Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean.

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.

Exacerbation of experimental autoimmune encephalomyelitis in rodents infected with murine gammaherpesvirus-68.

Viral infections have long been suspected to play a role in the pathogenesis of multiple sclerosis. In the present study, two different rodent models of experimental autoimmune encephalomyelitis (EAE) were used to demonstrate the ability of murine gammaherpesvirus-68 (gammaHV-68) to exacerbate development of neurological symptoms. SJL mice received UV-inactivated gammaHV-68 or intranasalgammaHV-68, followed by immunization against proteolipid-protein peptide 139-151. Infected mice became moribund within 10 days post-immunization, whereas mice exposed to UV-inactivated gammaHV-68 recovered. In the second model, Lewis rats were exposed to UV-inactivated gammaHV-68 or to gammaHV-68, followed by passive transfer of encephalitogenic T lymphocytes specific for myelin basic protein. Consistently, infected rats had higher clinical scores, and this result was observed during acute or latent gammaHV-68 infection. It is unlikely that this gammaHV-68-induced exacerbation was due to significant viral replication within the central nervous system since nested PCR, viral plaque assays, and infectious-centers assays demonstrated no detectable virus in spinal cords or brains of infected rodents undergoing EAE. Taken together, these studies demonstrate increased clinical symptoms of EAE in rodents infected by a gammaherpesvirus that has a limited ability to invade the central nervous system.

Reduced CTL response and increased viral burden in substance P receptor-deficient mice infected with murine gamma-herpesvirus 68.

One component of the protective host response against mucosal pathogens includes the local production and increased expression of certain neuropeptides and their receptors. The present study further demonstrates this fact by investigating the contribution that substance P receptor expression makes toward immunity against a gamma-herpesvirus infection. Following intragastric inoculation with murine gamma-herpesvirus 68 (gamma HV-68), expression of substance P and its receptor was increased in mucosal and peripheral lymphoid organs in wild-type strains of mice. These results suggested that this receptor/ligand pair might be an important component of the host response against this viral infection. Such a hypothesis was supported by the demonstration that mice, genetically deficient in substance P receptor expression, showed an increased viral burden when compared with syngeneic C57BL/6 mice. Furthermore, substance P receptor-deficient mice showed a reduced CTL response against gamma HV-68, suggesting a mechanism to explain this increased viral burden. Such limitations in the Ag-specific CTL response in substance P receptor-deficient mice could result from lowered expression of IL-12 during viral infection. Consistent with this hypothesis, increases in mRNA encoding IL-12 and secretion of this cytokine into sera of infected, wild-type animals were markedly reduced in substance P receptor-deficient mice. These studies demonstrate that genetic elimination of substance P receptors in mice results in an increased gamma-herpesvirus burden and an altered host response.

Bacterium-induced CXCL10 secretion by osteoblasts can be mediated in part through toll-like receptor 4.

Two common pathogens known to cause bone infection, Salmonella and Staphylococcus aureus, were investigated to determine their abilities to induce chemokine expression in cultured mouse and human osteoblasts. While these cells are responsible for bone formation, we were surprised to find that they could respond to bacterial infection by upregulating expression of the chemokine CXCL10 (IP-10). However, there were significant differences in the abilities of the gram-negative bacterium Salmonella and the gram-positive bacterium S. aureus to induce expression of CXCL10. Reverse transcription-PCR and enzyme-linked immunosorbent assay analyses showed high levels of Salmonella-induced CXCL10 mRNA and protein expression, respectively, whereas the osteoblast response to S. aureus was significantly less. Consistent with these findings, Salmonella-derived lipopolysaccharide (LPS), but not S. aureus-derived peptidoglycan, could induce expression of CXCL10. An antibody against toll-like receptor 4 (TLR4) could block the LPS-induced CXCL10 production, demonstrating the functional expression of TLR4 by osteoblasts. Despite the inducible nature of TLR2 mRNA expression by bacterium-infected osteoblasts, peptidoglycan failed to stimulate CXCL10 secretion. Immunofluorescent staining of bacterium-infected calvaria (i.e., skull bone) demonstrated the presence of CXCL10 in osteoblasts. The fact that osteoblasts did not express CXCR3 mRNA, whereas T lymphocytes can express high levels of this receptor, suggests that osteoblast-derived CXCL10 may recruit T lymphocytes to the sites of bone infections.