ß-glucan-based adjuvants for hepatitis B vaccination: particulate design for prophylactic and therapeutic needs

The hepatitis B virus (HBV) killed 887 000 people in 2015. The World Health Organization (WHO) set the goal to eliminate HBV as a public health threat by 2030. The major hurdles include the high prevalence in developing countries due to limited vaccination coverage and mother-to-child transmission and, the ineffective HBV clearance from hepatocytes with currently available antivirals. Hence, therapeutic vaccines may stimulate both the neonate immature or the chronic hepatitis B exhausted immune systems, to either avoid HBV persistence or promote HBV clearance. Additionally, new vaccines can be designed to provide the antigen with increased stability to temperature variations, benefiting HBV vaccine coverage in developing countries, where the cold chain to vaccine transportation is not readily available. Thus, this thesis aimed to develop new powerful adjuvants to include in new vaccines to meet hepatitis B current needs. β-glucan particles were elected to mimic pathogen three-dimensional structure and chemical composition. The capacity to induce HBsAg-specific Th1 antiviral protection through several vaccination strategies was the main goal. Indeed, this was the first time that nonmodified natural β-glucans were used as adjuvants for HBsAg. The new formulations of HBsAg vaccines were tested through subcutaneous (SC) and oral routes, while plasmid DNA (pDNA) vaccines only through the SC route. Different β-glucan adjuvants were developed and included β-glucan particles (GPs), prepared from alkaline/acid treatment of Saccharomyces cerevisiae, β-glucan/chitosan particles (GenGluChiPs), prepared by a precipitation technique followed by genipin crosslink and, polyplexes prepared by pDNA complexation with cationic PβAE and PDMAEMA polymers (Pol) in the presence of β-glucan (GluPol) or combined with GPs (GPsPol). Notably, GenGluChiPs were produced by a new precipitation technique to combine two polymers that do not interact with each other. GPs were tested in all vaccination schedules while GenGluChiPs only for HBsAg SC vaccination and GluPol only for pDNA SC vaccination. Additionally, chitosan particles were developed for comparison purposes either by precipitation/coacervation (ChiPs) or precipitation followed by genipin crosslink (GenChiPs). Both were positively charged and had a mean diameter near 900 nm.GenGluChiPs, also positively charged, measured approximately 1.3 μm. On the other side, alginate coated ChiPs (AlgChiPs), used for oral vaccination, were negatively charged and had a mean diameter close to 1.5 μm. GPs were electrically neutral and measured between 2 μm and 4 μm. Pol and GluPol were highly positive with a mean diameter of 180 nm. Regarding the oral vaccination study (Chapter 2), although both AlgChiPs and GPs were efficiently internalized by intestinal Peyer’s patches, the oral vaccination schedule resulted in 60 % mice seroconversion, easily surpassed by a SC priming prior the oral boosts. The presence of HBsAg-specific IgA on mucosal surfaces and IFN-γ in the liver were the major advantages found. Interestingly, in vitro studies showed that only ChiPs were able to induce mast cell activation, evaluated by cell degranulation and β- hexosaminidase release. Concerning pDNA vaccination study (Chapter 3), although the excellent Pol transfection results, further enhanced by the combination with GPs (GPsPol) in fibroblast and macrophage cell lines, the SC vaccination either with Pol, GluPol or GPsPol resulted in only 40 % seroconversion and low antibody titers. The mechanistic study with GenChiPs, GenGluChiPs and GPs showed that the increased TNF-α secretion from mice spleen cells was associated to β-glucan (GenGluChiPs and GPs), while RANTES secretion was associated to chitosan (GenChiPs and GenGluChiPs), suggesting an immunological advantage of the newly developed GenGluChiPs. However, in the human monocyte study, the TNF-α production was consistently observed for all the particles. The mice immunization study with HBsAg to validate GenGluChiPs adjuvant (Chapter 4) showed high serum anti-HBsAg IgG, mostly subtype IgG1 followed by IgG3. No signs of cell-mediated immunity were found after two vaccine doses. However, in another study with three vaccine doses (Chapter 5), the GPs adjuvant induced a strong and varied HBsAg-specific cell-mediated immunity observed by the secretion of cytokines related with Th1, Th2, Th17, Th22 and Treg-biased immune responses. For the first time, these studies allowed the validation of GPs as great adjuvant to include in HBsAg vaccines, also revealing a therapeutic value against viral infections. Overall, the work herein developed and described represents an important contribution to the knowledge of both β-glucan and chitosan/β-glucan particle adjuvant mechanisms, with a great impact for future studies.