Historické, sociální a medicínské aspekty infekčních chorob „V neustálém cyklu proměn malé se stává velkým a velké nepatrným“ Lao‘c „Historie je svědkem času, světlem pravdy, esencí paměti, učitelkou života, poslem z minulých věků“ Marcus Tullius Cicero Středověk Černá smrt – Mor 1346-1353 Yersinia pestis 33 Epidemie – epidemiologická opatření Vyneste své mrtvé !!! Třicetiletá válka 1618-1648 „Štěstí vyrůstá z úcty a neštěstí z násilí“ Lao‘c Napoleonské války 1797 -1815 Generálové Mráz, Hlad a Tyfus „Dobré vojsko je prostředek rodící neštěstí“ Lao‘c Chřipkové epidemie ve 20. století vysoká koncentrace mladých lidí stresové faktory reservoár patogenu – prasata, drůbež, koně migrace a promíchávání obyvatelstva zranění koncentrace nemocných v polních lazaretech zajatecké tábory Podmínky pro vznik pandemie španělské chřipky 1918-1919 Whole live, genetically modified or atenuated microorganisms Replication Antigenic structures Dead Microorganisms Antigenic structures Exogenous antigens toxins Homogenates or fractionated antigens Subunits Peptides T and B epitopes DNA Vaccine genetic information on protein Ag structure, adjuvant protein (cytokines) self adjuvant structures (CpG motifs) No replication, no Ag structure (DNA) Pure Proteins, Polysaccharides, Glycoproteins, TH2 TH1 Vaccinology Biology – Medicine Immunology, Immunopathology, Cell Biology, Virology, Bacteriology, Parasitology, Cancer Biology, Molecular Biology… Chemistry: -Bio -Physical -Polymer -Organic Colloids, Proteins, Lipids, Peptides, DNA, Polymers, Polysaccharides, … Industrial technologies Fermentation, Purification of Biopolymers, Organic Synthesis, Production of Colloid Systems, Sterilisation, Lyophilisation, … Economy – Government - Health Policy - Anti-vaccination Movement Reverse vaccinology Production of recombinant protein/antigen Synthesis of peptide antigen/epitop A need for adjuvans and delivery systems owing to low immunogenity Liposome based recombinant vaccines Classification of liposomes Morphology of liposomes Morphology of liposomes Viewed by cryoelectron microscopy Classes of liposomes based on their functionality  Conventional liposomes nonspecific interaction with melieu  Stealth liposomes sterically shielded , low non-specific interactions, long circulating  Targeted liposomes specific interaction via coupled ligand  Polymorphic-Cationic change their phase upon interaction with specific agents, pH or temperature sensitive Liposome-based products  Antimicrobial drugs Amikacin (MiKasome) Amphotericin B (AmBisome) Econazole, Nystatin  Anticancer drugs Doxorubicin (Doxil), Daunorubicin (DaunoXome),Vincristine (VincaXome), CisPlatin, Paclitaxel  Vaccines Newcastle disease, Avian rheovirus, Hepatitis A, Trivalent influenza (Epaxal-Berna)  Dermatological preparations Adjuvans and recobinant vaccines Reverse vaccinology Production of recombinant protein/antigen Synthesis of peptide antigen/epitop A need for adjuvans and delivery systems owing to low immunogenity Block scheme of the peptidoglycan of bacterial cell wall N-acetylmuramic acid N-acetylglucosamine Pentaglycine bridge Peptide chain Basic repeating unit GMDP Minimal immunoadjuvant unit MDP O O OH AcHN OH O O HO AcHN O OH H MDP nH3C CH-CO-L-Ala-D-isoGln Peptidoglycan n = 10 - 100; GMDP n = 1 Muramyl glycopeptides N-acetylmuramic acid N-acetylglucosamine Muramyldipeptide O O OH AcHN OH O O HO AcHN O OH H MDP nH3C CH-CO-L-Ala-D-isoGln Peptidoglycan (n = 10 - 100) MDP ("Muramyldipeptide", i.e. minimal immunoadjuvant unit) GMDP (n = 1; "Glucosaminylmuramyldipeptide", i.e. basic repeating unit) O O OH AcHN HO HO CHCO-L-Ala-D-isoGlnCH3 MDP Analogs of MDP and GMDP developed as potential immunotherapeutics by pharmaceutical industry O O OH AcHN HO HO CHCO-L-Ala-D-GlnCH3 O(CH2)3CH3 Murabutid (Inst. Choay, France) O O OH AcHN HO HO CHCO-L-Ala-D-isoGln-L-Lys OH H3C(H2C)16OC CH3 Romurtid (Muroctasin) (Daiichi Pharmaceutical Co., Japan) O O OHAcHN HO HO CHCO-L-Ala-D-isoGln-L-Ala-NH-CH2CH2O-P-O-CH2CH3 OH O CHOOC(CH2)14CH3 CH2OOC(CH2)14CH3 MTP-PE (Ciba-Geigy, Switzerlan) MTP-PE NovaArtis (Ciba-Geigy,Switzerland) Serious side effects e.g. pyrogenity, ulceration Analogs of muramylglycopeptides developed on IOCB in collaboration with VRI Transformations of MDP and GMDP molecules to norAbu-MDP and norAbu-GMDP analogs resp. led to decrease or elimination the pyrogenicity and enhancement immunoadjuvant activity O O OH AcHN HO HO CHCO-L-Ala-D-isoGlnCH3 O O OHAcHN HO HO CH2CO-L-Abu-D-isoGln O O OH AcHN HO O HO HO AcHN O OH CHCO-L-Ala-D-isoGlnCH3 O O OH AcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln MDP norAbu-MDP Less pyrogenic and higher immunoadjuvant than MDP GMDP Less pyrogenic and higher immunoadjuvant than MDP norAbu-GMDP Nonpyrogenic and higher immunoadjuvant than GMDP Registrated by Russian company Peptek O O OHAcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln O O OHAcHN HO HO CH2CO-L-Abu-D-isoGln Bulky lipophilic residue Bulky lipophilic residue Adjuvans based on lipophilic analogues of norAbuMDP norAbuGMDP Activity domain interaction with intracellular receptors Accesory domain Incorporation into lipid structures Ledvina M., Ježek J., Hříbalová V., Turánek J.: Liphilic analogues of N-acetyl-normuramyl-L-a-aminobutanoyl-Disoglutamine withs immunostimulatory activity, Czech Pat. CZ 296 720, 2006. Ledvina M., Turánek J., Miller A.D., Hipler K: Adjuvants, International Appl.(PCT. Appl.) No. 0804989.2, 2009. Lipophilic derivatives of norAbuMDP/GMDP All Compounds are Non-Pyrogenic Data: Rabbit Dose: 100nmol/kg (ca 0.1mg/kg) NMGP s.c. Temperature was measured in 1, 2, 3, 4, 6 and 24 hour 20 intervals. The preparation passes the test if the Sum +∆Tmax < 1.1 °C (3 rabbits per group) Structural subunits of peptidoglycan and their intracellular receptors Hypothesis - Low affinity of Nor-abu-MDP/GMDP for Cryopyrin could be responsible for low pyrogenicity of some derivatives of MDP Pyrogen Antimicrobial/anticancer peptides O O OH AcHN HO O HO HO HN O OH CH2CO-L-Abu-D-isoGln CH3(CH2)16C O N-L18-norAbuGMDP Stimulation of innate immunity of newborn kids against Cryptosporidium parvum J. Turánek et. al. Stimulation of innate immunity of newborn kids against Cryptosporidium parvum by application of immunomodulator ß-DGlcNstearoyl-(1 4)-norMurNAc-L-Abu-D-isoGln entrapped in liposomes. Parasitology 2005 131: 601-608 O O OH AcHN HO O HO HO HN O OH CH2CO-L-Abu-D-isoGln CH3(CH2)16C O N-L18-norAbuGMDP Lyophilised liposomal formulation of immunomodulator NorAbuMDP LiposIMM/MT05 Liposomal vaccines  Biodegradable and nontoxic carriers for preparation of corpuscular antigens  Simple incorporation or covalent attachment of peptide (T and B epitops) and protein antigens to the membrane or internal water phase  Preservation of natural conformation of membrane protein antigens Liposomal vaccines  Simultaneous incorporation of antigens and adjuvatns in one structure (MPLA, lipoproteins, glucans, MDP analogues, cytokines)  Systemic, intradermal and mucosal application  Oral delivery  Control of the MHC I or MHC II antigen presentation is possible (TH1/2 immune) Preparation of IRIV vaccine Epaxal-Berna  TEM micrograph Association of protein antigen with liposome Liposome based self-assembling immunogenic nanosystems O O OHAcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln O O OHAcHN HO HO CH2CO-L-Abu-D-isoGln Bulky lipophilic residue Bulky lipophilic residue Adjuvans based on lipophilic analogues of norAbuMDP norAbuGMDP Activity domain interaction with intracellular receptors Accesory domain Incorporation into lipid structures Ledvina M., Ježek J., Hříbalová V., Turánek J.: Liphilic analogues of N-acetyl-normuramyl-L-a-aminobutanoyl-Disoglutamine withs immunostimulatory activity, Czech Pat. CZ 296 720, 2006. Ledvina M., Turánek J., Miller A.D., Hipler K: Adjuvants, International Appl.(PCT. Appl.) No. 0804989.2, 2009. EU and USA patent application September 2010 O O OH AcHN HO HO CHCO-L-Ala-D-isoGlnCH3 MDP MDP New lipophilic adjuvants for liposomal vaccines nor-AbuMDP nor-AbuGMDP Monophosphoryl Lipid A (MPLA) D-(+)-trehalose 6,6'-dibehenate (TDB) Role of molecular adjuvants Liposome based self-assembling immunogenic nanosystems Surface modification- targeting Hyaluronic acid, chitosan, cationic lipids, glycolypids, dendrimers, functionalised lipids – covalent and noncovalent binding of antigens Synthetic and structuraly defined adjuvans (CpG, MPL-A, lipophilic MDP analogues) AF microscopy and TEM pictures of rHSP90 metallochelating liposomes AFM TEM 0.1 1 10 100 1000 10000 100000 0 10 20 30 40 micelles liposomes liposomes+hsp90 liposomes+gp120 Size (nm) Number(%) Immunogold labelled Mašek et al. J. Control. Release, 2011, 151 (2), 193-201. TEM of liposomal bound rOsp C 1 10 100 0 5 10 15 20 25 OspC (3.7 nm) 200 Liposomes (48.8 nm) Liposomes + OspC (54.3 nm) 505 Size (nm) %ofVolume Dendritic cell– presentation of antigen, regulation and direction of immune response Activation of anticancer immunity neuropeptides Intradermal immunisation Structure and cellular location of TLRs and NOD1 and NOD2 Structural subunits of peptidoglycan and their intracellular receptors Hypothesis - Low affinity of Nor-abu-MDP/GMDP for Cryopyrin could be responsible for low pyrogenicity of some derivatives of MDP Pyrogen Antimicrobial/anticance r peptides O O OH AcHN HO O HO HO HN O OH CH2CO-L-Abu-D-isoGln CH3(CH2)16C O N-L18-norAbuGMDP Liposome based self-assembling immunogenic nanosystems Surface modification- targeting Hyaluronic acid, chitosan, cationic lipids, glycolypids, dendrimers, functionalised lipids – covalent and noncovalent binding of antigens Synthetic and structuraly defined adjuvans (CpG, MPL-A, lipophilic MDP analogues) Confocal microscopy of DC phagocytosed fluorescence labelled liposomal vaccine and quantification of the process by flow cytometry Control Empty liposomes NorAbuMDP liposomes DiO C18 fluorescence probe HLA-DR Lissamine-rhodamine labelled liposomes O O OHAcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln O O OHAcHN HO HO CH2CO-L-Abu-D-isoGln Bulky lipophilic residue Bulky lipophilic residue 3D view of intracellular localisation of fluorescent liposomes in DC Intracellular localisation of liposomes Confocal microscope Leica SP2 Lyophilised liposomal formulation of immunomodulator NorAbuMDP LiposIMM/MT05 Infection diseases transmitted by tick  Human ehrlichiosis – anaplazmosis – bakteria Ehrlichia chaffeensis, Anaplasma phagocytophilum  Encephalitis - Flaviridae viruses  West Nile – Flaviridae viruses  Bartonellosis – bacterium Bartonella henselae  Babesiosis – protosoan Babesia  Rickettsiosis - bakteria Riskettsiaceae  Tularemia - bakterium coccobacil Francisella tularensis  Q fiver - Coxiella burnetti  Borreliosis – Borrelia b. surface antiges OspA, OspB, OspC, OspD, OspEa Lyme boreliosis  Pathogen: Borelia burgdorferi, B afzelii, B. garinii TEM of liposomal bound rOsp C 1 10 100 0 5 10 15 20 25 OspC (3.7 nm) 200 Liposomes (48.8 nm) Liposomes + OspC (54.3 nm) 505 Size (nm) %ofVolume 0 100000 200000 300000 400000 500000 600000 700000 titr experimentalgroups Titr anti-OspC IgP v séru ELISA titers for specific anti rOspC antibodies O O OHAcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln O O OHAcHN HO HO CH2CO-L-Abu-D-isoGln Bulky lipophilic residue Bulky lipophilic residue O O OHAcHN HO O HO HO AcHN O OH CH2CO-L-Abu-D-isoGln CH2CO-L-Abu-D-isoGln Bulky lipophilic residue Journal of Controlled Release 2011 submitted AlOH MDP MT05 MT06 naive sera IgG2a 2358 7683 4537 10770 343 0 20000 Titranti-OspC IgG2a norAbu-MDP-Lys (B30) MT06norAbu-MDP-Lys (L18) MT05 ELISA titers for specific anti OspC IgG subclasses Safety of synthetic recombinant liposomal vaccine against cirkovirus (pigs, i.d. application) Liposome based vaccine Mineral oil based vaccine Necrotic area Ulceration DNA vaccines Dna Vaccine Time Line  1983 In vivo expression of DNA (production of insulin in rats , DNA-liposome) (Nicolau et al.)  1992 Demonstration of immunogenicity (Tang et al.) Genetic immunisation  1993 Early protection studies  1994 Naming of technolgy, WHO organised the conference in Geneva  1995 First phase 1 human trials  1996 FDA „Points to Consider“, (first US patent, VICAL, San Diego,CA)  1998 HIV-1, malaria, influenza, herpes, and hepatitis B in human trials Principle of DNA Vaccination  Selection of antigen and construction of plasmid  Production of plasmid in industrial scale  Formulation of plasmid, development of carrier sytem, industrial scale production  Clinical trials FROM GENE TO PATIENT  FERMENTATION  CELL RECOVERY microfiltration, centrifugation  CELL LYSIS - lysozyme, NaOH and SDS treatment  CLARIFICATION AND CONCENTRATION Ammonium-acetate  PEG  Potassium-acetate  Isopropanol  Selective  (spermidine, polycations) membrane processes miF,UF,diaF  CHROMATOGRAPHY Size exclusion, Ion exchange HIC (Hydrophobic interaction chromatography) RPC (Reversephase chromatography)  PURE PLASMID Antigen DNAvaccineMarket Price of pruducts Industry Technolgy Clinical trials Plasmid Vector Adjuvant Unit Transcription Unit Origin GeneInsert Antibiotic Resistance Poly A Tail Promoter CpG Motifs CpG Motifs CpG Motifs CpG Motifs Antigen Cytokine ILs (2,4,12,15,18), GM-CSF,IFN-, MIP-1 CD86 (B7-1) Heat Shock Proteins CMV, RSV(No mamalian origin) Col E1 BGH, SV40 Targeted AG, Transcription Unit Electron micrograph of supercoiled plasmid HP-CGE analysis of plasmide topologies Optimalistion of a pDNA fermentation process Plasmid  The market for gene therapy products could exceed 45 billion USD by 2010,  A typical dose size (patients with melanoma) is 0,3 g but full treatments could require milligram quantities (a role for suitable carriers)  Large scale processes for plasmid preparation  Criteria of plasmid purity required for human application  Bacterial gDNA < 10ng per dose (< 10 ng gDNA/ gcDNA)  Bacterial proteins < 10ng per dose (undetectable by BCA assay)  RNA non seen on agarose gel or HPLC  Endotoxin unit < 0.1EU per g plasmid  Residual antibiotics !?. Manufacturers Cobra BioManufacturing UK Fit Biotech Finland Althea Qiagen (Germany, USA) Boehringer Ingelheim Austria Fermentas Lithuania PlasmidFactory Germany MoloGen Germany 1. Snadná a rychlá příprava plasmidu 2. Snadná izolace, kontrola a skladování 3. Možnost rychlé modifikace 4. Neobsahuje složky s neznámým efekty 5. DNA není imunogenní – možnost opakované aplikace 6. V základní formě indukce Th1 odpovědi Mechanism of DNA immunisation Keratinocyte gene gun MHC I MHC II Crosspresentation Injection somatic, MHC II negative cells Dendritic cell MHC I TH1 Response TH2 Response Crosspresentation antigen je exprimovaný na cytoplasmatických volných rybosomech Mechanismus aktivace CD8 T lymfocytů Mechanismus aktivace CD4 T lymfocytů DNA vakcinace aktivuje B lymfocyty k tvorbě specifických protilátek Adjuvantní působení DNA Coban et al, 2008 Mechanismus indukce imunitní CD8 T lymfocytární odpovědi na „exogenní“ antigen Přenesená „cross“ prezentace Přenesená prezentace „cross“ 1. TAP závislá a) korpuskulární antigen - fagosom (NOX2) – proteasom b) solubilní antigen – receptor – časný endosom - proteasom -ER - proteasom – TAP na endosomu – MHC I vazba antigenu na scavengerové receptory – MHC II 2. TAP nezávislá (vakuolární) TAP – závislá „cross“ prezentace korpuskulární antigen solubilní antigen MHCIIMHCI TAP závislá „cross“ prezentace – korpuskulární antigen Pro srovnání prezentace korpuskulárního antigenu na MHC II TAP závislá „cross“ prezentace – solubilní antigen Pro srovnání prezentace solubilního antigenu na MHC II TAP - nezávislá „cross“ prezentace U člověka „cross“ prezentují solubilní i korpuskulární antigen konvenční DC (mDC-2) typizované CD141+ (BDCA-3), CD11c+ u myší konvenční DC CD8+, CD11c+ DNA Vaccine Carriers  Naked DNA (i.m., unprotected against degradation) Pneumatic Jet Injection (particle free, mm-cm below the skin surface, strong shearing forces)  Needle (modified tatoo instrument), Electroporation  Gene Gun Delivery of Genes (epidermal, gold particles) TH2 response  Micro-Organisms:(Attenuated and genetically modified bacteria - Shigella, Salmonella, or Listeria) (mucosal immunisation, incorporation of pDNA into genome)  DNA-Containing Cochleates (oral)  Liposomes (all routes)  Polymers (biodegradable particles, mucosal, oral) Dendritické buňky  Obtížná transfekce  I vitro lipofekce velmi málo účinná  Účinnost in vitro elektroporace kolem 10-15% pro Langerhansovy buňky nebo DC odvozené z progenitorů; DC odvozené z periferních monocytů jsou resistentní k transfekci  Rychlá degradace pDNA v dendritických buňkách, vrozená resistence k intracelulárním patogenům (viry, bakterie), výjimka HIV-1  Některé virové promotory mohou být i signálem pro ohrožení, snaha používat somatické promotory (promotor cytoskeletálního proteinu fascinu) Svalové buňky  Poměrně dobře transfekovatelné – častý cíl pro genovou terapii (dlouhodobá transfekce, dlouhá doba života)  Nevirové vektory nejsou příliš účinné ( na rozdíl od dobré účinnosti např. na transfekci jater nebo plic)  U myší transfekce svalových vláken volným plasmidem v rozmezí 2-5 týdnů (inhibiční efekt extracelulární matrix na penetraci pDNA, použití hyaluronidázy, rychlá degradace pDNA, 90% během prvních minut po aplikaci) Způsoby přenosu DNA do buněk  Volná DNA  Virové vektory – retrovirové vektory – adenovirové vektory – adeno-asociované viry  Nevirové vektory – kationické liposomy – polymery  Fyzikální metody – gene-gun, jet-device – elektroporace – sonoporace  vektory odvozené od herpes simplex virů  hybridní virové vektory Faktory ovlivňující úspěšnost DNA vakcinace  immunogennost antigenu patogena  frekvence a způsob podání, forma DNA vakcíny  dávka pDNA  lokalizace antigenu (sekretovaný, membránový nebo cytoplasmatický)  věk, zdravotní stav  živočišný druh Formy aplikace DNA vakcín Osud DNA v závislosti na použitém chemickém systému Classification of liposomes Morphology of liposomes Morphology of liposomes Viewed by cryoelectron microscopy Classes of liposomes based on their functionality  Conventional liposomes nonspecific interaction with melieu  Stealth liposomes sterically shielded , low non-specific interactions, long circulating  Targeted liposomes specific interaction via coupled ligand  Polymorphic-Cationic change their phase upon interaction with specific agents, pH or temperature sensitive Artificial Virus •Fully synthetic particles •Deletion of undesirable structures Immunosuppressors Crossreacting antigens •Selective targeting •Production in cell free system •Resembles a live atenuated vaccine Cataionic lipids Cataionic lipids - Imperial College Cataionic lipids of the 3rd generation Teargeting groups: peptides or saccharides Encapsulation of plasmids into liposomes 100 nm 500 nm Interaction of cationic liposomes with DNA Routes of Application  Intramuscular  Intraepidermal  Mucosal: Oral, Nasal,Vaginal, Rectal, Lung, Sinovial  Intravenous  Intraperitoneal  Subcutaneous Aplikace DNA vakcíny do svalu Aplikace DNA vakcíny do jater Microenhancer Arrays Epidermal application Intradermal application Intranasal delivery Elektroporace  Princip elektroporace – při aplikaci elektrického napětí dochází ke změnám transmembránového potenciálu, po dosažení kritické hodnoty se membrána stává permeabilní, dochází k tvorbě hydrofilních pórů – průchod extracelulárních molekul do buňky Využití elektroporace Vnášení farmakologicky významných molekul (hydrofilní povahy) do buněk  Genová terapie a DNA vakcíny - In vivo a in vitro transfekce buněk (antigeny, růstové faktory, cytokiny, enzymy) vnášení DNA do buněk pro účely genové terapie  Protinádorová elektrochemoterapie - zvýšení selektivity a účinnosti chemoterapeutik (bleomycin) Elektroporace in vivo  Vliv na účinnost elektroporace – velikost, tvar a morfologie buněk – viskozita a vodivost extracelulární tekutiny (struktura extracelulární matrix)  Elektrické parametry – nízký počet (2-10) středně dlouhých (1-50 ms) pulsů při nízké frekvenci (1-2 Hz) – série velmi krátkých (200-500 s) pulsů při vysoké frekvenci (10-1000 Hz)  Typy elektrod – ploché elektrody, přiložené zevně ke svalu nebo kožnímu lemu – jehlovité elektrody pro intramuskulární aplikaci – Needle array – multilektrodové aplikátory s rotujícím elektrickým polem Srovnání účinnosti elektroporační transfekce pomocí šestielektrodového a dvouelektrodového aplikátoru Babiuk at al. Vaccine 2002, 20, Vliv intenzity elektrického pole na účinnost transfekce volný pDNA Hyauloronidáz a + Elektroporace McMahon MJ and Wells DJBiodrugs 2004, 18,155-165 Vliv extracelulární matrix na elektrotransfekci myšího svalu Typy elektrod Typy elektrod Electroporation Aplicators Transfekce in vivo u myší transfekce lýtkového svalu myší pomocí elektroporace pGFP Intenzita: 80 V/cm Doba pulzu: 20ms Počet pulzů: 6 Frekvence: 1s Transfekce in vivo u myší Elektroporace Bez elektroporace Balb/c, samice (stáří 8-9 týdnů), lýtkový sval 50µg pDNA (βGal), 48 h Transfekce in vivo s použitím elektroporace Parametry elektroporace: 150 V/cm 6 pulzů délka pulzu 20 ms frekvence pulzů 1s Zpracování svalu: po 72 h. vypreparování lýtkového svalu zmražení n-heptanem a nařezání 7 μm řezů fixace řezů detekce β-galaktozidázy pomocí X-gal + elektroporace - elektroporace 1 μg 1L 2L 3L 1L 2L 3L a - - - - - b - 25 - - - c - 23 - - - d 1 25 8 - - e 3 - 13 - - f 3 1 12 - - g - - - h - - - i - j 10 k 4 l 4 10 μg 1L 2L 3L 1L 2L 3L a 2 7 - - - b 11 14 - - - c 21 15 - - - 1 d 95 18 75 - - 1 e - 12 - - - 4 f - - 14 g h - 50 μg 1L 2L 3L 1L 2L 3L a - 1 65 - - b 19 16 75 - - 1 c 41 15 81 - 1 d 16 56 60 - 5 2 e 46 25 1 2 2 f 30 60 1 1 g 16 72 - - 5 h - 24 - - 2 i - - j - 0 10 20 30 40 50 60 70 1 μg 10 μg 50 μg Počettransfekovaných svalovýchvláken Volná DNA Elektroporace Dávka DNA 0 10 20 30 40 50 60 70 1 μg 10 μg 50 μg Počettransfekovaných svalovýchvláken Transfekce in vivo Tisíce DNA vakcín testováno u experimentálních zvířat Tři vakcíny jsou chváleny pro veterinární použití V současnosti (2011) není žádná DNA vakcína schválena pro klinické využití u lidí Aktuální situace u DNA vakcín Tisíce DNA vakcín testováno u experimentálních zvířat Tři vakcíny jsou chváleny pro veterinární použití V současnosti (2011) není žádná DNA vakcína schválena pro klinické využití u lidí Aktuální situace u DNA vakcín Klinické studie s DNA vakcínami •prevence infekcí • HIV, SARS, HPV, hepatitida B, chřipka, hemoragická horečka Ebola, západonilská horečka ... • malárie •terapie nádorových onemocnění • karcinom ledvin, pankreatu, prsu, prostaty, plic, močového měchýře, hepatocelulární karcinom, • melanom Počet klinických studií s DNA vakcínou přesahuje 500 (www.clinicaltrials.gov) Závěr  Přenosný elektroporátor pro práci v terénu není dostupný (jsou ve vývoji)  Proces je do určité míry bolestivý (lokální nebo úplná anestesie)  Transfekce pomocí elektroporace vykazují 10- 1000 násobně vyšší účinnost ve srovnání s volným plasmidem  Stále není optimalizovaný proces, velká variabilita výsledků  Testovány aplikace i.m., i.n. a i.d. Adjuvans for DNA vaccines  Aluminium and Calcium Salts (i.m.)  Liposomal lentinan (glucans) (p.o.)  Monophosphoryl lipid A (i.m.)  Muramyldipeptide analogues  Cytokines (induced, external or encoded by plasmid) Th1/Th2  CpG motifs (integral part of plasmide DNA)  Cholera toxin, HSP  8Br-cAMP - enhencer of CMV promoter The role of CpG motive in DNA vaccines  CpG - frequency 1/16 in bacteria and 1/60 in vertebrates (methylated in vertebrate DNA  Th1- like patern of cytokine (IL-12, INF)  Induction of strong CTL response  Immune response towards CpG - an evolutonary adaptation to augment innate immunity in response to bacterial infection  CpG DNA - T-cells independent and antigen nonspecific activation of B-cells, stimulation of B-cells to proliferate, secrete Ig, IL-6, IL- 12, protection of B-cells from apoptosis,experession of MHCII and B-7  -direct activation of monocytes and macrophages  -activation of dendritic cells to secrete various cytokines and chemokines Mechanism of immune stimulation by CpG DNA Dendritic Cell Conclusion for DNA Vaccines  CpG-DNA or CpG-ODN can be used as a vaccine adjuvants  Two active parts of pDNA vaccine  1. insert encoding the protein antigen (accesory protein - cytokines)  2. CpG-S motifs directly stimulating B-cells and Th1 cytokine expression Advantages of DNA Vaccines  Subunit immunisation with no risk for infection  Presentation by both MHC I and II  Ability to raise TH1 and TH1/2 response  Focused immune response  Immunisation of neonates (colostral immunity)  Easy of development (cloning of genes) and production  Stability of vaccine Disadvantages of DNA Vaccines  Limited to protein immunogens  Potential for atypical processing of bacterial and parasite proteins  Lack of long term experience with use of DNA vaccines  Patents: VICAL (San Diego,CA) , Wistar Institute (PA) University of Massachusetts (MA), GENEMEDICINE Inc. (TX) Safety Issues  Production of pDNA and its formulation according to Good Manufacturing Practice  WHO:Guidelines Assuring the Quality of DNA Vaccines, 1997, Tech Rep Ser No-17  Integration into host genome undetectable for naked plasmid (construcion of plasmid, avoidance of the sequences possibly mediating chromosomal integration, retroviral, lentiviral) (effect of carrier - liposomes, bacteria)  Tolerance (neonates ) only one case in mice immunised by MA  Autoimmunity risk no grater than that posed by live viral vaccines  Anti-DNA Antibodies (no evidence from the experiment with lupus-prone mice)  Effect of antibiotic resistance gene Futre Promise  Preparation of monoclonal antibodies  New approach and precise tool for the study of immune responses  Screening of protective effect of multiple different microbial antigenes and their combination (library immunisation)  Development of new efficient vaccines (HIV, malaria, cancer, tuberculosis, influenza, ebola, hepatitis B)  Development of vaccines for veterinary application  DNA vaccines are not yet licensed in many countries, therefore national authorities are not experienced with this kind of product and do not differentiate between gene medication and gene modification. Within the EU two opposite points of view are maintained as regards DNA vaccinated animals. Gene medication or genetic modification? Different regulations and interpretations The EU definition of GMO The EU difinition of genetic modification DNA vaccines -Problems  Safety of DNA vaccines – proved  Efficacy – still real problem in large animals  Field experiments in large animals – condemnation of cadavers – economical as well as ethical problem  GMO – artificial problem (pig, broiler, mule)  Food safety - ? Consumption of residual plasmid? Spreading of the antibiotics Plasmid Vector Adjuvant Unit Transcription Unit Origin GeneInsert Antibiotic Resistance Poly A Tail Promoter CpG Motifs CpG Motifs CpG Motifs CpG Motifs Antigen Cytokine ILs (2,4,12,15,18), GM-CSF,IFN-, MIP-1 CD86 (B7-1) Heat Shock Proteins CMV, RSV(No mamalian origin) Col E1 BGH, SV40 Targeted AG, Transcription Unit - minicircleAccesory and Adjuvant Unit Risk and uncertainity Real time PCR format – SYBER GREEN I Quantitech Syber Green kit (Qiagen Lightcycler (Roche) Cycling condition Initial actiovation 15 min. 95ºC 20 Denaturation 15 s 94ºC 20º Annealing 25 s 55ºC 20º Extension 10 s 72ºC 20ºC pCMV HSP 60 155 bp BGH Plasmid pVAX Hsp60 TM814 (pDNA) persistence: Real Time PCR (RTPCR) pUC ori pVAX Hsp60 TM814 4582 bp RT-PCR: Syber Green format, Lightcycler 161bp PCR product  specificity – matrix inhibition, validation isolation method  linearity (linear range) - 109 – 101 copies  detection limit – 3 copies (≈7.5 ag) / 500ng gDNA pDNA Muscle injection Necropsy, tissue sampling gDNA isolation (GuSCN:silica) Validation RTPCR 161bp y = -3,3029x + 36,538 R2 = 0,9859 PCR efficiency 2,00 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 log koncentrace Cp Quantification of the pDNA in bovine musles by the means of Real Time -PCR Calibration curve for pDNA in H2O Detection limit in water: 2 copies/l (per reaction) LQL: value below linear limit of quantification (10 copies/reaction) Detection limit in muscle tissue: (3 copies/ reaction) ≈3 000 Site of pDNA vaccine application in cow m. coccygeus ln. sacralis externus caudalis Vakccination of calves with the DNA vaccine against ringworm (trychophytosis) m. coccygeus Persistence of pDNA at the injection site: 1, 7, 28, 90, 180, 365 days after injection 1 7 0 1 2 3 4 5 6 7 8 9 10 liposome electroporation injection 28 90 180 365 detection limit Days Plasmidcopies(log)/500ngisolatedDNA pDNA: 10 g - i.m (calf muscle) RTPCR: 500 ng gDNA - injection site non-treated muscle - negative non-treated animals - negative Plasmid levels in calf muscle (injection site) after administration of 10 g pDNAX in 5 weeks old BalB/C mice. 1 7 0 1 2 3 4 5 6 7 8 9 10 pDNAX:CDAN-DOPE pDNAX electroporation naked pDNAX detection limit 14 28 Days after administration pDNAcopies(log)/500ngisolatedDNA Day 1 Day 28 Persistence of DNA vaccines in large animals Code Group (3-4 bulls/each group) 1. immunisation 2. immunisation 1 500 g DNA 400 l 500 l 2 500 g DNA + 500 g ML-455 400 l 500 l 3 500 g DNA + 2.5 mg CDAN-DOPE 600 l 800 l 500 g DNA – 1.21 x 10*14 molecules7 months HSP 60 TM 814 pVAX heat shock protein 60 (Hsp60) Trichophyton mentangrophytes Hsp60 TM LiposIMM-N-L18NAGMDP Applications: Stimulation of innate immunity (viral, bacterial, protozoal O O OH AcHN HO O HO HO HN O OH CH2CO-L-Abu-D-isoGln CH3(CH2)16C O N-L18-norAbuGMDP Diameter (nm) 5 0 100 500 Zeta potential (mV) -100 0 10 0 400 nm Mean 178 nm Mean –30.6 mV Site of pDNA vaccine application in cow m. coccygeus ln. sacralis externus caudalis Persistence of pDNAX in beef after i.m. administration Beef cattle groups Interval between 2nd immunisation and slaughter (days) pDNA copies at the injection site copies/ 500 ng DNA (n=5) pDNA copies opposite to- injection site muscle (n=4) pDNA copies distant muscle (n=3) pDNA copies DLNa total (n=6) pDNA 242