Cigarette smoking is prevalent worldwide and has consistently been the top preventable cause of many serious diseases., which result in huge mortality, morbidity, and economic loss, in recent decades. In recent years, nicotine vaccines that can induce production of nicotine specific antibodies in human have emerged as a promising medicine to treat tobacco addiction. In the past decade, there have been numerous nicotine vaccine candidates evaluated in human clinical trials, including NicVaxNicVAX®, TA-NICTA-NIC®, Nic002NIC002®, NiccineNiccine®, and SEL-068SEL-068®. . However, traditional nicotine vaccine designs haves many disadvantages, including low immunogenicity, low specificity, difficulty in integration of molecular adjuvants, and short immune response persistence. To overcome the above limitations, in this study, various nanoparticle-based vaccine delivery systemsvaccine componentss have been developed and evaluated as potential delivery vehicles for vaccines against nicotine addiction. Firstly, a nicotine vaccine was synthesized by conjugating bovine serum albumin (BSA)-nicotine complex to the surface of nano-sized cationic liposome. Significantly higher anti-nicotine antibody titer was achieved in mice by liposome delivered nicotine vaccine compared with nicotine-BSA vaccine. Secondly, a novel nanoparticle (NP)-based delivery platform was constructed by incorporating a negatively charged nanohorn into cationic liposome to improve the stability of liposome and reduce nanoparticle flocculation. Subsequently, nicotine vaccine was constructed by conjugating nicotine-BSA complex to the surface of the nanohorn supported liposome (NsL). Marked improvement in stability in vitro and significant increase in titer of anti-nicotine antibodies were detected in nanohorn supported liposome ( NsL) delivered vaccine than liposome delivered vaccine. In addition, NsL nicotine vaccine exhibited good safety in mice after multiple injections. Thirdly, lipid- poly(lactic-co-glycolic acid) (PLGA) hybrid NPs were constructed as vaccine delivery system. due to the fact that nanohorn is not currently approved for clinical use, we substituted the nanohorn with poly(lactic-co-glycolic acid) (PLGA) nanoparticles and constructed PLGA-lipid hybrid nanoparticles. Preliminary results showed that PLGA-lipid hybrid NPs nanoparticles exhibited improved stability, better controlled release of antigens, as well as enhanced uptake by dendritic cell (DC). A lipid-PLGA hybrid NPnanoparticle was also developed that was structurally responsive to low pH challenge. The lipid shell of the hybrid nanoparticle was rapidly disintegrated under a low pH challenge, which resembles the acidic environment of endosomes in DCsdendritic cells. The hybrid NPs exhibited minimal antigen release in human serum at physiological pH, but a faster release of antigen from this NP compared to non-pH sensitive NPs was observed in DC. In the final study, hybrid NPnanoparticles with various cholesterol concentrations were constructed. Slower and more controlled release of antigens in both human serum and phosphate buffered saline were detected in nanoparticles with higher cholesterol content. However, nanoparticles containing higher cholesterol showed poorer stability due to increase fusion among NPnanoparticles. It was later found that PEGylation of NPs can effectively minimize fusion caused size increase after long term storage, leading to improved cellular uptake. The findings from this study on the nanohorn-lipids based nicotine vaccine as well as lipid-PLGA hybrid NPs may provide solid basis for future development of lipid-PLGA based nicotine vaccine. / Ph. D.

Tobacco smoking has consistently been the leading cause of preventable diseases and premature deaths. Currently, pharmacological interventions have only shown limited smoking cessation efficacy and sometimes are associated with severe side effects. As an alternative, nicotine vaccines have emerged as a promising strategy to combating nicotine addiction. However, conventional conjugate nicotine vaccines have shown limited ability to induce a sufficiently strong immune response due to their intrinsic shortfalls. In this study, a lipid-poly(lactic-co-glycolic acid) (PLGA) nanoparticle-based next-generation nicotine vaccine has been developed to overcome the drawbacks of conjugate nicotine vaccines. Also, the influence of multiple factors, including nanoparticle size, hapten density, hapten localization, carrier protein, and molecular adjuvants, on its immunogenicity has been investigated. Results indicated that all these studied factors significantly affected the immunological efficacy of the nicotine nanovaccine. First, 100 nm nanovaccine was found to elicit a significantly higher anti-nicotine antibody titer than the 500 nm nanovaccine. Secondly, the high-density nanovaccine exhibited a better immunological efficacy than the low- and medium-density counterparts. Thirdly, the nanovaccine with hapten localized on both carrier protein and nanoparticle surface induced a significantly higher anti-nicotine antibody titer and had a considerably better ability to block nicotine from entering the brain of mice than the nanovaccines with hapten localized only on carrier protein or nanoparticle surface. Fourthly, the nanovaccines carrying cross reactive materials 197 (CRM197) or tetanus toxoid (TT) showed a better immunological efficacy than the nanovaccines using keyhole limpet hemocyanin (KLH) or KLH subunit as carrier proteins. Finally, the co-delivery of monophosphoryl lipid A (MPLA) and Resiquimod (R848) achieved a considerably higher antibody titer and brain nicotine reduction than only using MPLA or R848 alone as adjuvants. Collectively, the findings from this study may lead to a better understanding of the impact of multiple factors on the immunological efficacy of the hybrid nanoparticle-based nicotine nanovaccine. The findings may also provide significant guidance for the development of other drug abuse and nanoparticle-based vaccines. In addition, the optimized lipid-PLGA hybrid nanoparticle-based nicotine nanovaccine obtained by modulating the studied factors can be a promising candidate as the next-generation nicotine vaccine for treating nicotine addiction. / PHD

Existing therapeutic options for the alleviation of nicotine addiction have been largely ineffective at stemming the tide of tobacco use. Immunopharmacotherapy, or vaccination, is a promising, alternate therapy that is currently being explored. Results from previous studies indicate that nicotine vaccines (NVs) are effective in subjects that achieve high drug-specific antibody titers, though overall efficacy has not been observed. Consequently, improvement of these vaccines is necessary before they can achieve approval for human use. In this report, three separate approaches towards NV potentiation are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Rat and human physiological and pharmacological parameters were obtained from literature and used to construct compartmentalized models for nicotine and cotinine distribution. These models were then calibrated and validated using data obtained from literature. The final models verified the therapeutic potential of the NV concept, identified four key parameters associated with vaccine success, and established correlates for success that could be used to evaluate future NVs prior to clinical trials. In the second approach, conjugate NV scaffoldings were engineered by using wild-type (WT) and chimeric human papilloma (HPV) 16 L1 protein virus-like particles (VLPs). The chimeric protein was created by removing the last 34 C-terminal residues from the WT protein and then incorporating a multi-epitope insert that could universally target major histocompatibility complex (MHC) class II molecules. The proteins were subsequently expressed in E. coli and purified using a multi-step process. Comparisons between the separation outcomes revealed that the insert was able to modulate individual process outcomes and improve overall yield without inhibiting VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human leukocyte antigen (HLA) population frequency data and MHC epitope prediction software. The most immunogenic epitopes were concatenated with interspacing cathepsin cleavage sequences and the resulting protein was re-evaluated using the earlier methods. This work represents the first ever in silico design of chimeric antigens that could potentially target all of the major HLA DQ and HLA DR allotypes found in humans. / Doctor of Philosophy / Existing treatment options for addressing nicotine addiction have been largely ineffective at preventing tobacco use. Vaccination, on the other hand, is a promising, alternate treatment option that is currently being explored. Previous studies have shown that nicotine vaccines (NVs) are effective in the subjects that respond well to the vaccine. Effectiveness in the majority of vaccine recipients, however, has not been observed. Consequently, improvement of these vaccines is necessary before they can be used in humans. In this report, three separate approaches for improving NV effectiveness are explored. The first approach applied physiologically-based pharmacokinetic (PBPK) modeling to better assess NV potential. Parameters were obtained from literature and used to construct models that could predict NV effectiveness in rats and humans. These models were then calibrated and validated using data obtained from literature. The final models verified that NVs could work if certain conditions were met, identified four key parameters associated with vaccine success, and allowed for estimation of NV efficacy prior to their evaluation in humans. In the second approach, protein carriers for conjugate NVs were constructed using the human papilloma (HPV) 16 L1 protein. This protein is known for its ability to form virus-like particles (VLPs). Both a modified and an unmodified (wild-type) protein were constructed. The modified HPV 16 L1 protein was created by replacing the last 34 C-terminal amino acids with a polypeptide insert that could enhance the immunogenicity of the vaccine. The modified and unmodified proteins were then expressed in E. coli and purified. Results indicated that the insert was able to modulate individual process outcomes and improve overall process yield without preventing VLP assembly. In the third approach, commonly used carrier proteins were computationally mined for their MHC class II epitope content using human gene frequency data and MHC epitope prediction software. The epitopes that were predicted to be the most immunogenic were linked together with interspacing protease recognition sequences and the immunogenicity of the resulting protein was re-evaluated using the prediction software. This work represents the first computational design of antigens that could potentially allow a vaccine to be effective in a large portion of human population regardless of the genetic variability.