenced by the selective IgA responses to cholera toxin (CT). FASEB J. 18:A825, #558.23.
- Yang, X., Trunkle, T., Crist, K., and Pascual, D.W. 2004. Oral immunization with a Salmonella vaccine vector expressing Yersinia pestis capsular (F1) antigen elicits elevated mucosal IgA and serum IgG antibodies. 104 ASM meeting, New Orleans, LA, Abstract #1158.
- Pascual, D.W. 2004. Anti-Inflammatory vaccination using a Salmonella vaccine vector vector. 4th International Forum on Infection and Immunity.
- Kobayashi, R., Kohda, T., Kataoka, K., Ihara, H., Kozaki, S., Pascual, D. W., Staats, H. F., Kiyono, H., McGhee, J. R., Fujihashi, K. 2004. A mucosal vaccine prevent botulism. Clinc. Invest. Med. 27:196B (A# T47.200).
- Pascual, D.W. 2004. The role of tachykinins in bacterial infections. Front. Biosci. 9:3209-3217.
- Walters, N., Sura, M., Trunkle, T., and Pascual, D.W. 2005. Enhanced immunoglobulin A response and protection against Salmonella enterica serovar Typhimurium in the absence of the substance P receptor. Infect. Immun. 73:317-324.
- Pascual, D. W. and Bost, K. L. 2005. Neuropeptides for mucosal immunity. Mucosal Immunology, 3rd edition, Section C: Functional characteristics of mucosal cells and tissues, J. Mestecky, J. Bienenstock, M. E. Lamm, W. Strober,, J. R. McGhee, and L. Mayer eds., Academic Press, Inc., San Diego, CA. Chap. 38, pp. 737-748.
- Kobayashi, R., Kohda, T., Kataoka, K., Ihara, H., Kozaki, S., Pascual, D. W., Staats, H. F., Kiyono, H., McGhee, J. R., and Fujihashi, K. 2005. Botulinum neurotoxin-specific S-IgA antibodies prevent mucosal botulism. J. Immunol. 174:2190-2195.
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Progress 10/01/03 to 09/30/04
Outputs
To assess the effectiveness of each Salmonella construct in delivering the K99 fimbrial subunit to mucosal inductive tissues, outbred CD-1 female mice (n=8/immunization group) were orally immunized and mucosal secretions and serum were collected and analyzed for anti-K99 fimbria antibody titers. Six wk post-immunization, and approximately 3 wk after mating, the AP112-vaccinated females elicited the greatest IgA titers in milk, serum, and fecal samples when compared to AP114-, AP116-, and H647-vaccinated females (p<0.001). Maximal increases in serum IgG, IgG1, and IgG2b titers were obtained for AP112-vaccinated females. However, serum IgG1 and IgG2b antibody responses were sequentially reduced in mice orally immunized with AP114, AP116, and H647 strains. Serum IgG2a anti-K99 titers were equivalent for mice vaccinated with AP112 or AP114 strains, but were reduced for AP116-vaccinated mice. Interestingly, the IgG2b titers were not reduced between AP114 and
AP116-vaccinated mice. The observed increases of IgA and IgG1 K99-specific titers by AP112-immunized mice suggest that Th2-type cells supported this immune response.
Impacts
These results support the contention that variation in the expression of K99 fimbrial subunit within different compartments of Salmonella vaccine vectors alters the type of Th cell responses elicited.
Publications
- Wang, X., Hone, D.M., Haddad, A., Shata, M.T., and Pascual, D.W. 2003. M cell DNA vaccination for CTL immunity to HIV. J. Immunol. 171:4717-4725.
- Wang, X., Hillemeyer, P., and Pascual, D.W. 2003. Segregation of mechanisms for CTL killing between lungs and regional lymph nodes subsequent to intratracheal delivery of an adenovirus 2 vector. Viral Immunol. 16:525-539.
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Progress 10/01/01 to 09/30/02
Outputs
The investigations performed in my laboratory is to better understand bovine T and B cell responses. To further this effort, we have developed an immunization scheme using a prototype vaccine for bovine enterotoxigenic E. coli (ETEC). We have recently cloned and expressed the bovine cytokines, interferon-gamma and IL-4. We have developed polyclonal antibodies against these regeants to help in the development of detection assays for these cytokines. With these new reagents, we have developed bovine IFN-gamma and bovine IL-4 ELISA assays. We have recently cloned bovine IL-2 and and IL-10 genes into protein expression systems. Using purified recombinant proteins, we have generated polyclonal antibodies against bovine cytokines, and can now follow bovine T cell responses. Thus, we are continuing with these efforts to develop the necessary reagents to assist in analyzing bovine T cell responses.
Impacts
These studies are relevant for development of bovine vaccines and understanding bovine infectious diseases.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/03/02
Outputs
The focus of our studies is to assess the feasibility of attenuated Salmonella vectors as a vaccine delivery vehicle for cows similar to that described for humans and for chickens. We propose to use attenuated Salmonella vectors as a means of protecting newborn calves against bovine enterotoxigenic Escherichia coli (ETEC). We hypothesize that protective immunity to ETEC is dependent upon how the fimbriae is expressed, i.e., extracellular versus intracellular expression. Testing of immunogenicity of ETEC vaccine in heifers. Current studies are examining humoral immunity induced following oral immunization with the bovine ETEC vaccines in heifers for subsequent challenge studies in newborn calves. Nine heifers were divided into three groups: vector only (three orally immunized with H647), PBS (two given vehicle only), and Salmonella-K99 (four orally immunized with strain AP112). Heifers were given three oral doses of vaccine on days 0, 30, and 60. After 30 days
following the last dose, heifers were artificially inseminated. Nine of nine became pregnant, and successfully calved. Newborn calves were allowed to received colostrum for the first 12 hrs post-parturition, and then challenged with wild-type K99+ ETEC (strain B41). Calves were followed daily and in some instances, supportive fluids were provided. One of two calves from vehicle-treated heifers scoured; three of three calves from vector-immunized heifers scoured; and one of four calves from AP112-vaccinated heifers scoured. This one scouring calf was able to clear after one day the K99+ E. coli, but did later develop a rotavirus infection. Evaluation of fecal CFU revealed that those calves from PBS-treated and H647-vaccinated heifers shedded K99+ E. coli for greater lengths of time whereas calves from AP112-vaccinated heifers shedded K99+ E. coli for shorter duration. Thus, these initial studies suggest that Salmonella-K99 vaccine was effective for stimulating long-lasting immunity in
heifers for eventual protection of their newborn calves.
Impacts
It is anticipated that this vaccine could improve protective immunity for newborn calves against scours or E. coli-derived diarrhea.
Publications
- 1. Ascon, M.A., Walters, N., and Pascual, D.W. (2003) Surface expression of K99 fimbriae required for protective immunity. (Submitted).
- 2. Walters, N., Ascon, M.A., and Pascual, D.W. (2003) Oral immunization of heifers with Salmonella expressing K99 fimbriae protects newborn calves against oral challenge with wild-type enterotoxigenic Escherichia coli. (In preparation).
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Progress 10/01/00 to 09/30/01
Outputs
The investigations performed in my laboratory is to better understand bovine T and B cell responses. To further this effort, we have developed an immunization scheme using a prototype vaccine for bovine enterotoxigenic E. coli (ETEC). We have recently cloned and expressed the bovine cytokines, interferon-gamma and IL-4. We developed polyclonal antibodies against these regeants to help in the development of detection assays for these cytokines. With these new reagents, we have developed bovine IFN-gamma and bovine IL-4 ELISA assays. We have recently cloned bovine IL-2 and and IL-10 genes into protein expression systems. Using purified recombinant proteins, we have generated polyclonal antibodies against bovine cytokines, and can now follow bovine T cell responses.
Impacts
These studies have important implications for bovine vaccine development.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
The investigations performed in my laboratory is to better understand bovine T and B cell responses. To further this effort, we have developed an immunization scheme using a prototype vaccine for bovine enterotoxigenic E. coli (ETEC). We have recently cloned and expressed the bovine cytokines, interferon-gamma and IL-4. We developed polyclonal antibodies against these regeants to help in the development of detection assays for these cytokines. With these new reagents, we can now follow bovine T cell responses.
Impacts
These studies have important implications for bovine vaccine development.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
One objective was to orally immunize calves with a prototype Salmonella vaccine to determine the specific B cell (antibody) responses to K99 fimbriae from enterotoxigenic Escherichia coli (ETEC). Accomplishments: 1) We have successfully generated the proposed vaccine prototype for bovine ETEC. The Salmonella construct, designated AP112, when orally administered to mice elicits elevated mucosal and serum antibodies to the expressed ETEC K99 fimbriae. 2) Orally immunized dams with the AP112 vaccine induced protective immunity to neonatally challenged murine pups providing protection to >90% for 10 days whereas dams orally immunized with irrelevant, isogenic vaccine strains, failed to provide protective immunity. 3) Orally immunized calves with the AP112 vaccine induced both mucosal and systemic immunity to K99 fimbriae. A second objective was to develop molecular and antibody probes to bovine cytokines, interferon-gamma and interleukin 4 (IL-4) to assess T cell
responses subsequent to oral immunization with prototype Salmonella vaccines. Accomplishments: 1) We have recently cloned the bovine IFN-gamma cDNA into an expression cassette, and can obtain functional bovine IFN-gamma evident by its ability to induce bovine IgG2 antibodies upon addition to mitogen-stimulated bovine peripheral blood B cells. 2) We have generated a polyclonal antibody to bovine IFN-gamma, and have devised an ELISA for the detection of bovine IFN-gamma induced in mitogen and antigen-specific T cell cultures. 3) We have recently cloned the bovine IL-4 CDNA to develop similar anti-cytokine reagents described for bovine IFN-gamma.
Impacts
(N/A)
Publications
- Ascon, M.A., Hone, D.M., Walters, N., Rognlie, M., and Pascual, D.W. 1998. Directed mucosal immunization with Salmonella vector expressing enterotoxigenic Escherichia coli (ETEC) fimbriae provokes elevated IgG and IgA responses. FASEB J. 12:A909, #5263.
- Ascon, M.A. Hone, D.M., Walters, N., and Pascual, D.W. 1998. Oral immunization with a Salmonella vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity. Infect. Immun. 66:5470-5476.
- VanCott, J.L., Chatfield, S.N., Roberts, M., Hone, D.M., Hohmann, E., Pascual, D.W., Yamamoto, M., Yamamoto, S., and J.R. McGhee. 1998. Regulation of host immune responses by modification of Salmonella virulence genes. Nature-Med. 4:1247-1252.
- Dybing, J.K. and Pascual, D.W. 1998. IL-18 is important for protection against oral challenge with Salmonella typhimurium. FASEB J. 12:A569, #3303.
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Progress 01/01/97 to 12/31/97
Outputs
We propose to use attenuated Salmonella vectors to immunize young calves against bovine enterotoxigenic Escherichia coli (ETEC). ETEC is the second leading cause of diarrhetic death of newborn calves. We propose that if we can elicit immunity to the fimbriae and prevent attachment to host intestinal epithelium, we should be able to protect against ETEC. We have completed the Salmonella-K99 fimbriae as well as its associated chaperone proteins and spliced into our selection plasmid (non-antibiotic) dependent selection), and transformed susceptible delta-aroA Salmonella mutant. We are evaluating both bovine and murine antibody responses to K99 fimbriae. In both animal models, we detect elevated serum IgA and IgG antibody responses. To test efficacy of our construct, we are employing a neonatal challenge model whereby we will challenge newborn pups obtained from Salmonella-K99 or its isogenic strain (which lacks K99 fimbriae) to determine effectiveness in reducing
colonization. To assist in analyzing bovine T cell responses, we cloned bovine IFN-gamma cDNA and expressed it in our E. coli protein expression system. We now have recombinant bovine (b) IFN-gamma and have generated antibodies to bIFN-gamma. These antibodies have allowed us to develop a bIFN-gamma-specific ELISA to detect vaccine-induced IFN-gamma-dependent immune responses.
Impacts
(N/A)
Publications
- Ascon, M.A., Hone, D.M., Walters, N., Rognlie, M., and Pascual, D.W. 1998. Directed mucosal immunization with Salmonella vector expressing enterotoxigenic Escherichia coli (ETEC) fimbriae provokes elevated IgG and IgA responses. FASEB J. (in press)
- Ascon-Cabrere, M., Hone, D.M., and Pascual, D.W. 1998. Expression of K99 fimbriae by an attenuated Salmonella vector induces elevated mucosal IgA antibody responses. (in preparation)
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