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Nanotechnology and mucosal vaccines Dr. José Crecente Campo jose·[email protected] (Mª José Alonso’s group) Agenda • Introduction – What is a vaccine? – History of vaccination – Adjuvants • Mucosal vaccines – Mucosal immunology – Nanosystems for mucosal vaccines – Nasal • Conclusions What is a vaccine? A vaccine typically contains an agent that resembles a disease-causing microorganism and stimulates the body's immune system to recognize the agent as a threat, destroy it, and keep a record of it. www.wikipedia.org http://www.historyofvaccines.org History of vaccines Classic vaccines Live attenuated microorganisms Inactivated pathogenic agents Advances in antigen development Recombinant proteins Peptides (epitopes) Nucleic acids Potent immune response X Instability X Adverse side Safer No need of adjuvant effects X Less immunogenic Easier to produce X Lower probability of cross-presentation Cheaper Need potent adjuvants! Gonzalez-Aramundiz, J. V., Cordeiro, A. S., Csaba, N., de la Fuente, M., & Alonso, M. J. (2012). Nanovaccines: nanocarriers for antigen delivery. Biologie aujourd'hui, 206(4), 249-261. Cordeiro, A.S.; Alonso, M.J.; De la Fuente, M. Nanoengineering of vaccines using natural polysaccharides. Biotechnology Advances 33 (2015) 1279–1293 Adjuvants Advances in adjuvant development Immunostimulatory molecules TLR agonists Bacterial toxins Particulate delivery systems Micro Nano Saponins Cytokines Macromolecules Nanocarriers Polysaccharides Chitosan, dextran, mannan, beta glucans Recognized by receptors in APCs Pathogen-like size and structure Co-encapsulation of other adjuvant molecules Mucosal administration Biocompatible / Biodegradable Versatile materials Cordeiro, A.S.; Alonso, M.J.; De la Fuente, M. Nanoengineering of vaccines using natural polysaccharides. Biotechnology Advances 33 (2015) 1279–1293 Adjuvants Alum (1930s, Aluminium salts) Emulsions Name Composition Commercial name MF59 Squalene O/W Flu and Focetricia (Seasonal and pandemic influenza) AS03 Squalene + tocopherol O/W Pandemrix (Pandemic influenza) AF03 Squalene O/W Humenza (Pandemic influenza) Montanide ISA 51 Mineral oil W/ O CimaVax EGF* (Non-small lung cancer) Liposomes Name Composition Commercial name Inflexal V (seasonal influenza) Virosome liposomes Epaxal (Hepatitis A) BLP-25 or emepepimut-S Biomira liposomal peptide 25 - De Gregorio et al. Vaccines for the twenty-first century society. Nature Reviews Immunology, 2011, 11, 865-872 - Di Pasquale et al. Vaccine Adjuvants: from 1920 to 2015 and Beyond. Vaccines 2015, 3, 320-343 Stimuvax (MUC-1 positive cancer) Agenda • Introduction – What is a vaccine? – History of vaccination – Adjuvants • Mucosal vaccines – Mucosal immunology – Nanosystems for mucosal vaccines – Nasal • Conclusions Mucosal vaccines Systemicmucosal response Natural entrance Needle-free vaccination Mucosal vaccines Easier production Neutra, M. R., & Kozlowski, P. A. (2006). Mucosal vaccines: the promise and the challenge. Nature Reviews Immunology, 6(2), 148-158. Mucosal immunology Large surface Exposed enviroment MALT Inductive/Effector Lycke, N. (2012). Recent progress in mucosal vaccine development: potential and limitations. Nature Reviews Immunology, 12(8), 592-605. Mucosal vaccines Challenge Challenges Enzymes Barriers Pinto J. Et al., Int. J. Pharm. (2013) Vicente et al.; J Drug Deliv Sci (2010) Nanotechnology Protection Penetration Targeting Ag release Sustained Stimulation Adjuvants Solution Nanosystems for mucosal vaccines SYNTHETIC POLYMERS Polyesters: PLGA, PLA, PGA Polyethyleneimine PEI Acrylic acid based polymers … NATURAL POLYMERS Chitosan Hyaluronic acid Alginate, dextran, starch … Cordeiro, AS, Alonso, MJ (2015). Recent advances in vaccine delivery. Pharmaceutical Patent Analyst, in press. Nasal NALT -Large surface area -Reduced enzymatic activity -Moderately permeable epithelium -Highly vascularized mucosa -Absence of first-pass metabolism -Induce systemic and mucosal immunity -Easy to administer -Increase residence time -Mucoadhesive compounds/help penetration Potential irritation/irreversible damage longterm Nasal PLA PLA-PEG NPs TT-I125 Intranasal Rat nasal mucosa Tobío et al., 1998 Tobío et al. Stealth PLA-PEG Nanoparticles as Protein Carriers for Nasal Administration. Pharmaceutical Research, Vol. 15, No.2, 1998. Tobío et al. Improved immunogenicity of a core-coated tetanus toxoid delivery system. Vaccine 18 (2000) 618, 622 Nasal PLA PLA-PEG NPs TT-I125 Intranasal Tobío et al., 1998 Hypothesis of PEG contribution: Improved stability in the mucosal fluids Increased transport across mucosal barriers Protection/controlled release of the encapsulated antigen Tobío et al. Stealth PLA-PEG Nanoparticles as Protein Carriers for Nasal Administration. Pharmaceutical Research, Vol. 15, No.2, 1998. Tobío et al. Improved immunogenicity of a core-coated tetanus toxoid delivery system. Vaccine 18 (2000) 618, 622 Nasal Chitosan nanoparticles preparation Ionic gelation technique TPP Antigen Chitosan Chitosan NP 1st report on chitosan nanoparticles: Calvo et al. J. Appl. Pol. Sci, 1997 More than 6000 articles, 100,000 citations Nasal 2005: “GRAND CHALLENGES IN GLOBAL HEALTH” GC 1: Create effective single-dose vaccines GC 3: To develop needle-free delivery systems for vaccines Nanostructures as delivery vehicles for rHBs Antigen Nasal MULTIFUNCTIONAL NANOCAPSULES Antigens: rHbsAg, influenza HI Polymeric coating Polysaccharides, polyaminoacids Oils: squalene, Miglyol, Vitamin-E Surfactants: PEG-stearate, Tween 80, TPGS Nasal HB antigen Chitosan Miglyol + Imiquimod Phospholipids Vicente, S., Peleteiro, M., Díaz-Freitas, B., Sanchez, A., González-Fernández, Á., & Alonso, M. J. (2013). Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: a needle-free vaccination strategy. Journal of Controlled Release, 172(3), 773-781. Nasal In vitro uptake of chitosan nanocapsules by macrophages HB antigen Chitosan Miglyol + Imiquimod Phospholipids Cytoskeleton Nuclei Nanocapsules Vicente, S., Peleteiro, M., Díaz-Freitas, B., Sanchez, A., González-Fernández, Á., & Alonso, M. J. (2013). Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: a needle-free vaccination strategy. Journal of Controlled Release, 172(3), 773-781. Nasal Anti-HBsAg IgG (mUI/mL) Immune response generated by rHBs Ag loaded nanocarriers following intranasal vs. i.m. administration nanocapsules 10µg i.n. (0, 28, 56) HBsAg-Alum 1µg i.m. (0, 28, 56) 10000 1000 100 Protective levels 10 1 62 92 122 Time (days) Sara Vicente et al. PLoS ONE 8(4): e62500, 2013. 152 Nasal Immune response generated by rHBs Ag loaded nanocarriers following intranasal administration S. Vicente et al. Journal of Controlled Release 172 (2013) 773–781 Nasal Nanocapsules for Single-Shot Vaccination Anti-HBsAg IgG (mIU/mL) 10000 Day 73 Day 103 Day 133 Day 163 Day 193 1000 100 10 1 NC 10µg (single-dose) Sara Vicente et al. PLoS ONE 8(4): e62500, 2013. NC 10µg (0,28) HBsAg-Alum 10µg (0,28) Nasal Elimination of cold chain Ease of transport and storage Simple manual resuspension Thermostable 1 2 3 4 5 6 Completely preserved integrity of rHbsAg after one year of storage rHBsAg 25ºC 25ºC 4ºC HIV-vaccine (NIH project) Formulation 1 Evaluation Group 1: prime rVSVpcs, boost NPs Group 2: prime NPs, boost NPs Cocktail with 12 peptides NPs Previous results Hypothesis Vaccine targets 1)Protease cleave sites 12 peptides 2)Vital proteins for replication 2 proteins (2 plasmids) Natural immunity to HIV virus Formulation 2 Cocktail with 2 plasmids NPs Non published results. Jorge Pinto et al. Intranasal administration for NPs Group 3: prime rVSV prot1prot2, boost DNA-NPs HIV vaccine Formulation 1 Chitosan NPs with 1 peptide Pilot Plant Microfluidics - Scale up (small batches) - GMP production - Hihgly advanced characterization techniques NANOVAC Team COLLABORATORS Prof. África González (UVI) Dr. Belén Díaz-Freitas (UVI) Mercedes Peleteiro (UVI) Dr. Ma Luo (U. Manitoba)