Debra Rood

Correlates of Immune Protection in Chickens Vaccinated with Mycoplasma gallisepticum Strain GT5 following Challenge with Pathogenic M. gallisepticum Strain Rlow

ABSTRACT Colonization of the avian respiratory tract with Mycoplasma gallisepticum results in a profound inflammatory response in the trachea, air sacs, conjunctiva, and lungs. A live attenuated M. gallisepticum vaccine strain, GT5, was previously shown to be protective in chickens upon challenge; however, the mechanisms by which this vaccine and others confer protection remain largely unknown. The current study evaluated several potential correlates of GT5 vaccine-mediated immune protection following challenge with the pathogenic M. gallisepticum strain Rlow. GT5-vaccinated chickens developed mild tracheal lesions, consisting of few and scattered, discrete, lymphofollicular aggregates in the lamina propria. In addition, low numbers of aggregated B, CD4+, and CD8+ cells were observed to infiltrate the trachea, in stark contrast to the large numbers infiltrating the tracheas of sham-vaccinated chickens challenged with Rlow. Lymphofollicular aggregates were rarely observed prior to day 12 postchallenge in sham-vaccinated chickens. Instead, they contained an increasingly more cellular inflammatory response characterized by expansion of the lamina propria by lymphoplasmacytic and histiocytic infiltrates. This was due in part to expansion of interfollicular zones by large numbers of infiltrating CD4+ and CD8+ cells and a sizeable population of immunoglobulin A (IgA)- and IgG-secreting plasma cells. GT5-vaccinated chickens also had higher serum IgG concentrations, and significantly higher numbers of M. gallisepticum-specific IgG- and IgA-secreting plasma/B cells within the trachea, than did sham-vaccinated chickens. These responses were observed as early as day 4 postchallenge, indicating the importance of antibody-mediated clearance of mycoplasma in GT5-vaccinated chickens.

A modified live Mycoplasma gallisepticum vaccine to protect chickens from respiratory disease

The aim of this study was to assess the efficacy of a modified live Mycoplasma gallisepticum vaccine (GT5) for the protection of chickens against infection and respiratory disease. GT5 was constructed by the reconstitution of the avirulent high passage R (R(high)) strain with the gene encoding the major cytadhesin GapA. GT5 expressed GapA on its surface yet retained the phenotypic characteristics of the parental R(high) strain. Birds vaccinated with GT5 were protected upon challenge with the virulent low passage R (R(low)) strain as evidenced by a complete absence of tracheal lesions 2 and 4 weeks post-challenge, in contrast to sham immunized/challenged control birds. Modest amounts of IgG, and little, if any secretory IgA or IgM anti-M. gallisepticum were found in tracheal washings following vaccination. However, copious amounts of specific IgA were found following challenge, especially in sham immunized birds. This suggests that the tracheal IgG elicited by GT5 vaccination may have been responsible for blocking the initial colonization of R(low), thereby resulting in protection.

Vaccination of BALB/c mice with an avirulent Mycoplasma pneumoniae P30 mutant results in disease exacerbation upon challenge with a virulent strain.

ABSTRACT Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

Administration of embryonic stem cell-derived thymic epithelial progenitors expressing MOG induces antigen-specific tolerance and ameliorates experimental autoimmune encephalomyelitis

Tolerance induction, and thus prevention or treatment of autoimmune disease, is not only associated with the persistent presence of self-antigen in the thymus, but also relies on a functional thymus; however, the thymus undergoes profound age-dependent involution. Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T cell development. We have reported that mouse embryonic stem cells (mESCs) can be induced in vitro to generate thymic epithelial progenitors (TEPs) that further develop into functional TECs in vivo. We show here that transplantation of mESC-TEPs expressing self-antigen myelin oligodendrocyte glycoprotein (MOG) in mice results in enhanced T cell regeneration, long-term expression of MOG in the thymus, prevention of experimental autoimmune encephalomyelitis (EAE) development, and remission of established EAE. Our findings indicate that transplantation of ESC-TEPs expressing disease-causative self-antigen(s) may provide an effective approach for the prevention and treatment of autoimmune disease.

Use of Inactivated Escherichia coli Enterotoxins To Enhance Respiratory Mucosal Adjuvanticity during Vaccination in Swine

ABSTRACT In order to augment responses to respiratory vaccines in swine, various adjuvants were intranasally coadministered with a foot-and-mouth disease virus (FMDV) antigen to pigs. Detoxified Escherichia coli enterotoxins LTK63 and LTR72 enhanced antigen-specific mucosal and systemic immunity, demonstrating their efficacy as adjuvants for nonreplicating antigens upon intranasal immunization in swine.

Recombinant IL-7/HGFβ Hybrid Cytokine Enhances T Cell Recovery in Mice Following Allogeneic Bone Marrow Transplantation

T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the β-chain of hepatocyte growth factor (HGFβ) (IL-7/HGFβ), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFβ gene in which the IL-7 and HGFβ genes are connected by a flexible linker to generate rIL-7/HGFβ protein. Here, we show that rIL-7/HGFβ treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFβ hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFβ increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFβ enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFβ treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFβ-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFβ-treated BMT mice. Therefore, rIL-7/HGFβ may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.

Tbx1 Modulates Endodermal and Mesodermal Differentiation from Mouse Induced Pluripotent Stem Cells

The T-box transcriptional factor (Tbx) family of transcriptional factors has distinct roles in a wide range of embryonic differentiation or response pathways. Tbx1, a T-box transcription factor, is an important gene for the human congenital disorder 22q11.2 deletion syndrome. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem-cell-based therapies. In this study, we generated iPSCs from Tbx1(-/-) and Tbx1(+/+) fibroblasts and investigated the spontaneous differentiation potential of iPSCs by detailed lineage analysis of the iPSC-derived embryoid bodies. Undifferentiated Tbx1(-/-) and Tbx1(+/+) iPSCs showed similar expression levels of pluripotent markers. The ability of the Tbx1(-/-) iPSCs to generate endodermal and mesodermal lineages was compromised upon spontaneous differentiation into embryonic bodies. Restoration of Tbx1 expression in the Tbx1(-/-) iPSCs to normal levels using an inducible lentiviral system rescued these cells from the potential of defective differentiation. Interestingly, overexpression of Tbx1 in the Tbx1(-/-) iPSCs to higher levels than in the Tbx1(+/+) iPSCs again led to a defective differentiation potential. Additionally, we observed that expression of fibroblast growth factor (FGF) 10 and FGF8 was downregulated in the Tbx1(-/-) iPSC-derived cells, which suggests that Tbx1 regulates the expression of FGFs. Taken together, our results implicated the Tbx1 level as an important determinant of endodermal and mesodermal lineage differentiation during embryonic development.

c‐Met signalling is required for efficient postnatal thymic regeneration and repair

We have reported that in vivo administration of the hybrid cytokine rIL‐7/HGFβ or rIL‐7/HGFα, which contains interleukin‐7 (IL‐7) and the β‐ or α‐chain of hepatocyte growth factor (HGF), significantly enhances thymopoiesis in mice after bone marrow transplantation. We have shown that the HGF receptor, c‐Met, is involved in the effect of the hybrid cytokines. To address the role of c‐Met signalling in thymocyte development and recovery, we generated conditional knockout (cKO) mice in which c‐Met was specifically deleted in T cells by crossing c‐Metft/ft mice with CD4‐Cre transgenic mice. We show here that although the number of total thymocytes and thymocyte subsets in young c‐Met cKO mice is comparable to age‐matched control (Ctrl) mice, the cKO mice were more susceptible to sub‐lethal irradiation and dexamethasone treatment. This was demonstrated by low recovery in thymic cellularity in c‐Met cKO mice after insult. Furthermore, the number of total thymocytes and thymocyte subsets was markedly reduced in 6‐ to 12‐month‐old cKO mice compared with age‐matched Ctrl mice, and the thymic architecture of 12‐month‐old cKO mice was similar to that of 20‐month‐old wild‐type mice. In addition, c‐Met deficiency reduced cell survival and the expression of Bcl‐xL in double‐positive thymocytes, and decreased cell proliferation and the expression of cyclin E and cyclin‐dependent kinase 5 in single‐positive thymocytes. Our data indicate that c‐Met signalling plays an important role in thymic regeneration after thymic insult. In addition, T‐cell‐specific inactivation of c‐Met accelerates age‐related thymic involution.

FOXN1 recombinant protein enhances T‐cell regeneration after hematopoietic stem cell transplantation in mice

A prolonged period of T‐cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T‐cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell‐penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T‐cell regeneration in patients following HSCT.