von Gabain, Alexander

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For more information be invited to: www.intercell.comVACCINE TECHNOLOGY IIAlbufeira, June 1 - 6, 2008Towards a therapeutic HCV vaccine - a preclinical and clinical learning curveAlexander von GabainJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 1» The treatment of chronic HCV patients is currently based on (pegylated)-Interferon and Ribavirin- Significant side effects- Not all infected patients can be treated - Significant costs of treatment (up to 30.000 USD per year)- Long duration (up to 48 weeks)» Sustained virus response rates are between 50 and 60%,for genotype 1 only 43-46% 1,21. Fried M. et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med., Vol. 347, 13, 13 Sep 2002.2. Manns M.P. et al. PINF alfa-2b plus ribavirin compared with INF alfa-2b plus ribavirin for initial treatment for chronic hepatitis C: a randomized trial. Lancet, Vol. 358 (9286), Sep 2001Chronic Hepatitis C: Standard of CareJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 2» Stronger, broader, quicker and more sustained CD4 and CD8 T-cell responses in self-limited course of acute hepatitis C» Response to antiviral therapy may be associated with increased T-cell responses» Viral persistence in chronic hepatitis C is associated with immune evasion? impaired function of HCV-specific T-cells? mutational T-cell epitope escape» Chimp modelsDiepolder 1995, Missale 1996, Rehermann 1996, Lamonaca 1999, Gruener2000, Thimme 2001, Wedemeyer 2002, Lauer 2002&2004, Cox 2005, Boettler2005, Spangenberg 2005,...HCV: importance of T-cell responsesJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 3Th 2 CytokinesIL-4, -5, IL-10, IL-13Endogen. processed antigensSolubleAntigenAPCTh 1 CytokinesIFNγ, TNFαCD 8CD 4MHC IIMHC IHepatocyteB-cells AntibodiesClonal ExpansionTc1 and Tc2Cytokines, CytotoxicityNKNKTThe T-cell system and Hepatitis C virus infectionIFN-gamma ELIspotHLA-tetramer(CCR7, CD45RA)IFN-gamma ELIspotLymphoproliferationJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 4C E1 NS5NS4NS3NS2E2|--- Envelope   ---| |---------------------------- Non-structural proteins   --------------------------------|CoreThe IC41 HCV vaccine: 5 synthetic peptides adjuvanted with Poly-L-ArginineCore23-44A*0201DRB1*1101Core132-140A*0201NS31073-1081A*0201NS31248-1261A*0201DRB1*0101DRB1*0401DRB1*0404DRB1*0701DRB1*1101DRB1*1501NS41764-1786A*0201DRB1*0101DRB1*0401DRB1*0404DRB1*0701DRB1*1101>80% conserved regions in HCV genotypes 1, 2, 3Intercell peptide #Class I Class IIHCV-GenomeJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 5987654321P o s itio n***H LA  b in din g***T C R  re c ep to rVTWCVGNICW ild ty peISMASTG en . 1M ,LT ,S ,AG en . 6LMG en . 5MAG en . 4T ,IDGT ,S ,AG en . 3LIS ,AT ,SG en . 2(*)Conservative (green) and non-conservative (red) amino acid exchanges in each position of the NS2-1073 peptide among the different genotypes of the Hepatitis C Virus. indicates the positions important for HLA binding or for the TCR receptor recognition.Fytili et al. ,Vaccines 2008Sequence variability in the NS3-1073 CTL epitope*JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 6Ex vivo Elispot IC41 vaccinated healthy volunteer020406080100G1 G2 G3 G4G5 G6020406080100120140160G1 G2 G3 G4G5 G6Cross-genotype recognition of twenty-eightNS3-1073 peptide variantsIFN-γ ELISPOT USING T-CELLS INDUCED AGAINST WILDTYPEIn vitro T-cell lineFytili et al. ,Vaccines 2008JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 7Treatment scheduleVaccin-ations-4 0 4 8 12 16 20 24 28 32Study weekRandom-izationStudy period» Screen-ing» Treat-ment» Follow upImmunol-ogicalchecksStudy designControl groupsTreatment groups5 Hepatitis C Peptides**0.005.002.502.505.00Poly-Arginine**2.000.001.252.002.001212121212No. ofpatientsTotal 60BCGHK* Study period: end 2002 -mid 2004** Different dose levels44TREATMENT SCHEDULE AND STUDY DESIGN (IC41-201)IC41-1: 60 chronic HCV patients, standard IFN/riba therapy non-responders/relapsersKlade et al.Gastroenterology2008Phase II in non-respondersJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 8Interferon gamma ELIspot using frozen PBMCAssay standard:control cellsHIV vs. CMV peptidesHIVPositive Controls:CMV, EBV, Flu-peptidesCon ACMVEBVFLUHBV ConAIpep83Ipep1426Ipep84Ipep87Ipep89Ipep1051Ipep1334Ipep1874/75ELISPOT: > 3x OVER BACKGROUND, AT LEAST 15 PER MIO. PBMCJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 9CLASS I AND II RESPONSE RATES (ELISPOT)IC41/Dose H*Peptide only (5 mg)Poly-Arginine only (2 mg)0255033.3%0.0%0.0%Response rate in %CD4+ T-cellsPoly-Arginine only (2 mg)Peptide only (5 mg)IC41/Dose H*0255025.0%0.0%0.0%Response rate in %CD8+ and CD4+ T-cells* 2.5 mg peptides;2.0 mg Poly-ArginineIC41 induces Th1/Tc1 type immune responses in non-responder patientsPhase II in non-respondersKlade et al.Gastroenterology2008,Firbas et al., 2006JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 10-4 0 4 8 12 16 20 24 32 44IC41 induces T-cell proliferation in non-responder patients654321Dose Group H*Control PeptideControl Poly-ArginineWeek of study Median response stimulation indexMEDIAN CLASS II T-CELL PROLIFERATION: DOSE GROUP Hvaccinationsend ofvaccination* 2.5 mg peptides;2.0 mg Poly-ArgininePhase II in non-respondersKlade et al.Gastroenterology2008JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 11Results of concluded Phase II study –IC41 already showed trend in efficacyGroup results of 1 Log responders in Phase II trial*12.50/2.00H 8%00.00/2.00B 0%0 0%5.00/0.00C5.00/2.00K 17%22.50/1.25G 0%0Resp.DosageGroup NResults of patient with viral load reduction in high dose group*ELISPOTSpots per 200,000 PBMCHCV-RNA0-4 0 4 8 12 16 20 24 28 32 4415510Week of studyClass I6 vaccinationsBaselineHCV-RNA (Log IU/ml)4-4 0 4 8 12 16 20 24 28 32 44756Week of study6 vaccinationsThreshold viral load effectPHASE  II NON RESPONDERS (IC41-1)Class I responses of >10spots/200,000 are associated with transient viral load reductionsKlade et al.GastroenterologyIn revisionPhase II in non-respondersJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 12Evidence for mutational T-cell epitope escape in a patient responding to IC41-1 vaccination* Published and presented at the EASL Meeting in Vienna, April  2006RESULTS OF PATIENT WITH VIRAL LOAD REDUCTION*ELISPOTSpots per 200,000 PBMCHCV-RNA0-4 0 4 8 12 16 20 24 28 32 4415510Week of clinical trialClass I6 vaccinations monthlyBaselineHCV-RNA (Log IU/ml)4-4 0 4 8 12 16 20 24 28 32 44756Week of clinical trial6 vaccinations monthlyThreshold viral load effectImpaired recognition of an HCV T cell epitope evolving in a single patient during vaccination 0%50%100%150%IC41 (Ipep89) V-variant I-variant% IFN-gamma ELIspotsCINGVCWTVCINGVCWSVCINGVCWS ICINGVCWS ICINGVCWSVPhase II in non-respondersJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 13IC41-2: Combination with standard therapyPEGINF/RBV0 4 8 12 16 20 24 28V232V336V440V544V648V752V860V972V10IC41 6 vaccinations s.c. in 4 weeks intervalsRelapse rate wk  52 / 60 / 72Weeks after start ofstandard therapyV visitImmunological Assays wk 28 / 48 / 52 / 60 / 72Patients with chronic hepatitis C of genotype I scheduled for standard treatment for 48 weeks already treated for 28 weeks and responded at week 12Heiner WedemeyerChristoph Kladeet al.AASLD 2007JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 14Sustained responders show a stronger and more frequent T-cell response – Target Population*INTERFERON γ ELISPOTS IN RELAPSED PATIENTS (N= 8) VS. SVR (N=14)051015202530V2 V3 V4 V5 V6 V7 V8 V9 V10200420054004400740085001500250035004700570089006051015202530V2 V3 V4 V5 V6 V7 V8 V9 V1010012001200340037004700770099009spots / 200.000 PBMCRelapse patientsspots / 200.000 PBMCsustained responders•*Target Population N = 23, for 1 patient missing HCV-RNA data between V8–V10• 9009: RNA rebound Heiner WedemeyerChristoph Kladeet al.AASLD 2007JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 15Conclusions from non-responder patients (IC41-1) and late add-on to PEG-IFN/RBV (IC41-2)» favorable safety profile in chronic HCV patientswith or without PEG-IFN/RBV standard therapy» optimal vaccine dose (2,5 mg peptides / 2,0 mg poly-L-Arg)» Th1/Tc1 type responses in chronic HCV patients,no apparent negative influence through PEG-IFN/RBV» several transient 1 log Hepatitis C - RNA responders at optimal dose» RNA responses associated with strongest CD8+ responses achieved» T-cell immunogenicity requires optimization(rate, strength, breadth, sustainability)JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 16Improving immunicity of IC41 in HLA-transgenic mouse model0200400600800100012001400s.c. i.d. i.d. + "imiquimod" -lot H (AB) lot H (AB) lot H (AB) -HHD.2 mice injected  with nr. of IFN-γ -producing cells / million CD4+ T cell-enriched SPC's0200400600800100012001400s.c. i.d. i.d. + "imiquimod" -lot H (AB) lot H (AB) lot H (AB) -HHD.2 mice immunized with nr. of IFN-γ -producing cells / million CD4+ T cell- depleted SPC's~ 600~ 700~ 800~ 400~ 1500legend:mediumIpep 83Ipep 84Ipep 87Ipep 89Ipep 1426Ipep 1334Ipep 1874Ipep 1875pRIpep 1274CD8+ T-cells CD4+ T-cellsHHD.2 miceDose/100µl/mouse:100µg/peptide +400µg pR(lot H in-house mixture AB)exp. scheme:day 0, 14, 28,42, 56, 70s.c. or i.d.injectionday 7 after 6th inj.IFN-g ELIspot(spleen cells) day 29 after 6th injAPC transferday 30 after 6th injFACS analysisspleen cells0102030405060708090100I C4 1 s. c. I C4 1 i. d. I C4 1 i. d. +Aldar a% killingIn vivo CTL assayIC41 i.d. + „Imiquimod“IC41 s.c. IC41 i.d.legend:% killed APCIpep 1274 (irrel.)% killed APCIpep 87 (rel.)TEST APPLICATION SITES  ± IMIQUIMODJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 17IC41-3 Study concluded January 2008OPTIMAL VACCINATION SCHEDULE IN TREATMENT NAIVE PATIENTS8 vaccinations*           0» 50 Chronic HCV patients,  treatment naive, HCV Genotype 1.Desired subset with low viral load at baseline» Endpoints: ? Decline in HCV-RNA? T-cell response» Status ? Participating countries: Romania, Poland, Germany? End of recruitment on track for February 2007» First vaccination on September 26 2006, first data Q2/20072 4 6 8 10 12 14 16 38weekFU 1 FU 2* Bi-weekly; intradermal; topical Aldara®(3M)Phase II in naïve patientsJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 18Primary endpoint met – a weak, but statistically significant HCV-RNA reductionOVERVIEW IC41-3 PHASE II DATAStatisticallysignificant0 2 4 6 8 10 12 14-2 16V1 V2 V3 V4 V5 V6 V7 V8V0 V9Point EstimateWeeks* 46 patients** 25 patients*** 95% confidence intervals1st vaccinationlast vaccinationTotal study group*Statisticallysignificant0 2 4 6 8 10 12 14-2 16V1 V2 V3 V4 V5 V6 V7 V8V0 V9WeeksVisitsChange fromBaseline***(Log HCV-RNA/copies/ml) 1st vaccinationlast vaccinationHigh viral load patients (>2 mio copies/ml)**-1,1-1,0-0,9-0,8-0,7-0,6-0,5-0,4-0,3-0,2-0,10,00,10,20,3-1,1-1,0-0,9-0,8-0,7-0,6-0,5-0,4-0,3-0,2-0,10,00,10,20,3Visits..- .- .- .- .- .Change fromBaseline***(Log HCV-RNA/copies/ml) ..- .- .- .- .- .JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 19Conclusions from IC41 trials» Favorable safety profile in chronic HCV patientswith or without PEG-IFN/RBV standard therapy» Optimal vaccine dose / schedule identified» Th1/Tc1 type responses in chronic HCV patients,no apparent negative influence through PEG-IFN/RBV» Antiviral activity demonstrated in patients with strongest CD8+ responses, and treatment group with optimal vaccinationJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 20HCV therapeutic vaccination: Forward StrategyDevelopment of second generation vaccine»More & better peptides (HLA-restriction, efficacy)» Improved T-cell adjuvant (IC31®)Future plans: combination therapy» plus PEG-IFN/RBV» plus novel small molecule inhibitorJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 21Overlapping set of synthetic peptides derived from viral genome121110987654321242322212019181716151413363534333231302928272625Viral genomeVirusDefined T-cell epitopes for therapeutic vaccinesEpitope Capture: Binding to human HLA receptorsReacting with T-cell derived from humans with positive disease outcomeInduced in HLA-transgenic mice71115 36Identification of further T-cell peptidesReviewed inScharnagl & Klade2007, Exp Rev Vaccines 6:605-15.Schalich & Klade2008, Biol ChemKubitschke & Klade2008,in preparationT-CELL EPITOPE IDENTIFICATION PROGRAM JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 22Identification of HCV vaccine candidate peptides beyond IC41++++?DR4(?)(from Day et al.)DR1, 4, 7, 11A2Ipep 1547 +++++++++++++++(+)+EpitopeCaptureA24?(DR4 / Ipep 1650)?(Ipep1800, IVS)DR1, 4, 7, 11A2, A11(?), Cw7Ipep 1846DR7DR1, 7, 11DR1, 4, 7DR1, 4DR1, 7, 11(?)DR11Class II epitopes??(Ipep1801)???(Ipep1605, IVS)?Human PBMC screening?(DR4 / Ipep 1563)B35Ipep 1799A24(?)(A2 no final data)A2, A3, A11Ipep 1798B8Not applicableA24Ipep 1827(as expected negativefor A2, B7, DR4B60Ipep 1624A24?B7, (A2)A2, B7Ipep 1829?(B7 / Ipep 1506)A2, A3, B7Ipep 1835tg mice screeningAdditionalpredicted epitopesClass IepitopesPeptideHLA-COVERAGE: 80-90% IN EUROPE, USA AND JAPANIVS: in vitro stimulation of PBMC from HLA-matched healthy donors PCT/EP2003/009482Otava & KladeAASLD 2004Kubitschke & Kladein preparationJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 23» KLK: antimicrobial peptide H-KLKL5KLK-OH? Type 2 immune responses (+ proteins)? Depot formation at injection site» ODN1a: oligodeoxynucleotide oligo-(dIdC)13phosphodiester, ssDNA? Type 1 induction? Activation of APC (Dendritic Cells)? TLR-9 / MyD88-dependent signalingHLA-DRCD40CD83CD86 CD80Potent and sustainedTh-1 / type 2responsesT cellB cellIC31®: a TLR agonist comprising two chemically defined biodegradable components? Enhancement of antigen and ODN1a uptake by APC JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 24IC31®: Induction of potent type 1 cellular immune responses0100200300400500600700800IFN-γ producing cells(per 106 spleen cells)PEPTIDE-SPECIFIC IFN-γ PRODUCTIONOVA257-264 mTRP-2181-188AlumCpG 1668IC31®day 7 after singleinjectionIFN-γ ELISpot050100150200250IFN-γ producing cells(per 106 spleen cells)naiveor mTRP-2181-188CTL - EFFECTOR CELLSmTRP-2181-188+ IC31®Specific killing of APCEXAMPLE: IMMUNIZATION WITH MODEL PEPTIDESrelevant peptideirrelevant peptideno peptideJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 25Protective immunity of a novel TB subunit vaccine adjuvanted with IC31®PRECLINICAL EVALUATION – SURVIVAL (GUINEA PIG)** 3x i.m. injection, 4-week intervalAerosol infection; 16 weeks after first injectionBCGAg85B/ ESAT-6 + IC31®Naive/ SalineWeeks post infection% survival0 10 20 30 40020406080100120&JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 26Protectivity is linked to IFN- γ producing T-cellsindicative for Th-1 driven immunity0,00,51,01,52,0Log 10 resistanceRESIDUAL BACTERIA (lung) IFN-γ productionBCG IC31®&0100020003000400050006000IFN-γ(pg/ml)BCG IC31®®DEFINITION OF PROTECTION MARKERS (MOUSE MODEL)JUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 27Induction of antigen-specific T-cells in humans vaccinated with the novel TB subunit vaccinesDATA FROM TB PHASE I STUDY: STRONG TH-1 INDUCTION&IFN-γ in T-cell supernatants (Ag85B/ESAT-6-specific ELISA;Estimated Marginal Means)Frequency of IFN-γ prod. T-cells (Ag85B/ESAT-6-specific ELISpot;Estimated Marginal Means)visit 6 months 6 monthsvisitJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 28Dramatic improvement of IC41 by replacing poly(Arg) with IC31® (IC410)exp. scheme:day 0, 14, 28i. fp. injectionday 34APC transferday 35FACS analysis(LNC)      ELIspot(spleen cells)01002003004005006007008009001000IC41 lotK PD03127 IC410 -HHD.2 mice immunized (f.p.) with nr. of IFN-g producing cells/million CD4depleated SPC'slegend:mediumIpep 83Ipep 84Ipep 87Ipep 89Ipep 1426Ipep 1334Ipep 1874Ipep 1875pRIpep 1274Dose/100µl/mouse:IC41:200µg/peptide +400µg pR43(lot K, batch PD03126)IC410:50µg/peptide +35nmol KLK+1.4nmol ODN1a(inhouse mixture)naiveIC410 i.fp.IC41 i.fp.020406080100IC41 IC410 naive% killingIpep 89naiveIC410 i.fp.IC41 i.fp.legend:% killed APCIpep 1274 (irrel.)% killed APCIpep 87, 89 (rel.)IFN-γ PRODUCTIONCD8+ T CELL EFFECTOR FUNCTIONJUNE 2, 2008VACCINE TECHNOLOGY IIPAGE 29AcknowledgmentsAlexander von GabainErich TauberElisabeth SchullerKaren LingnauINTERCELL HCV study groupMichael P. MannsHeiner WedemeyerChristoph SarrazinThomas BergHolger HinrichsenGrazyna CholewinskaStefan ZeuzemHubert BlumUlrich SpenglerRudolf StauberBernd Jilma

Towards a Therapeutic Hepatitis C Vaccine – a Preclinical and Clinical Learning Curve

https://dc.engconfintl.org/cgi/viewcontent.cgi?article=1002&amp;context=vaccine

Towards a Therapeutic Hepatitis C Vaccine – a Preclinical and Clinical Learning Curve

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