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 – Highly effective – Need for increased potency – Potential risk – Designer – Manufacturing challenge e.g., Type of TH • Recombinant proteins – Specificity: avoid deleterious or diversional – Potent 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 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 into short (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 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 r r 50 HI Tite HI Tite 25 (neutralizing surrogate) (neutralizing (neutralizing surrogate) (neutralizing

0 HA DNA Vaccine 50µg 50µg Irrelevant 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 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 + •• HepatitisHepatitis BB •• CanceCancerr •• (()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: : • • Amplify • Co-stimulatory antigen molecules mRNA • Targeting molecules

 or  CpG Content: (immunostimulatory sequences) Sequential 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, 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 • • Colon • • • Prostate • Rabies • Leishmania • Myeloma • ,C,D • Schistosoma • Lymphoma • Ebola • Taenia ovis • E7-Induced • Herpes Simplex • Toxo. gondii • Fibrosarcoma • Papilloma • CMV • Rota • Measles • House Dust Mite • LCMV • Peanut • St. Louis Enceph • Experimental Airway Hyperresponsiveness Bacteria • B. Burgdorferi • C. tetani • M. Tb • Diabetes • S. typhi • EAE (MS model)