Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

The protein monomer of bacterial flagella, FliC, is known to stimulate human innate immunity through activation of Toll-like receptor five. Linking native Salmonella FliC with various antigens has demonstrated an increased immune response as compared to single antigen presentation. To drastically reduce production time and allow for a more cost effective recombinant vaccine adjuvant, a synthetic construct was created that enables genetic linkage of FliC to other known antigens. The construct contains the necessary components for immune system stimulation while the non-essential regions were replaced with commonly used restriction enzyme recognition sites to aid in ligation with other antigens and cloning into various expression vectors and hosts. After synthesis in the inducible expression vector pJ404, the construct was transformed into competent BL21 E. coli and expression was confirmed through SDS-PAGE, Western blot, and MALDI MS/MS. The cells were adapted to fermentation media and re-screened for expression, and upon confirmation a 20-liter fermentation was conducted. The resulting samples were analyzed for expression within the insoluble and soluble cellular fractions to further optimize fermentation conditions. Once purified, this synthetic FliC will serve as a platform technology for the standardized co-expression of the TRL5 activator with a variety of antigens in both prokaryotic and eukaryotic systems.

Adjuvants are compounds that enhance immune responses to antigens present in a vaccine. They are particularly important in subunit vaccines; without adjuvants, these vaccines are often poorly immunogenic. A novel adjuvant platform developed at VIDO-InterVac is comprised of CpG-ODN or poly I:C, innate defense regulator peptides, and a new class of adjuvant called polyphosphazene. The polyphosphazenes have demonstrated a great potential as a safe and effective adjuvant. In particular, the polysphosphazenes poly[di(carboxylatophenoxy)-phosphazene] PCPP and poly[di(sodium carboxylatoethylpehnoxy)-phosphazene](PCEP) have been used in numerous animal studies where they not only have been shown to enhance the quality and quantity of the adaptive immune response, but also were shown to induce parenteral and mucosal immune responses with many different antigens, demonstrating their versatility. However the mechanisms by which the polyphosphazenes stimulate the innate immune response are only partially understood. Antigen presenting cells (APCs) are capable of facilitating the uptake of antigen and directing the immune response. Based upon the proposed mechanism of action of another adjuvant, we chose to investigate whether porcine monocytes could be induced to secrete pro-inflammatory cytokines IL-1 and IL-18 in response to stimulation with polyphosphazenes PCEP and PCPP. The release of these cytokines is thought to be mediated by the Nod-Like Receptors (NLRs), which are cytosolic pattern recognition receptors expressed in APCs. It is suggested that these receptors act in conjunction with TLR transcription pathways to control caspase-1 and release associated pro-inflammatory cytokines IL-1and IL-18 (Kawai and Akira, 2011). We first investigated the relative gene expression of three Nod-like receptor genes: nod1, nod2 and nlrp3 in various populations of porcine peripheral blood mononuclear cells (PBMCs) and found that monocytes, dendritic cells and B cells express increased relative levels of these receptors as compared to T cells. Subsequently, we evaluated the relative NLR expression in several porcine mucosal and lymphoid tissues and observed genes to be most significantly expressed in nasal mucosa, bronchial mucosa, and lung while limited in tissues associated with Peyer’s patches, jejunal wall. Both the mesenteric lymph node and bronchial lymph node exhibited similar patterns and levels of expression of nod1, nod2 and nlrp3. To characterize the activation of NOD1, NOD2 and NLRP3 receptors in response to stimulation with polyphosphazenes, porcine monocytes were stimulated with PCEP or PCPP in both the presence and absence of a second signal (poly I:C and CpG-ODN, TLR-7 and -9 agonists respectively). We found that PCEP and PCPP alone did not significantly upregulate nod1, nod2 and nlrp3, nor genes for cell activation markers such as CD80 and CD86. However monocytes cultured with the combination of CpG-ODN, Poly I:C and PCPP appeared to moderately express IL-18, CD80 and CD86. The secretion of pro-inflammatory cytokines from cultured monocytes was determined with Enzyme Linked Immunosorbent Assays (ELISA). It was found that IL-1was secreted in significantly higher quantities in the supernatant of cells stimulated with both polyphosphazene and TLR ligands, as opposed to those cultured with polyphosphazene alone. Assays for IL-18, IL-6, IL-10 and IL-12 did not detect a significant presence of these proteins in the supernatant. Furthermore, we found that a soluble caspase inhibitor did not significantly reduce the production of IL-1by monocytes, and was likely attributable to cell death at high concentrations. Taken together, these results suggest that porcine monocytes, B cells and dendritic cells express elevated levels of the NLRs as compared to T cells. Additionally, areas of the respiratory tract appear to express increased levels of these receptors relative to mucosal and lymphoid tissues of the gastrointestinal tract. Neither PCPP or PCEP alone were capable of inducing significant production of IL-1 or IL-18 by cultured monocytes, however stimulation of these cells with a combination of CpG-ODN, poly I:C and polyphosphazene resulted in the secretion of IL-1.

Field and greenhouse experiments were conducted at Southern Illinois University-Carbondale in 2006 and 2007 to evaluate the herbicide tembotrione for postemergence grass control. Tembotrione inhibits the p-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme, which aids in the formation of essential plant constituents for photosynthesis. Tembotrione efficacy was examined in the greenhouse on large crabgrass, giant foxtail, shattercane, and fall panicum. Significant activity that could translate to commercial levels of weed control in the field was found on all species except fall panicum. Greenhouse studies also compared the efficacy of tembotrione, mesotrione, and topramezone which represent the three HPPD-inhibiting herbicides commercially available in U.S. corn production. Tembotrione and topramezone have more activity on these grasses than mesotrione. Tembotrione was also tank-mixed with either nicosulfuron or foramsulfuron to evaluate fall panicum response. Activity on fall panicum was similar weather nicosulfuron or foramsulfuron was applied alone or with tembotrione. In the field, it was also determined that nicosulfuron or foramsulfuron could be added to tembotrione to control fall panicum. The addition of atrazine to nicosulfuron and tembotrione did not negatively effect fall panicum control. It was also observed in both the field and greenhouse that utilizing methylated seed oil provided more activity than crop oil concentrate.

No description available.

Development of new vaccination technology has been hindered by a lack of new adjuvants to enable development of protective immunity using different vaccine delivery methods. A vaccine delivery system using oral adjuvants would be applicable across species for both individual and mass vaccination in both the medical and veterinary fields. We sought to create an oral nanoparticle (NP) vaccine delivery system that is easy to produce and uses polymers as oral adjuvants with killed virus. Our hypothesis was gelatin and chitosan would enhance viral uptake and stimulate immune cells to produce protective immunity. This would allow the safer killed form of each virus to be used in place of modified live (MLV) viruses and avoid undesirable side effects like immunosuppression. The research objectives were to 1. Fabricate and characterize gelatin NPs encapsulating inert materials of similar size and shape to the viruses of interest for fabrication proof-of-concept 2. Modify the NP delivery system to minimize immune cell cytoxicity for the vaccine delivery application 3. Fabricate and characterize FPV and HEV viral nanoparticles' stability, cellular uptake/infectivity, and released viruses' ability to replicate 4. Compare the abilities of the killed HEV nanovaccine, killed HEV with loose gelatin and chitosan polymers (no nanoparticle), and a live HEV commercial vaccine to induce textit{in vivo} seroconversion, protective immunity, and side effects during clinical and challenge studies in turkeys We proved our hypothesis to be correct in addition to demonstrating matching the encapsulation material size to empty NP size leads to preferred encapsulated NP formulation parameters, chitosan stabilizes the NP formulation bypassing the need for cytotoxic crosslinkers, and paraformaldehyde is able to kill virus prior to vaccine formulation while still preserving virus morphology sufficiently for immune cell recognition. This development constitutes one of the first novel adjuvants discoveries in 70 years and opens the door for conversion of injectable vaccines to oral delivery across species. / Doctor of Philosophy / Most vaccinations use needles to inject a live but weakened virus into the body, which causes a mild infection. The body learns to protect itself using this weak form of the virus, so that when the body encounters a stronger form of the same virus later on in life, the body is able to quickly kill the virus and recover from the infection. If we could package a dead virus with the right mixture, we could get the body to recognize the dead virus and learn to protect itself just as the body does with the weaker, live virus. This would avoid the mild infection and the unpleasant symptoms associated with the live virus injection and allow us to use a safer dead virus vaccine. Additionally, with the right package, we could drink our vaccines instead of using injections. Here, we tried to create a drinkable, safer dead virus packaged with gelatin and shellfish fiber in a vaccine that allows the body could recognize the dead virus and learn to protect itself similarly to the live virus vaccine. The goals of this work were to: 1. Practice putting plastic beads of similar size and shape to viruses in gelatin packages to understand how to safely package viruses in gelatin as part of making the new drinkable vaccine 2. Adjust the process of making the gelatin plastic bead packages to work well with cells in the laboratory as a second step toward making safe vaccines 3. Use the packaging process to package a dead chicken virus and a dead turkey virus separately with gelatin and shellfish fiber and measure each packaged virus 4. Test the dead packaged turkey virus vaccine with gelatin and shellfish fiber, the dead unpackaged turkey virus with loose gelatin and shellfish fiber, and a live turkey virus that is currently used as a vaccine in turkeys to see which allows the body to protect itself without causing side effects We showed that using plastic beads of similar size to empty gelatin and shellfish fiber packages creates the preferred packaged plastic bead measurements. The shellfish fiber kept the packages intact and from falling apart, so no additional chemicals were needed. The preservative used to kill the virus worked while still keeping the virus recognizable to the body. This new packaging for vaccines is a breakthrough in vaccine development and will allow us to change injectable vaccines to drinkable vaccines in other animals and humans.

Toxoplasma gondii, protozoaire intracellulaire obligatoire, est l’agent responsable de la toxoplasmose, infection qui revêt un caractère sévère au cours de toxoplasmoses cérébrales ou congénitales en médecines humaine et vétérinaire. Aucun vaccin n’est actuellement disponible ; le développement de stratégies vaccinales efficaces est donc d’actualité. Le potentiel vaccinant de deux protéines de rhoptries de T. gondii, RON4 et ROP18, protéines injectées dans la cellule au site de l’invasion lors de l’étape d’attachement à la cellule, a été évalué dans deux stratégies vaccinales contre la toxoplasmose chronique chez la souris: vaccination ADN par voie intramusculaire et vaccination par voie nasale avec des protéines recombinantes. L’immunisation avec des plasmides optimisés exprimant RON4, la partie N-terminale ou la partie C-terminale de RON4 co-administrés avec un plasmide exprimant l’adjuvant GM-CSF ou l’immunisation par voie nasale avec une protéine recombinante RON4 associée à la toxine cholérique, induit des réponses systémiques humorale et cellulaire (mixte Th1/Th2) mais ne confère pas de protection. Dans nos conditions expérimentales RON4 n’est pas un candidat vaccin potentiel. Des stratégies pour augmenter son immunogénicité par voie nasale et pour orienter la réponse cellulaire vers un profil Th1 pourraient cependant être envisagées. L’immunisation avec des plasmides bicistroniques exprimant à la fois ROP18 sous forme sécrétée et le GM-CSF ou ROP18 cytosolique et le GM-CSF, induit des réponses humorales et cellulaires (Th1) similaires et ne confère pas de protection significative. La co-administration d’un plasmide exprimant l’IL-12 n’augmente pas les réponses immunes avant infection mais a néanmoins contribué à augmenter la réponse cellulaire après infection. L’immunisation par voie nasale avec une protéine recombinante ROP18 associée à la toxine cholérique, induit une réponse systémique humorale (Th1/Th2) et confère une protection significative (réduction de la charge parasitaire de 50%). La co-administration de l’adjuvant poly I:C augmente la réponse cellulaire mais n’a pas d’effet sur la protection. Nos résultats suggèrent que ROP18 est un candidat vaccin potentiel, des stratégies pour améliorer son effet protecteur sont à envisager. / Toxoplasma gondii, an obligate intracellular protozoan, is the etiologic agent of toxoplasmosis. This infection has severe consequences during cerebral or congenital toxoplasmosis both in human and veterinary medicines. No vaccine is currently available, so the design of efficient vaccine strategies is still a topical question. In this study, RON4 and ROP18, two rhoptry proteins of T. gondii which are discharged into the host cell at the invasion site, immediately following intimate contact with the host cell, were evaluated in two vaccine strategies against chronic infection in mice: DNA vaccination by the intramuscular route and recombinant protein vaccination by the nasal route. DNA immunization with optimized plasmids encoding full length RON4, or only the N-terminal, or the C-terminal part of RON4 plus a plasmid encoding the adjuvant GM-CSF or nasal immunization with a recombinant RON4 protein plus cholera toxin induced systemic humoral and cellular responses (mixed Th1/Th2) but failed to confer protection. Strategies intended to enhance the immunogenicity of RON4 by the nasal route and to enhance the Th1 immune response against RON4 could be more effective.DNA immunization with ROP18 expressed as a secreted or a cytosolic form by bicistronic vectors which encode both the antigen and the adjuvant GM-CSF induced similar humoral and cellular (Th1) responses but did not confer significant protection. Co-administration of a plasmid encoding the adjuvant IL-12 did not enhance the immune responses before challenge but was able to prime a cellular immune response that was boosted by the parasite infection. Nasal immunization with a recombinant ROP18 protein plus cholera toxin induced systemic humoral responses (mixed Th1/Th2) and conferred partial protection (50% brain cysts reduction). Co-administration of the adjuvant poly I:C enhanced the cellular response but did not potentiate the protection. Our data suggest that ROP18 is a potential vaccine candidate against toxoplasmosis. Strategies to improve the protective effect of ROP18 should be investigated.

Daniel R Frederick

The problem investigated Four studies investigated the phenomenon of cancer-related fatigue (CRF) in women who had received adjuvant treatment for early-stage breast cancer, with a view to reducing the diagnostic uncertainty surrounding the syndrome and thus facilitating progress in both clinical management and aetiological research. Procedures and results A cross-sectional study of 109 women compared a ???cancer-specific??? self-report questionnaire (FACT-F) (canvassing fatigue symptoms) and a more generic questionnaire (SPHERE) (identifying depression and fatigue). Thirty-seven percent of women reported fatigue. Overall in 20%, fatigue was associated with psychological distress. Seventeen percent of women had fatigue but no depression. A qualitative study utilised focus groups to identify and compare the distinctive features of CRF with those of women with chronic fatigue syndrome (CFS). A similar set of symptoms was found in both groups, including overwhelming fatigue, un-refreshing sleep and subjective concentration problems. However, women with CFS also reported myalgia and arthralgia. Using the Structured Clinical Interview for Neurasthenia- SCIN, the third study compared the symptoms of three groups of women with fatigue: those with CRF, CFS or major depression. The detailed ???interviewer guide??? provided explicit directions for evaluating and classifying symptoms. This study confirmed the core symptom of ???profound fatigue unrelieved by rest???, and additional features that distinguished between the clinical diagnoses. The fourth study compared features of the evolution of clinically-identified fatigue syndromes in women from two prospective cohort studies; women with post-cancer fatigue (PCF) and women with post-infective fatigue syndrome (PIFS). Major conclusions A syndrome of PCF, present at least six months following adjuvant treatment and unexplained by medical or psychiatric disorder was investigated. The characteristics of PCF and those of CFS are very similar, with the fatigue state having indistinguishable descriptors. Longitudinal evaluation of the symptom complexes of PCF and PIFS suggests divergent pathways may be relevant. Co-morbid features like sleep disturbance; physical deconditioning and mood disturbance may be implicated as factors in the evolution and prolongation of PCF. These studies provide a basis for a more uniform and rigorous classification system - a necessary first step towards advancing the field both in investigating aetiology and new intervention strategies.

The problem investigated Four studies investigated the phenomenon of cancer-related fatigue (CRF) in women who had received adjuvant treatment for early-stage breast cancer, with a view to reducing the diagnostic uncertainty surrounding the syndrome and thus facilitating progress in both clinical management and aetiological research. Procedures and results A cross-sectional study of 109 women compared a ???cancer-specific??? self-report questionnaire (FACT-F) (canvassing fatigue symptoms) and a more generic questionnaire (SPHERE) (identifying depression and fatigue). Thirty-seven percent of women reported fatigue. Overall in 20%, fatigue was associated with psychological distress. Seventeen percent of women had fatigue but no depression. A qualitative study utilised focus groups to identify and compare the distinctive features of CRF with those of women with chronic fatigue syndrome (CFS). A similar set of symptoms was found in both groups, including overwhelming fatigue, un-refreshing sleep and subjective concentration problems. However, women with CFS also reported myalgia and arthralgia. Using the Structured Clinical Interview for Neurasthenia- SCIN, the third study compared the symptoms of three groups of women with fatigue: those with CRF, CFS or major depression. The detailed ???interviewer guide??? provided explicit directions for evaluating and classifying symptoms. This study confirmed the core symptom of ???profound fatigue unrelieved by rest???, and additional features that distinguished between the clinical diagnoses. The fourth study compared features of the evolution of clinically-identified fatigue syndromes in women from two prospective cohort studies; women with post-cancer fatigue (PCF) and women with post-infective fatigue syndrome (PIFS). Major conclusions A syndrome of PCF, present at least six months following adjuvant treatment and unexplained by medical or psychiatric disorder was investigated. The characteristics of PCF and those of CFS are very similar, with the fatigue state having indistinguishable descriptors. Longitudinal evaluation of the symptom complexes of PCF and PIFS suggests divergent pathways may be relevant. Co-morbid features like sleep disturbance; physical deconditioning and mood disturbance may be implicated as factors in the evolution and prolongation of PCF. These studies provide a basis for a more uniform and rigorous classification system - a necessary first step towards advancing the field both in investigating aetiology and new intervention strategies.