NewNew TechnologiesTechnologies andand VaccineVaccine DevelopmentDevelopment MargaretMargaret A.A. Liu,Liu, M.D.M.D. Courtesy of T Sharrar, Smithsonian Institution Courtesy of T Sharrar, Smithsonian Institution NeedNeed forfor NewNew VaccinesVaccines (Million) (Million) Disease Annual New Cases Annual Deaths DiarrhealDiarrheal DiseasesDiseases 1,3001,300 2.52.5--44 AcuteAcute RespiratoryRespiratory DiseasesDiseases 3.73.7 TuberTubercculosulosisis 77--88 22--33 HIHIVV 5.85.8 33 MalariaMalaria 500500 1.51.5--33 IssIssuueses forfor LiveLive AttenuatedAttenuated VirusVirus VaccinesVaccines •• NaturaNaturall ininfecfecttionion maymay notnot indinduucece immuimmunnityity oror optimaloptimal immuneimmune responsesresponses •• SomeSome virusesviruses causecause deleteriousdeleterious immuneimmune responsesresponses •• PotentialPotential reversionreversion toto virulencevirulence –– ConcernConcern forfor HIHIVV •• DecreasedDecreased efficacyefficacy duedue toto prepre--exexistingisting antibodiesantibodies –– InfluenzaInfluenza •• DecoyDecoy antigensantigens onon thethe virusvirus ComparisonComparison ofof VaccineVaccine TechnologiesTechnologies • Live attenuated viruses • DNA vaccines – Highly effective – Need for increased potency – Potential risk – Designer immune response – Manufacturing challenge e.g., Type of TH • Recombinant proteins – Specificity: avoid deleterious or diversional antigens – Potent antibody response – Stability – Non-native forms – Safety – Not induce CTL – Generic manufacturing • Viral vectors – Cost – Risk – Resistance / pre-existing antibody – Inflammation HIVHIV CladeClade (Strain)(Strain) DiversityDiversity H G J A E C D B F HIV and the Pathogenesis of AIDS, ASM Press (1998), J.A. Levy HeterogeneityHeterogeneity ofof HIVHIV StrainsStrains ExogenousExogenous PPrroteinotein ResultsResults inin GeneratGeneratiionon ofof TT CellCell HelpHelp ButBut NotNot CTLCTL CD4 + Helper T cell CD4 Acidified (TH) Endosome MHC Class II Glycoprotein T cell Receptor Exogenous Endocytosis Antigen Golgi Apparatus Nucleus Modified from Mc Donnell WB and Askari FK, NEJM 334:42 (1996) DNADNA VaccineVaccine GeneratGeneratiionon ofof CTLCTL byby DNADNA VaccinesVaccines CD8 + DNA Proteasome Cytotoxic cleaves protein T cell Vaccine into short peptides (CTL) Cytosolic Antigen CD8 MHC Class 1 Glycoprotein T cell Receptor mRNA DNA Modified from Mc Donnell WB and Golgi Askari FK, NEJM Nucleus Apparatus 334:42 (1996) 19181918 FluFlu PandemicPandemic 20 Million Deaths Courtesy of T Sharrar, Smithsonian Institution InitialInitial DemonstrationDemonstration ofof EfficacyEfficacy ofof DNADNA VaccinesVaccines •• GenerationGeneration ofof CCTLTL byby DNADNA vaccinevaccine •• ProtectionProtection byby DNADNA vaccinevaccine againstagainst infectiousinfectious challengechallenge •• CrossCross--strainstrain protectionprotection H1N1 (1934) H3N2 (1968) Ulmer JB, Donnelly JJ…Liu MA, Science 259: 1745 (1993) DNADNA VaccineVaccine ProtectsProtects AgainstAgainst CrossCross--StrainStrain InfluenzaInfluenza ChallengeChallenge NP DNA Vaccine Control DNA 100 80 60 40 % Survival % Survival 20 0 0 2 4 6 8 10121416182022242628 Day After Infection Fu T-M…Liu MA and Donnelly JJ, J Virol 71:2715 (1997) AdditionAddition ofof IrrelevantIrrelevant PlasmidPlasmid DNADNA IncreasesIncreases AntigenAntigen--SpecificSpecific ImmuneImmune ResponsesResponses 75 Non-Human Primates r r 50 HI Tite HI Tite 25 (neutralizing surrogate) (neutralizing surrogate) 0 HA DNA Vaccine 50µg 50µg Irrelevant Plasmids 0 200µg Donnelly JJ…Liu MA, Ann Rev Imm 15:627 (1997) ImmuneImmune ResponsesResponses ofof DNADNA VaccinesVaccines ResultsResults from:from: •• SpecificSpecific immunityimmunity agagaaiinstnst encodeencodedd antigeantigenn •• NonNon--ssppecificecific immuneimmune effectseffects ofof plplasmasmiidd backbonebackbone DNA Vaccine (Bacterial Plasmid DNA) Innate Immune Responses: IL-6, IL-12, IL-18, NK α α Cell IFN-α, TNFα Cell APC IFN-γ Modified from Krieg, AM, Current Op Imm 12: 35 (2000) PlasmidPlasmid NonNon--SpecificSpecific StimulationStimulation DueDue to:to: •• PuPuCGPyPyPuPuCGPyPy sequencessequences –– ““CpGCpG momottiiffss”” •• PotentialPotential meansmeans toto increaseincrease // decreasedecrease // oror changechange naturenature ofof immunogenicityimmunogenicity ofof DNADNA VaccinesVaccines Krieg AM…Klinman DM, Nature 374:546 (1995) Klinman DM…Krieg AM, PNAS 93:2879 (1996) Sato Y…Carson DA and Raz E, Science 273:352 (1996) Klinman DM…Ishijatsubo Y, JI 158:3635 (1997) HIVHIV HIVHIV EnvelopeEnvelope gp120 gp41 DifferentDifferent FormsForms ofof HIVHIV EnvelopeEnvelope UsedUsed forfor ImmunizationsImmunizations Monomer gp120 Soluble Oligomer Membrane Bound gp140 gp160 Recombinant protein Recombinant protein DNA vaccine BB cellcell Antibodies CD8 + DNA Proteasome Cytotoxic Vaccine cleaves protein into short peptides T cell (CTL) Cytosolic Antigen CD8 MHC Class 1 Glycoprotein T cell Receptor mRNA DNA Modified from Mc Donnell WB and Golgi Askari FK, NEJM Nucleus Apparatus 334:42 (1996) ClinicalClinical TrialsTrials ofof DNADNA VaccinesVaccines •• HIHIVV – Therapeutic and prophylactic – Multiple vaccines / multiple trials •• InfluenzaInfluenza •• MalariaMalaria – Multiple vaccines / multiple trials – Antigen + cytokine genes •• HepatitisHepatitis BB •• CanceCancerr •• (Gene(Gene Therapy)Therapy) SecondSecond GenerationGeneration DNADNA VaccinesVaccines •• IncreasIncreaseded potencypotency •• “Designer”“Designer” immuneimmune responseresponse •• OralOral deliverydelivery AreaArea ofof MucosalMucosal Surfaces:Surfaces: 1½1½ BasketballBasketball CourtsCourts EncapsEncapsulatedulated DNA:DNA: MicroparticlesMicroparticles DNADNA VaccineVaccine RepliconsReplicons RapiRapidlydly ProduceProduce MoreMore ProteinProtein AntigenAntigen Replicon DNA Nucleus m7 G 5' nsProteins Antigen 3' A(n) Replicase Antigen mRNA “Designer“Designer GeneGene Vaccines”Vaccines” Genes Encoding: Replicon: • Cytokines • Amplify • Co-stimulatory antigen molecules mRNA • Targeting molecules or CpG Content: (immunostimulatory sequences) Sequential Immunization with DNA then Protein Generates Optimal Antibody Responses Prime Boost Percent seroconversion DNA — 90% DNA DNA 100% DNA PROTEIN 100% PROTEIN — 0% PROTEIN PROTEIN 50% PROTEIN DNA 90% 0 1000 2000 3000 4000 5000 6000 Anti-Gag Ab titers ProtectionProtection ofof BALB/cBALB/c micemice afterafter immunizationimmunization withwith plasmidplasmid DNADNA and/orand/or recomrecombinantbinant MVAMVA Immunization 1 Immunization 2 % Protection* DNA DNA 0 MVA MVA 20 DNA MVA 100 MVA DNA 0 *5 animals/group Antigens used: PbCSP + PbTRAP J. Schneider, …, A.V.S. Hill, Nature Medicine 4:397-402 DNADNA Vaccines:Vaccines: ToolTool forfor FFuunctionalnctional GenomicsGenomics//ProteomicsProteomics Genome In Vitro Expression Select Ligate Genes In Vivo Expression/Function or Immunogenicity CharacteristicCharacteristic ofof DNADNA VaccinesVaccines •• AbleAble toto generategenerate CTL,CTL, antibodies,antibodies, TTH – Cross-strain protective CTL – Advantages of antigen structure for antibodies • Transmembrane protein • Native glycosylation – TH intrinsically TH 1 • Can co-deliver cytokines to augment or alter TH phenotypes – Mechanisms for CTL and TH generation elucidated – Ability to stimulate desired immune responses not induced by wild-type disease – Avoid certain limitations/concerns of viral vectors CharacteristicsCharacteristics ofof DNADNA VaccinesVaccines •• SecondSecond generationgeneration DNADNA VaccinesVaccines – Increased potency – Oral/Mucosal delivery – Facile manipulation of immune responses •• PotentialPotential advantagesadvantages forfor clinicalclinical usageusage – Ability to generate T cell immunity: critical for many unconquered diseases – Key characteristics relevant to globally-needed vaccines • Generic technology • Stability • Manufacturing ease • Cost • Potential duration of immune response DiseaseDisease ModelsModels inin WhichWhich DNADNA VaccinesVaccines HaveHave DemonstratedDemonstrated EfficacyEfficacy Infectious Diseases Cancer Viruses Parasites/Protozoa • Breast (Her2/neu) • HIV • Malaria • Colon • Influenza • Mycoplasma • Prostate • Rabies • Leishmania • Myeloma • Hepatitis B,C,D • Schistosoma • Lymphoma • Ebola • Taenia ovis • E7-Induced • Herpes Simplex • Toxo. gondii • Fibrosarcoma • Papilloma • CMV Allergy • Rota • Measles • House Dust Mite • LCMV • Peanut • St. Louis Enceph • Experimental Airway Hyperresponsiveness Bacteria • B. Burgdorferi • C. tetani Autoimmune Disease • M. Tb • Diabetes • S. typhi • EAE (MS model).