A Biodegradable Microparticle Vaccine Platform using Femtomole Peptide Antigen Doses to Elicit T-cell Immunity against Chlamydia abortus
Type of DegreePhD Dissertation
General Veterinary Medicine
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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 10^8 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 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.