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New Strategies Towards Vaccine Design Challenging HIV

New Strategies Towards Vaccine Design Challenging HIV. Ralf Wagner Univ. Prof. for Medical Microbiology and Gene Therapy, University of Regensburg. Starting point. Targets of immune response. envelope proteins: Env structural proteins: Gag, Pol regulatory proteins: Tat, Rev, Nef

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New Strategies Towards Vaccine Design Challenging HIV

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  1. New Strategies Towards Vaccine DesignChallenging HIV Ralf Wagner Univ. Prof. for Medical Microbiology and Gene Therapy, University of Regensburg

  2. Starting point Targets of immune response • envelope proteins: Env • structural proteins:Gag, Pol • regulatory proteins: Tat, Rev, Nef • accessory proteins: Vpr HIV Clades Immune response + + • A (Africa) • B (Europe, N & S-America) • C (Asia, Africa …) • E (Asia) • F (Africa) • G (South America, Africa) • neutralizing AB, V1/V2, IgG/IgA • CD4+ T cell response • CD8+ CTLs • broad, cross clade, • high quality, polyfunctional • mucosal immunity • long term memory • feasable immunization regimen immunogen design molecular virology Delivery / Combination of Multivalent HIV/AIDS Vaccine Strategies Bacterial Vectors DNA rAd,NYVAC Adjuvants (CpG, PEI, MF59) Peptides Proteins Pseudovirions Viral Vectors ? ex vivo analysis basic in vivo / mice preclinic / rhesus macaques clinic / phase 1/2/3

  3. tev vif tat rev LTR LTR pol vpr gag env nef vpu Sequence analysis of C (CN54) Knowledgeon molecularepidemiology Asia Prevalence Incidence Virus population Regional clustering Host genetic background China B C C B C B C C B B C B India Subcloning pCR-script antigens regional strains next generation mosaics mosaic antigens CHIVAC EU FP5 / 6 Shao Yiming, Hans Wolf

  4. E K Y F R S/ A V K G D R F Y K T L R A W Y R Q I F H Y F Knowledgeon specifictargets/ epitopes - Controlofvirusreplication 100 80 60 % spec. Lysis 40 20 0 3 4 5 6 7 8 9 kb 1 2 0 LTR LTR Infectivity tev (35) (1) (22) (8) conservative vif tat rev HLA-B14 CTL-Epitope pol vpr env gag nef 298 299 300 301 302 303 304 305 306 vpu non- conservative HLA-B14-restricted CTL-epitope (aa298-306) Infectivity p17 MA p24 CA p6 p7 100 80 p2 p1 60 % spec. Lysis MHR 40 20 major homology region 0 D R F Y K T L R A Focus IR to conserved proteins and epitopes limits immunological escape True for both T and B cell responses DFG

  5. AA Human HIV-1 Ala GCC GCA Arg AGG AGA Asn AAC AAT Cys TGC TGT Gln CAG CAA Glu GAG GAA Gly GGC GGA Northern constitutive export syngag syn-gag Knowledgeon virusreplication (I) UTR-wtgag-RRE UTR-wtgag-RRE gag-RRE cis activerepressor sequenzes N C N C N C N C degradation - - - - + + + + wt-gag RRE splicing- machinery REV ran ? CRM1 y SD REV wt-gag RRE UTR BMBF

  6. HIV Vaccines • easy expression of late lentiviral genes • RNA nuclear export 50x • no Rev, no RRE • no 5‘-UTR, no Y constitutive nc export ? 1 Risk for Recombination RNA Packaging syn-gag cis activerepressor sequenzes Increased safety Increased yields Enhanced immunogenicity degradation 2 wt-gag RRE splicing machinery • protein production • DNA vaccines • viral DNA delivery (SFV) • bacterial DNA delivery (Listeria) • adenoviral vectors • herpesviral vectors (EHV) • lentiviral vectors REV ran ? CRM1 y SD 2 REV 3 wt-gag RRE UTR Knowledgetospecifygene design RNA- and Codon Optimization

  7. 1st Generation (EuroVacc): T CellVaccine Broad, Polyfunctional Mimic LTNP Profile EU FP 5, 6

  8. tev vif tat rev LTR LTR pol vpr gag env nef vpu Clade Chassis Status Developer / Manufacturer GagPolNef C (CN54) DNA-C GMP, B, T, I, Fill. UREG / Cobra Env C (CN54) DNA-C GMP, B, T, I, Fill. UREG / Cobra GagPolNef / Env C (CN54) NYVAC-C GMP, B, T, I, Fill. Sanofi Pasteur GagPolNef / Env C (CN54) MVA-C GMP, B, T, I, Fill Esteban GagPolNef / Env C (CN54) vTT-C GMP, B, T, I, Fill Y. Shao / S-CDC GagPolNef / Env B NYVAC-B GMP, B, T, I, Fill. Sanofi Pasteur GagPolNef / Env B MVA-B GMP, B, T, I, Fill. Esteban Gene Design, Protein Design and Vectors Sequence analysis of C (CN54) RNA and codon optimized Myr- (A) ΔFS Pr - (DN) scNef Gag Pr RT-N C N RT-C RT 160 kDa readthrough polyprotein, cytoplasmic, not processed soluble secreted monomeric gp120 Env gp120

  9. Clinical Analysis - Responders (gIFN+ T cells) Study design: 2 x 20 HIV negative volunteers London (MRC; J.Weber, S. McCormack), Lausanne (CHUV; G. Pantaleo) P < 0.001 P = 0.001 P < 0.001 P = 0.019 NYVAC C alone DNA C + NYVAC C Percentage of responders NYVAC-C DNA-C Higher percentage of responders in DNA-C prime / NYVAC-C boost group (>90%) compared to NYVAC-C group (<40%) Durability DNA-C / NYVAC-C >> NYVAC-C

  10. A Volunteer # EU11, DNA C + NYVAC C Gated on CD4 T cells 0% 0% 0.02% 0% 0.34% Neg TNF- CD4 IL-2 0% 0% 0.04% 0.07% 0.11% 6.6% 0.09 Env 0.01% 0.03% CFSE IFN-γ IFN-γ B Volunteer # EU11, DNA C + NYVAC C Gated on CD8T cells 0% 0.01% 0% 1.10% 0% 0.01% Neg CD107a TNF- CD8 IL-2 0% 0% 0% 0.07% 0.03% 0.20 1.21% 0.08% 1.94% Env 0.03% 0.17 0.22 CFSE IFN-γ IFN-γ IFN-γ Clinical Analysis - Responders (gIFN+ T cells) T cell responses are broad (mean 4,2 epitopes) and polyfunctional CD4 and CD8 responses are polyfunctional (IL-2, gIFN, TNFa) … mimicking profile seen in LTNPs

  11. Rhesus Env Gag Pol Nef Clinical Analysis - Responders (gIFN+ T cells) Human Env Gag #SFC/10e6 PBMC Pol Nef Magnitude of Env >> Gag Specific T Cell Responses

  12. TVDC Re-Design I: T CellImmunogens ImmunogenFormulation PoxviralVector (Chassis)

  13. CTL T TH ? CTL MHC-II MHC-I translation ? ? Pr55Gag MHC-I MHC-II membrane targeting NYVAC-C DNA-C NYVAC-C DNA-C TH cell membrane 100 nm Myr+ (AG) +FS Pr - (DN) DC particle release Gag scNef Pr55Gag Pr RT-N C N RT-C RT No co-stimulatory signals No MHC II presentation No T cell help CTL VLP 200 nm Re-Design – T cell Immunogens

  14. gp120 gp140 gp140 gp140 Re-Design – T Cell Immunogens & Plasmid Expression Gag PolNef Env encoded protein features PTVDC (VRC8400-Vector) Myr+ (AG) +FS Gag 95% PolNef 5% VLP Pr - (DN) Gag scNef +++ + - Pr RT-N C N RT-C RT Myr+ (G) ΔFS Gag +++ - - Gag VLP scNef soluble, cytoplasmic - ++ - Pr RT-N C N RT-C RT gp140 trimeric secreted Env - - +++ EuroVac (Cobra-Vector) Myr- (A) ΔFS Pr - (DN) scNef + + - no VLP Gag Pr RT-N C N RT-C RT - - +++ monomer Env

  15. Re-Design – T Cell Immunogens & Plasmid Balb/C, DNA 1 mock 2 GPN (Myr-,FS-) 3 GPN (Myr-,FS-) + gp120 4 GPN (Myr+,FS+) 5 GPN (Myr+,FS+) + gp120 6 Gag (Myr+) + PN 7 Gag (Myr+) + PN + gp120 Assembly and release as Pr55-Gag VLP supports the induction of Gag specific, CD8+ T cells

  16. Re-Design – Immunogen Formulation Balb/C, DNA 1 mock 2 GPN (Myr-,FS-) 3 GPN (Myr-,FS-)+ gp120 4 GPN (Myr+,FS+) 5 GPN (Myr+,FS+)+ gp120 6 Gag (Myr+) + PN 7 Gag (Myr+) + PN+ gp120 Mixture of 3 DNAs, 2 legs Co-administration of Env togeher with Gag (Pol) reduces Gag specific T cell responses

  17. Re-Design – Immunogen Formulation Balb/C, DNA Trivalent DNA 8 9 modify ratio left : right leg Spatial separation / ratio modification (formulation) leads to balanced and high level T cell response

  18. Re-introduction of -1 ribosomal frameshift leads to ~ 20x increased Gag expression, NOT on cost of PolNef Gag alone expresses equallywell Level of Gag expression and VLP formation crucial to level of induced T cellresponses Co-administration of Envsuppresses Gag specific T cellresponses Recommendations: DNA: 3 plasmidsencoding Gag, PolNef and Envoptimizemolar ratios (trivalent) NYVAC: 2 virusesexpressingGagPolNef and Env (bivalent) Conclusions Immunogens - Formulation

  19. gp140 gp140 gp140 Re-Design – Poxviral Vector Design C N M K F E P O I G L J H D A B aprox. 15Kb NYVAC aprox. 10Kb NYVACKC Myr+ (AG) +FS Pr - (DN) scNef Gag bivalent 2 viruses RT-N Pr C N RT-C RT Env Fragmented genes Intact genes • Growth in human primary keratinocytes • Highly attenuated (much less pathogenic compared to NYCBH) • No effect on DCs maturation • Potentiation of antigen direct and cross-presentation • Induction of higher levels of trangene expression • Induction of longer persistence of transgene expression • Stimulation of antigen-specific memory responses Collaboration Bert Jacobs, Karen Kibler

  20. TVDC Re-Design II: Assessimmunogenicityofnextgenerationimmunogens in NHP „Optimal“ immunizationschedule

  21. TVDC NHP Study Objectives: • To evaluate the immunogenicity of: • 2ndgenerationimmunogens, DNA and NYVAC • replicationcompetent NYVAC-KC and replicationdeficient NYVAC • clade C TV1 gp120 (formulated with MF59) in combination with DNA/NYVAC regimens • To compare scarification vs. i.m. route of administration (NYVAC-KC) • To evaluate the effects of DNA priming on T-cell and antibodyresponses

  22. 2nd Generation DNA and NYVAC constructs Trivalent DNA-C: Env ZM96, Gag ZM96, Pol-Nef CN54 Bivalent NYVAC-C: Env ZM96, chimeric Gag ZM96/Pol-Nef CN54 Bivalent NYVAC-C-KC: Env ZM96, chimeric Gag ZM96/Pol-Nef CN54 All constructs RNA- and Codon optimized (expression yields, vector stability) Re-Design II – NHP Study Immunogens:

  23. Re-Design – Opt Clinical Study Protocol (Rhesus) N G ICS Protein NYVAC-C-KC (Scar) ELISpot 8 1 Ab NYVAC-C-KC (IM) DNA-C (IM) Protein DNA prime groups 8 2 0 4 8 12 20 24 28 32 36 48 NYVAC-C (IM) Protein 8 3 NYVAC-C + Protein (IM) NYVAC-C (IM) 8 4 0 4 8 12 20 24 28 32 36 48 No DNA groups NYVAC-C + Protein (IM) NYVAC-C-KC (IM) 8 5 0 4 8 12 20 24 28 32 36 48

  24. Immunogenicity of DNA priming (post 3 DNA injections, w12) vs. NYVAC alone (post 2 injection, w8) 4 weeks after last injection Re-Design – Opt Clinical Study Protocol (Rhesus)

  25. DNA Prime Induces Polyfunctional and Balanced CD4 and CD8 T-Cell Responses Re-Design – Opt Clinical Study Protocol (Rhesus)

  26. AUP444. T-Cell Responses Following DNA Prime/NYVAC Boost IFN-g ELISpot 15050 14050 13050 12050 D3/N-KC(im)/3P 11050 D3/N-KC(scar)/3P 10050 9050 8050 D3/N(im)/3P SFUs per 106 Blood Mononuclear Cells 7050 6050 5050 4050 3050 2050 1050 50 P557 P554 P567 P554 P567 P557 P557 P554 P567 P539 P559 P544 P564 P554 P545 P563 P548 P567 P557 P544 P564 P563 P548 P539 P545 P559 P544 P564 P545 P539 P559 P563 P548 P537 P544 P564 P536 P562 P539 P559 P563 P534 P547 P534 P547 P568 P552 P546 P558 P545 P548 P537 P547 P536 P562 P537 P534 P547 P537 P534 P568 P552 P546 P558 P536 P562 P568 P552 P546 P558 P555 P549 P536 P562 P532 P540 P566 P532 P540 P566 P568 P552 P546 P558 P566 P549 P540 P566 P555 P549 P555 P532 P549 P540 P555 P532 Week 48 Week 50 Week 57 Week 57 Week 22 Week 57 Week 50 Week 48 Week 50 Week 22 Week 48 Week 22 2 wks Post 1st NYVAC 16 wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost 7 wks Post 3rd Protein Boost 2 wks Post 1st NYVAC 16 wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost 7 wks Post 3rd Protein Boost 2 wks Post 1st NYVAC 16 wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost 7wks Post 3rd Protein Boost Group 1 – NYVAC-C-KC Group 2 – NYVAC-C-KC IM Group 3 – NYVAC-C IM NYVAC-C KC: replication defective in human cells NYVAC-C KC: replication competent in human cells

  27. T-Cell Responses After NYVAC+gp120 Immunization IFN-g ELISpot 5800 5225 4650 Nkc2NkcP3 4075 3500 N2NP3 SFUs per 106 Blood Mononuclear Cells 2925 Primary Immunization Boost 2350 Primary Immunization Boost 1775 1200 625 50 P542 P556 P542 P556 P560 P569 P553 P561 P550 P551 P542 P556 P560 P569 P553 P561 P550 P551 P542 P556 P560 P569 P553 P561 P550 P551 P538 P543 P530 P565 P541 P531 P535 P533 P538 P543 P530 P565 P541 P531 P535 P533 P538 P543 P530 P565 P541 P531 P535 P533 P560 P569 P553 P561 P550 P551 P538 P543 P530 P565 P541 P531 P535 P533 Week 48 Week 50 Week 57 Week 28 Week 57 Week 48 Week 50 Week 28 4 wks Post 2nd NYVAC-C-KC (IM) + 2 NYVAC-C-KC/Protein Boost 24 wks Post 2nd NYVAC-C-KC (IM) + 2 NYVAC-C-KC/Protein Boost 2 wks Post 2nd NYVAC-C-KC (IM) + 3 NYVAC-C-KC/Protein Boost 7 wks Post 2nd NYVAC-C-KC (IM) + 3 NYVAC-C-KC/Protein Boost 4 wks Post 2nd NYVAC-C (IM) + 2 NYVAC-C/Protein Boost 24 wks Post 2nd NYVAC-C (IM) + 2 NYVAC-C/Protein Boost 2 wks Post 2nd NYVAC-C (IM) + 3 NYVAC-C/Protein Boost 7 wks Post 2nd NYVAC-C (IM) + 3 NYVAC-C/Protein Boost Group 4 – NYVAC-C-KC Group 5 – NYVAC-C NYVAC-C KC: replication competent in human cells NYVAC-C: replication defective in human cells

  28. Key time points: Groups #1-3 (DNA prime) atweek 22 Groups #4,5 (no DNA) atweek 28 Value Exact P Group 1 vs. Group 2 vs. Group 3 0.97 Group 4 vs. Group 5 0.003 Group 4 vs. Groups1-3 <0.001 Group Comparison of IFN-g ELISpot Responses by Wilcoxon Rank Sum Test

  29. CD4+ and CD8+ T Cell Function Groups G 1-3 G 4, 5 G 2 T • + + + • + + - • + - + • + - - • - + + • + - • - + G 1-3 G 4, 5

  30. Breadth of T-Cell Responses in DNA/NYVA-KC/gp120 Group #2 Breadth of T-Cell Responses in NYVAC-KC/gp120 Group #4

  31. No significant differences in the magnitude of vaccine-induced T-cell responses between the DNA prime groups Significant differences in the magnitude between: DNA prime and no DNA groups and NYVAC and NYVAC-KC in the no DNA prime groups DNA prime groups induced more polyfunctional CD4 and CD8 T-cell responses DNA prime groups had dominant Gag/Pol while no DNA groups predominant Env T-cell responses Conclusions I – T-Cell Response

  32. Is the Profile of T-Cell Response and ImmunizationRegimen (DNA Prime vs. No DNA) Associated to BetterAntibodyResponse?

  33. Vaccine-Induced Antibody Response Neutralizing antibodies ADCC Binding antibodies (total IgG) Plasma Env IgA antibodies Plasma IgG gp70 V1V2 antibodies Montefiori Lab

  34. Neutralizing Antibody Titers All for TZM.bl cellsKruskal-Wallis Rank Sum Test 5 Groups 1-3-DNA Prime Week 36 (after 2nd protein) Groups 4,5-No DNA Week 26 (after 2nd NYVAC/protein) 4 3 Log10 Titer 2 1 BaL.26 Bx08.16 MN.3 MW965.26 SF162.LS SS1196.1 TV1.21 HIV Isolate

  35. ADCC Activity in NYVAC-KC/gp120 (Gr.#4) Vs. NYVAC/gp120 (Gr.#5) ADCC Activity in DNA/NYVAC-KC/gp120 (Gr.#2) Vs. DNA/NYVAC/gp120 (Gr.#3) 25,000 4,000 20,000 3,000 15,000 Titer Titer 2,000 10,000 1,000 5,000 0 0 Wk 22 Wk 14 Wk 30 Wk 26 Wk 36 Wk 14 Wk 22 Wk 30 Wk 26 Wk 36 Wk22: after 3 DNA / 1 NYVAC Wk32: after 3 DNA / 1 NYVAC / 1 protein Wk32: after 3 DNA / 1 NYVAC / 2 protein Wk 14: after 2 NYVAC / 1 NYVAC + protein Wk 26: after 2 NYVAC / 2 NYVAC + protein Group 5 Group 3 Group 4 Group 2

  36. Cross-Clade Plasma IgG Antibody Titers Groups 1-3-DNA Prime Groups 4,5-No DNA IgG Titer (Mean AUC) Weeks

  37. Plasma IgG gp70 V1/V2 Antibody Titers 100000 Groups 1-3-DNA Prime Groups 4,5-No DNA 10000 1000 IgG antibody (Titer) 100 10 1 Wk 36 Wk 30 Wk 26 Wk 22 Wk 14 Wk 0

  38. NYVAC-KC and NYVAC plus gp120 groups compared to DNA prime groups showed: GreaterNAbactivityagainstTier 1 viruses and SHIVs (TZM.blassay) Greater ADCC activity Earlier and greater cross-clade IgGantibodytiters (more than 6 Mo after 1st immunization) Earlier, but comparable plasma EnvIgA and IgG V1/V2 responses Greater magnitude in T-cellresponses do not necessarily translate in betterNAbresponses Conclusions II – Antibody Response (prior to week 48 boost)

  39. What Are the Changes in the Profile of AntibodyResponsesAfter - Protein (Grs. #1-3) and - NYVAC+Protein (Grs. #4/5) Boost atWeek 48 ?

  40. Neutralizing Antibody Titers All forTZM.BL After Boost at Week 48 Kruskal-Wallis Rank Sum Test

  41. ADCC Titers After Boost at Week 48 Kruskal-Wallis Rank Sum Test

  42. Durable IgG anti-Env Breadth to Week 51

  43. Plasma IgA Responses to TV1 and gp70 V1V2

  44. No DNA NYVAC-KC and NYVAC groups (NYVAC+gp120 boost) showed: Substantialtransientincrease in T-cellresponse Stablyelevated but no increase in antibodyresponse DNA groups (only gp120 boost) showed: - Slighttransientincrease in T-cellresponse - Substantialincrease in antibodyresponse Conclusions III – Vaccine-Induced Responses at Week 48 boost

  45. T-Cell Polarizing Vaccine Regimen Strong T-cell Polarization Phase Weak Induction of Antibody Late Increase of Antibody Response 100 T-cell Magnitude of Response 10 Antibody 0 1 2 5 7 8 12 Months DNA NYVAC Protein Protein

  46. Antibody Polarizing Vaccine Regimen Weak T-cell Polarization Phase Strong Antibody Polarization Phase Late Increase of T-cell Response 100 Magnitude of Response 10 T-cell Antibody 0 1 3 6 12 Months NYVAC NYVAC + Protein NYVAC + Protein NYVAC + Protein

  47. Future Plans – Optimization of vaccine regimens Acceleration of the induction of the antibodyresponse Acceleration of prime/boostimmunizationschedule of T-cellpolarizing vaccine regimens Circumvent the interference of potent vaccine-induced T-cellresponsewith the induction of potentantibodyresponse Development of immunizationregimensresulting in a synergisticeffectbetween T-cell and antibodypolarizing vaccine regimens Increasebreadth of T and B cellresponses

  48. Re-Design III: Broaden T Cell Responses Improve B Cell Responses

  49. MGARASVLSGGELDRWEKIRL RPGGKKKYKLKHIVWASRELE RFAVNPGLLETSEGCRQILGQ LQPSLQTGSEELRSLYNTPIVQNIQGQMVHQ CLASSIFIER 6 4 negative effect 3 no effect s(x) 5 1 3 Re-Design – T Cell Responses (CutHiVac, Replivax) 1. Initialization Align epitopes to reference sequence and find all single point amino acid substitutions 3. Optimization Los Alamos HIVDatabase Reference Sequence A. Assign each epitope a numericalvalue (=score) to assess its importance to be in the optimized immunogene C. Find a set of compatible epitopes with maximal total score. 2. Structur/Function Conservation (VLP) B) Analyse incompatibilities between epitopes A. Classify all substitutions according to their effect on the functionality of the protein D. Return immunogen sequence: Freeze all substitutions in reference sequence B. Remove all epitopes which induce “harmful” amino acid substitutions E. Remove all used epitopes Objective: Broaden /focus T cell responses by incorporaing T cell epitopes into Gag and maintain VLP formation (cross presentation)

  50. Re-Design – B Cell Responses (IAVI, Replivax) Single integrations Flp-In stable cell-lines Antibiotic selection for 25 days „Linked“ cellularEnv-library Phenotype Genotype • Syntheticbiologytechniquestogenerate different typesofEnvlibraries (randomized, SeqPer) and • … toefficientlysubclonelibsinto pQL13 enabling • inducibleexpressionofEnv; cotranslationalcouplingofselectionmarker; couplingofphenotypeandgenotype • HT generationofstablecell-lines (<10E5) bybulktransfectionofEnvlibrariesclonedinto pQL13 • FACS basedscreeningprocedure • bNMAbs, germlineversions • Transfection: pQL13 + helperplasmid • bnMAB binding Cells Laser • QL cloning pQL13 • FACS-sorting Env library Analysis by deep sequencing • NestedPCR Objective: Exploit bnMAbs / germ line versions to select Env‘s with new properties and the potential to induce broad neutralization

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