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Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Campylobacter est responsable de la majorité des gastro-entérites bactériennes dans le monde. Sa membrane externe contient des porines qui permettent les échanges entre la bactérie et le milieu extérieur. Elles sont une des voies principales d’entrée pour les antibiotiques et les nutriments. Le premier objectif de mon travail était de participer à la caractérisation des porines MOMP et Omp50. Nous avons purifié ces protéines dans le cadre de collaborations pour l’étude des propriétés biophysiques de ces porines (M. Wintherhalter, Université Jacob, Allemagne) et la détermination de leur structure par cristallisation (J. Naismith, Université St Andrews, Écosse). La deuxième partie de ma thèse a été consacrée à l’étude et la caractérisation du produit du gène Cj1169c qui est en opéron avec le gène codant Omp50. Nous avons cloné et exprimé le gène Cj1169c chez Escherichia coli, ce qui a permis de purifier la protéine et de produire des anticorps spécifiques. Ces anticorps ont été utilisés pour étudier la prévalence de Cj1169c chez les espèces les plus fréquentes. Nous avons montré qu’elle était présente uniquement chez C. jejuni et C. lari et absente chez C. coli. En deuxième lieu, nous avons caractérisé sa production en fonction de conditions de culture et montré qu’elle dépendait du pH et de la température. Nous avons ensuite démontré que cette protéine est située dans le périplasme de Campylobacter. Puis, en raison de la présence de deux résidus de cystéines dans sa séquence, nous avons étudié son comportement en présence et en absence d’agents réducteurs de ponts disulfures. De plus, des résultats préliminaires suggèrent que cette protéine interagit avec Omp50. / Campylobacter is a leading cause of bacterial gastroenteritis worldwide. Its outer membrane contains porins which are pore forming protein that transport hydrophilic compounds like nutrients and antibiotics into the bacteria. My first objective was to purify the two porins MOMP and Omp50 from C. jejuni, in order to allow the study of the biophysical properties of porins (Collaboration with Mr. Wintherhalter, Jacob University, Germany), and to determine the structure of these porins by crystallization (Collaboration with J. Naismith, St Andrews University, Scotland).The second part of my thesis was devoted to the study of the product of the gene Cj1169c which is operonic with the Omp50 coding gene. We cloned and expressed the gene Cj1169c in Escherichia coli, purified the protein and produced Cj1169c-specific antibodies. We studied the prevalence of this protein in the most frequent species of Campylobacter and found the protein only in C. jejuni and C. lari never in C. coli. Then, we also studied the Cj1169c production in the function of several growing conditions, and we found that it depended on the pH and temperature. Besides, we demonstrated by several methods that this protein is located in the periplasmic compartment of the bacteria. Furthermore, because it contains two cysteins residues in its primary sequence, we studied the Cj1169c behavior in the presence and absence of disulfide bond reducing agents. In addition, preliminary results suggest that this protein interacts with Omp50.

Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E. Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models. Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization. Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority. / Kemi/biokemi

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.