A Biodegradable Microparticle Vaccine Platform Using Femtomole Peptide Antigen Doses to Elicit T-Cell Immunity Against Chlamydia Abortus
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A Biodegradable Microparticle Vaccine Platform using Femtomole Peptide Antigen Doses to Elicit T-cell Immunity against Chlamydia abortus by Erfan Ullah Chowdhury A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 10, 2016 Keywords: Chlamydia abortus, T-cell Vaccine, Low Antigen Dose, Peptide Antigens, Microparticle Delivery, Spray Drying Copyright, 2016 by Erfan Ullah Chowdhury Approved by Bernhard Kaltenboeck, Chair, Professor, Pathobiology Zhanjiang Liu, Assoc. Vice President and Assoc. Provost, Professor, School of Fisheries Stuart B. Price, Associate Professor, Pathobiology Chengming Wang, Professor, Pathobiology Abstract Successful vaccination against Chlamydia spp. has remained elusive, largely due to a lack of vaccine platforms for the required Th1 immunization. Modeling of T helper cell immunity indicates that Th1 immunity requires antigen concentrations that are orders of magnitude lower than those required for Th2 immunity and antibody production. We hypothesized that the C. abortus vaccine candidate proteins that we identified earlier, DnaX2, GatA, GatC, Pmp17G, and Pbp3, mediated protection in an A/J mouse model of C. abortus lung infection if administered each at low, 1-20 femtoMole doses per mouse. This immunization significantly protected the mice from lethal challenge with 108 C. abortus organisms. Additional experiments proved that particulate delivery of antigens was required for optimum immunity. As a delivery vehicle, we constructed spray-dried microparticles of 1-3 µm diameter that were composed of biodegradable poly-lactide-co-glycolide polymers and the poloxamer adjuvant Pluronic L121. These microparticles, when administered at 10 µg per mouse dose, were effectively phagocytosed by macrophages and protected C3H/HeJ mice from lethal challenge with C. abortus, and thus were effective immune stimulators (biological response modifiers). We further hypothesized that i) 20-mer peptides overlapping by 10 amino acids could substitute for whole protein antigens when embedded in a 1-3 µm diameter microparticulate vaccine; ii) such phagocytosed biodegradable microparticles would ii intracellularly release peptides and adjuvant from such microspheres in antigen presenting cells and would enable controlled generation of Th1 immunity; and iii) inclusion of Q-VD- OPh, an inhibitor of apoptosis, could suppress a co-emerging inflammatory Th17 response and enhance a protective Th1 response. A dose of 2.00 femtoMoles of each peptide per mouse significantly reduced disease after lethal C. abortus challenge inoculation, but failed to effectively eliminate chlamydiae. In contrast, the inclusion of Q-VD-OPh in the subcutaneously or intranasally administered 0.20 femtoMoles peptide vaccine resulted in effective elimination of C. abortus and completely reversed the disease outcome to a fully protected healthy phenotype. We hypothesized that the 50:50DL-PLG-PEG lactide-co-glycolide polymer, used as rapidly degrading vaccine carrier, had released substantial acidity intracellualry in antigen presenting cells that induced an immunosuppressive apoptotic signal. Thus, the enhanced apoptosis of antigen presenting cells required release of Q-VD-OPh from vaccine microparticles to abolish apoptosis and Th1 immunosuppression. Therefore, we investigated the use of an alternative polymer carrier, the slowly degrading and minimally acid-releasing polylactide polymer DL-PL R202S. This carrier, when used with 0.5-1.25 femtoMole dosage of C. abortus peptide antigens, induced highly significant protection against chlamydial challenge without triggering an unwanted Th1 suppressive or inflammatory immune response in the C. abortus respiratory mouse disease model. In summary, we have developed a fully synthetic biodegradable microsphere vaccine for controlled release of adjuvant and ultralow doses of peptide antigens. This vaccine platform may be commercially useful for discovery and production of vaccines. iii Acknowledgments I have completed this dissertation with the direct and indirect contribution of many people, whom I would like to thank sincerely. Dr. Bernhard Kaltenboeck, my major advisor in the PhD dissertation committee. I would like to express the deepest appreciation to him for his unmeasurable help, guidance, and support throughout my doctoral studies. Dr. Kaltenboeck has the attitude and substance of a genius and is able to convey convincing spirit of adventure in regards to scientific research. My research project based on completely novel ideas and I started from a black hole. Since we had to go through with many optimization approaches and conducted many experiments to confirm the findings, it took a lot of time to complete the study. During this time the motivation and support as I received from Dr. Kaltenboeck was unutterable. Dr. Kaltenboeck, thank you so much for your knid support and I will always be grateful for having the opportunity to study under you supervision. Dr. Stuart B Price, who is an advisor in my graduate committee. He was also my mentor in the Bacterial Pathogenesis course. I learned a lot from him and I would like to thank him for his advice and suggestions throughout my doctoral studies. Dr. Zhanjiang Liu is on my graduate committee and was my mentor in the Molecular Genetics and Biotechnology course. I highly appreciate his guidance throughout this study. Dr. Chengming Wang is also on my graduate committee. Since I joined Dr. Kaltenboeck’s lab as graduate student, I have been learning from Dr. Wang in many ways. Thank you so much Dr. Wang for all of your suggestions and advice. iv Dr. Frederik van Ginkel, who had been on my graduate committee, has recently passed away completely unexpectedly. He was also my mentor in the Cellular and Molecular Immunology course. I would like to thank him a lot for his guidance in my designing immunological studies. Dr. Joseph Giambrone, who is the University Reader of my dissertation and is also one of our collaborators in the vaccine research. I learned a lot from him about chicken disease models. I also highly appreciate his time to review this dissertation. Drs. Ram Gupta and Courtney Ann Ober from the Auburn University Department of Chemical Engineering were extremely helpful in getting us started in spray drying the vaccine microparticles. And Drs. Fernando Osorio and Hiep Vu at the Virology Center of the University of Nebraska in Lincoln were outstanding collaborators in conducting the pig PRRSV vaccine trials. Mrs. Dongya Gao, Dr. Kh Shamsur Rahman, and Dr. Yen-Chen Juan, who are my wonderful current colleagues. Their immense help and support throughout my doctoral study, particularly with the mouse work, was beyond the level of expression. I especially thank Dongya for her help with PCR, Shamsur for his help with ELISAs, and Yen-Chen for her help with the immunofluorescence study. I would also like to thank to all of my previous lab colleagues - Drs. Anil Poudel, Yihang Li, and SudhirAhluwalia for their help and support. I especially thank Anil for his tremendous support in mouse studies. Lastly, and most importantly, I want to thank my father – Azhar Chowdhury, my mother – Masia Chowdhury, and my brothers – Ekram Chowdhury, Zafar Chowdhury, Samim Chowdhury, and Ershad Chowdhury. They have always been there for me and I am thankful for everything they have helped me to achieve. I especially want to thank to my mother, with her tireless efforts, after the death of my father, has allowed me to reach my current position. I want to give special thanks to my wife Shamima Chowdhury for her continued love, care, and support to make this possible. I also want give special thanks to my daughter Eshal Chowdhury, her warm affection gave me the spirit to complete this study. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Abbreviations ......................................................................................................... xi Chapter 1 ............................................................................................................................1 1.1. Chlamydia – Background and taxonomy .........................................................1 1.2. Biology and molecular pathogenesis ...............................................................6 1.3. Chlamydial infection and significance...........................................................20 1.4. Host immunity against chlamydial infection .................................................30 1.5. Chlamydial vaccine .........................................................................................44 1.6. Research rationale and objective ...................................................................55 References ..............................................................................................................59 Chapter 2 ........................................................................................................................120