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Use of virus-like particles therapeuticParticles vaccination Vaccines Based on for Virus-Like Regulation of anti-viral cell responses against addiction andBother chronic diseases 1 • The great success of the HBV and in particular HPV vaccines the Use of virus-like particles for demonsrates therapeutic vaccination Regulation of anti-viral cell responses addiction andBother chronic diseases poweragainst of VLPs • All inacticated viruses are essentially VLPs • Do we need anything more to say? 2 Harnessing the Immune System Clinical Use Antibodies • Prophylactic Vaccines • Self-specific Antibodies • Allergies • Drug-specific Antibodies T Cells • Cancer • Chronic Viral Infections 3 4cLD0056_screenshow Mechanism of action of classical vaccines A selection of currently used vaccines and their effector mechanisms Pathogen Pox Polio Hepatitis B Hepatitis A Measles Rubella Mumps Tetanus Pertussis Tuberculosis 4 Effector Mechanism Antibody Antibody Antibody Antibody Antibody Antibody Antibody Antibody Antibody T cells Types of vaccines currently used and/or in development • Live attenuated (eg Polio Sabine or Rubella) • Inactivated (eg Polio Salk or Hepatitis A) • Isolated pathogen-components (eg bacterial conjugate vaccines, Tetanus, Diptheria, Pertussis (toxins)) • Recombinant (eg Hepatitis B, detoxified version of Diphteria) • DNA 5 Safety of the various vaccine types Safety/Specificity The “less alive” the safer Live attenuated Inactivated Isolated pathogencomponents 6 Recombinant proteins (DNA) Immunogenicity of the various vaccine types Immunogenicity The “less alive” the less immunogenic Live attenuated Inactivated Isolated pathogencomponents 7 Recombinant proteins DNA Challenge The problem that has to be solved Make Attenuated Pathogens as Safe as Recombinant Proteins or Make Recombinant Proteins as immunogenic as Attenuated Pathogens 8 Make Attenuated Pathogens as safe as Recombinant Proteins Ways to attenuate pathogens • Specific deletions in pathogen genome (eg virulence factors) rather than passaging • Recombinant versions of “safe” live vectors (eg yellow fewer vaccine that expresses dengue fever proteins (Aventis Pasteur)) • Replications-incompetent vaccines (eg Alphavirus-based vaccines) • Reassortant viruses (chimeras between human and non-human viruses) • Attenuation of pathogens by making them more immunogenic (eg IL-2 expressing vaccinia virus; listeriolysin expressing BCG) 9 Make Attenuated Pathogens as safe as Recombinant Proteins Immunogenicity • However: Degree of Attenuation/Safety 10 •The danger to hit immunocompromised people (Polio in AIDS patient, Tuberculosis Vaccine in IL-12-deficient patients) • Reversion to virulent form (All Polio cases in Europe are vaccine-induced) • If attenuation is based on impaired replication, there is a strong in vivo pressure for the reversion to more virulent forms (Polio ÅÆ HIV) • Even minimal side-effects are not tolerated (Rotashield) Challenge The problem that has to be solved Make Attenuated Pathogens as safe as Recombinant Proteins or Make Recombinant Proteins as immunogenic as Attenuated Pathogens 11 B cell responses The immunology behind it 12 B cell responses How can we optimze B cell responses? 13 B cell responses Optimizing Th cell responses 14 B cell responses Optimizing Th cell responses: But which Th cells 15 TH1 IL-12, IFNγ TH2 IL-4 Treg TGFβ/IL-10 THfo IL-21 B cell responses Optimizing Th cell responses 3 strategies for enhancing Th cell responses 1) Formation of a depot Æ Long-lived T cell stimulation 2) Targeting dendritic cells 3) Activation of dendritic cells 16 B cell responses Depot formation Typical Adjuvants forming a depot: • • • 17 Chemical precipitates (Alum) Oil-in-Water/Water-in-oil (IFA, Montanide MF59 etc) Protein precipitates (IC31) B cell responses Targeting dendritic cells Typical adjuvants targeting dendritic cells Æ Make them particulate • • • • • • 18 Liposomes Virosomes Microparticles ISCOMs/Iscomatrix IC31 VLPs B cell responses Activating dendritic cells Typical adjuvants activating dendritic cells • • • • • • • • • 19 CpGs, IC31 (TLR9) MPL (TLR4) RNA (TLR7/8) TLR-2,3,4,5 ligands Peptidoglycan (nods) Alum (Nalp3) QS21 Saponin Heat-shock proteins B cell responses 20 B cell responses • T help • Epitope density of Ag • Complement • Toll-like receptor ligands 21 Scientific Rational Science 262, 1448-1451 22 Organization: high low absent Antibody response +++ + - Induction of auto-antibodies +++ - - Role of epitope densitiy and organisation How important is Antigen-repetitiveness on a viral surface? Which is the minimal epitope density we need for effective crosslinking of the B-cell receptor? Study of antigen density versus antigen amount Antigen HBc subunit crosslinker Antigen HBc subunit crosslinker 23 . Role of Epitope Densitiy & Organization Antigen organization is critical for IgG responses 10 v. low low medium high 8 high 6 medium 4 low v. low 2 0 d7 d18 d25 d50 d81 days after immunisation 24 d126 d220 Role of epitope densitiy and organisation Why is epitope reptitiveness so important Our immune system exploits structural features for the discrimination of self and foreign. Viruses have small genomes and therefore consist of a small number of proteins. Viruses cannot help but have to express highly repetitive and highly ordered arrays of antigens on their surface 25 B cell responses • T help • Epitope density of Ag • Complement • Toll-like receptor ligands 26 Role of epitope densitiy and organisation Role of CD21 in B cell activation versus isotype switch The model: CD21 enhances BCR-signalling, increasing B cell activation 27 Role of epitope densitiy and organisation Role of Antigen Organization and CD21 for IgG responses IgG-titers in CD21 -/- 7 6 5 4 3 2 1 0 High Medium Low d14 d46 days after immunisation 28 d95 E LIS A tite r (-log2 , 4 0 x pre dilute d s e ra ) ELISA titer (-log2, 40 x prediluted sera) IgG-titers in Controls 7 6 5 4 3 2 1 0 High Medium d14 d46 days after immunization d95 Role of epitope densitiy and organisation B cell response CD21 reduces the number of BCRs that need to be engaged 29 +CD21 no CD21 Nr of cross-linked BCRs B cell responses Optimal Vaccines should An Optimal Vaccine should be: • Particulate (DC Targeting) • Repetitive (BCR receptor cross-linking) • Fix Complement (CD19/CD21) • Contain TLR-ligands or alike for DC and/or B cell activation • Economical and GMP-compatible Production • Stable 30 Virus-Like Particles Based on RNA-Phages VLPs derived from bacteriophages are: • Particulate Æ Target dendritic cells • Contain RNA Æ Activate dendritic cells • Are highly repetitive • Fix complement via natural Abs plus alternative pathway 31 In addition, they are • very stable • cheap to produce (3 g/l Bacterial Culture) 4cLD0056_screenshow Generating Repetitive Antigen Arrays Induction of Antibodies antigen N N O N O N O O O O N N O N O N O O O O N N N N O O O O O Cys O N N O O O O O O N N O O O N N N N O O O N N O N N O O O N O O O O O O O O O N O N O N O O N N O O 32 diameter = 30 nm N N O O O O N N MW = 379.36 N O O SMPH chemical linker From Mice to Men High Antibody Titers in Both Species Mouse Human Antibody titer (Endpoint) Antibody titer (OD50) 10000 1000 Factor 100 100 10 33 10000 1000 100 10 Peptide Adjuvants 100000 none Peptide Alum PeptidePeptideProtein Carrier VLP Alum none 10μg i.m. 50μg i.m. 10μg s.c. 50μg s.c. Antibody Response in Humans Anti-Nicotine Ab Responses 1000000 Antibody titer 100000 10000 1000 100 10 1 0 1 2 3 4 5 month 34 Months after Immunization Against Nicotine 6 If You`re in a Rush High Antibody Levels are Reached in a few weeks 100 μg, weekly regimen 100 μg, biweekly regimen 20000 ELISA titer Anti-nicotine IgG response 25000 100μg, monthly regimen (Ph. II) p<0.0001 15000 10000 5000 0 0 1 2 3 4 5 6 7 weeks 35 8 9 10 11 12 13 Factor 10.2 increase in antibody titers Influenza A-Virus Structure and Classification of Strains Classification of Influenza A Strains: Hemagglutinin (HA): H 1-16 Neuraminidase (NA): N 1-9 e.g. H3N2 ; H5N1 Human isolates of the last century: - H1-3, - N1-2 - RNA Virus, 8 segments – 10 Proteins 36 Universal Influenza A Vaccine Variability of HA versus M2 in Influenza A major pandemics Antigenic “drift” and “shift” from 1918 - 2000 1918 1957 1968 Representative portion of HA 1918 1957 1968 Complete extracellular domain of M2 37 Preclinical Testing of VLP-M2 Generation of M2-Immunodrug Antigen Linker Carrier (M2 peptide) (SMPH) (AP205) O N N Cys N O O SLLTEVETPIRNEWGCRCNDSSD-GGGC 38 O O O O Lys Preclinical Testing of VLP-M2 Induction of M2 specific antibodies M2 specific titers anti-M2 IgG 40'000 30'000 20'000 10'000 0 d14 d21 Strong immune responses against M2 are induced by VLP-M2 without adjuvant 39 Preclinical Testing of VLP-M2 Survival and „Fever“ Post 4xLD50 Dose Survival „Fever“ (Strain: PR8 H1N1 – mouse adapted) Æ VLP-M2 vaccinated mice are protected from a lethal challenge with Influenza A 40 Isotype-Switching ssRNA ELISA titer (-log2 x 40) IgG2a in mice and IgG1 in humans 6 5 4 3 2 1 0 Mice IgG1 IgG2a IgG2b IgG3 OD at 1:50 dilution Antibody Isotype 41 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Human IgG1 IgG2 IgG3 IgG4 Isotype-Switching Does RNA in Qb Play a Role? Digestion of RNA using RNAse 42 Isotype-Switching TLRs drive isotype switching ELISA titer (-log2 x 40) VLP/ssRNA 6 5 4 3 2 1 0 IgG1 IgG2a IgG2b IgG3 Antibody Isotype ELISA titer (-log2 x 40) VLP no RNA 6 5 4 3 2 1 0 IgG1 IgG2a IgG2b Antibody Isotype 43 IgG3 Isotype-Switching Toll-like recpetor 7 is Essential for Qb/ssRNA 10000 Qb/ssRNA IgG2a 1000 100 TLR7+/- TLR7-/- TLR7+/- TLR7-/- 10000 IgG1 1000 100 44 No IgG2a in TLR7-/- mice Influenza M2 Survival: Isotypes are the key 100 with RNAÆIgG2a % survival 80 AP205-M2 60 no RNAÆIgG1 AP205-M2 40 20 0 0 2 4 6 8 10 12 14 days after infection 45 16 18 20 22 TLR7 signalling regulates IgA (and IgG2a) responses against Qβ-VLP Mucosal Ab levels (BAL) IgG2a IgA 1000 IgG2a ELISA Titer IgA ELISA Titer 100 10 10 1 1 wild type 46 100 wild type (empty VLP) TLR7+/+ TLR7-/- Myd88-/- wild type wild type (empty VLP) TLR7+/+ TLR7-/- Myd88-/- CD8+ T cells Induction of CD8+ T cells Induction of Cytotoxic T cells TLR ligands make all the difference! 47 CD8+ T cells Induction of CD8+ T cells p33-VLP 7 days 48 CD8+ T cells + RNA VLPPGp33 VLP 25 20 15 10 5 0 C57BL/6 49 +CpG Empty % p33 specific CD8 T cells Induction of CD8+ T cells VLPCpG- VLPPGp33 p33 VLP MyD88-/- VLPCpGp33 Malaria vaccine Introduction: Life cycle of Plasmodium falciparum 50 Malaria vaccine Introduction: MSP119 51 MSP1 is expressed as 200 kDa protein -> several proteolytic processing steps -> 42 kDa C-terminus -> further proteolysis -> 19 kDa C-terminus MSP119 and MSP142 are leading vaccine candidates against blood stages of p. falciparum. MSP119 is highly conserved compared with other regions of the MSP1 antigen Characterization of the natural immune response of individuals from areas of endemic malaria led to the conclusion that MSP119 is most probably the target of a protective immune response Under certain conditions, recombinant MSP119 can induce immunity in monkeys and in mice. A high antibody titer appears to be essential Malaria vaccine MSP1.19: Structure N C Morgan WD et al., J. Mol. Biol. (1999) 289, 113-122 52 Malaria-vaccine Conformation of coupled MSP1.19 ? Quadruple ELISA, anti-MSP1, mAb 2F10 1:100 0.14 1:300 HRP Y Y 5 OD (450 nm) 0.12 Y Y 0.06 1:243000 1:729000 0.04 1:2187000 1:6561000 1:19683000 MSP1-Qb Qb MSP1 alone Quadruple ELISA, anti-MSP1, mAb 12.10 1:100 0.8 1:300 0.7 1:900 0.6 1:2700 0.5 1:81000 0.4 1:243000 0.3 1:729000 0.2 1:2187000 0.1 1:6561000 0 1:19683000 MSP1-Qb Qb MSP1 alone antigen 53 no antigen antigen OD (450 nm) 5) Goat anti-mouse IgG HRP 4) Mouse anti-MSP1 3) MSP1-Qb [Qb]=[1 mg/ml] (titration) 2) Rabbit anti-Qb polyclonal serum 1) Goat anti-rabbit IgG 1:81000 0 2 1 1:2700 0.08 0.02 4 3 1:900 0.1 no antigen Malaria-vaccine Anti-MSP119 antibody isotypes in mice ELISA titer (-log3 x 100) anti-MSP1.19 Isotypes, d35 6 5 4 3 2 1 0 IgG1 IgG2a MSP1-Qb MSP1-Qb, Alum Antigen injected 54 MSP1,Alum Concusions Not if, but which VLPs to take? • Efficient Production • Preferably NOT derived from a human pathogen - Interference with diagnostic kits - Potential interference with vaccine against the the pathogen and/or pathogen-induced disease (Immunopathology) • Not too small (a high enough number of epitopes needs to be coupled for optimal B cell responses) • 55 Packaging of natural TLR-ligand