Impact of RNA- and Codon Optimization on Safety and Immunogenicity of HIV Candidate Vaccines

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Cory Roberts
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Molecular Microbiology and Gene Therapy Unit Institute

1 Impact of RNA- and Codon Optimization on Safety and Immunogenicity of HIV Candidate Vaccines Ralf Wagner Molecular Microbiology and Gene Therapy Unit Institute of Medical Microbiology and Hygiene University of Regensburg

molecular virology Immune response neutralizing AB mucosal immunity CD4+ T cell response CD8+ CTLs broad, cross clade, high quality, polyfunctional long term memory Delivery /

2 Integrated Vaccine Strategies Targets of immune response HIV Clades A (Africa) B (Europe, N & S-America) C (Asia,. ) E (Asia) F (Africa) G (South America, Africa) + + envelope proteins: Env structural proteins: Gag, Pol regulatory proteins: Tat, Rev, Nef accessory proteins: Vpr immunogen design molecular virology Immune response neutralizing AB mucosal immunity CD4+ T cell response CD8+ CTLs broad, cross clade, high quality, polyfunctional long term memory Delivery / Combination of Multivalent HIV/AIDS Vaccine Strategies Proteins Peptides DNA Viral Vectors Pseudovirions rad, NYVAC Novel Adjuvants (CpG, PEI, MF59) Bacterial Vectors? basic immunology / mice preclinic / rhesus macaques clinic / phase 1/2 (Bojak, Deml & Wagner, 22) Overview

Pr55 gag Necessary Component of HIV Vaccines Pr55 gag Intracellular targeting Gag-assembly Nc transport p17 MA p24 CA p7 p6 p6* PR RT Gag-assembly Capsid-reorganization Cyp A incorporation

,1999, Paulus et al., 24; Ludwig et al., 27) Patients with strong Gag-Helper/CTL-Response control acute HIV infection better (Rosenberg et al., 1997; Ogg et al.

, 1999, many others) Recombinant and chimeric Gag VLPs induce Th1 / CTL responses in Balb/C mice; Cross priming (Wagner et al., 1993, 1994, 1996; Bojak et al., 22, 23, 24, Deml et al.

3 Pr55 gag Necessary Component of HIV Vaccines Pr55 gag Intracellular targeting Gag-assembly Nc transport p17 MA p24 CA p7 p6 p6* PR RT Gag-assembly Capsid-reorganization Cyp A incorporation Capsid disassembly RNA-packaging Reverse transcription particle release vpr incorporation PR-activation (IC 5 13 µm) Pr16 gag-pol (Beissinger et al., 1996, Fitzon et al.,1999, Paulus et al.,1999, Paulus et al., 24; Ludwig et al., 27) Patients with strong Gag-Helper/CTL-Response control acute HIV infection better (Rosenberg et al., 1997; Ogg et al., 1999, many others) LTNP HIV infected individuals control HIV replication by T cells recognizing highly conserved epitopes within Gag (Harrer et al., 1998, Wagner et al., 1999, many others) Recombinant and chimeric Gag VLPs induce Th1 / CTL responses in Balb/C mice; Cross priming (Wagner et al., 1993, 1994, 1996; Bojak et al., 22, 23, 24, Deml et al.,1997a,b, 25) Gag VLP partially protective in Rhesus monkey model (Wagner et al., 1998; Notka et al., 1999a, b, Ludwig & Wagner, 27) Gag specific T-cells have been linked to better control of HIV replication Rationals for Immunogen Selection Molecular Virology

Late Lentiviral Gene Expression is Complex early RNAs late RNAs AAA RRE HIV-1 CRM1 NES Rev Ran GTP AAA Rev-dependent RNA export Inhibitory Sequences Splice Sites Cytoplasm cytoplasmat.

4 Late Lentiviral Gene Expression is Complex early RNAs late RNAs AAA RRE HIV-1 CRM1 NES Rev Ran GTP AAA Rev-dependent RNA export Inhibitory Sequences Splice Sites Cytoplasm cytoplasmat. RNAs ~9 kb ~4 kb ~2 kb late + Rev -early strong splice donor and weak splice acceptors capture RNAs within splicing machinery cis-located AU-rich repressor elements (INS) retain RNAs within the nucleus 1% 27% AAAAAA splicing machinery & INS binding proteins PABP? hnrnp C? AU-rich INS AAA Intrinsic risks: Ψ-sequence, recombination, RRE/Rev,... Bypassing Rev/RRE Regulation

5 Understanding Late Lentiviral Gene Expression early RNAs late RNAs AAA RRE HIV-1 Rev-dependent RNA export CRM1 NES Rev Ran GTP AAA Inhibitory Sequences Splice Sites Cytoplasm AA cytoplasmat. RNAs mammals HIV-1 late Ala GCC GCA Arg AGG AGA ~9 AsnkB AAC AAT Cys TGC TGT ~4 kb Gln CAG CAA Glu GAG GAA ~2 kb Gly GGC GGA + Rev -early Influence of various cis-acting sequences [INS, UTR (SD), RRE (SA)] wtgag UTR-wtgag wtgag-rre UTR-wtgag-RRE ΔSD1-wtgag-RRE syngag UTR-syngag syngag-rre UTR-syngag-RRE Bypassing Rev/RRE Regulation Thema

Facilitating Late Lentiviral Gene Expression 1 2 2 syn-gag? cis active repressor degradation sequenzes wt - gag splicing machinery 3?

6 Facilitating Late Lentiviral Gene Expression syn-gag? cis active repressor degradation sequenzes wt - gag splicing machinery 3? ψ SD UTR RRE wt-gag constitutive nc export REV ran CRM1 REV RRE Northern Rev gag actin N syngag-rre UTR-wtgag-RRE UTR-wtgag-RRE C N C N C N C ΔSD1-wtgag-RRE RNA-and Codon Optimization

7 Consequencs of Modulated Late Lentiviral Gene Expression syn-gag? cis active repressor degradation sequenzes wt - gag splicing machinery 3? ψ SD UTR RRE wt-gag constitutive nc export REV ran CRM1 REV RRE HIV Vaccines facilitated gag-pol expression RNA nuclear export 5x no Rev, no RRE no 5 -UTR, no Ψ Risk for Recombination RNA Packaging Increased safety? Increased yields Enhanced Immunogenicity? Protein Production (CHO, PerC6) DNA Vaccines Bacterial DNA Delivery (Listeria) Adenoviral Vectors Herpesviral Vectors (EHV) Lentiviral Vectors Poxviral Vectors? Graf et al., 2; Wagner et al., 2; Deml et al., 21; Bojak et al., 23, Wild et al., 24, 27 RNA-and Codon Optimization

8 Impact of RNA & Codon Optimization in Poxviral Vectors wt - gag hu opt - gag hu opt cpg max -gag vaccinia - gag M NYVAC-Gag DNA-Gag WB - Antibody: CD-4/1 (anti p24 CA) NYVAC: No major impact of RNA & codon optimization on transgene expression DNA: Significant impact of RNA & codon optimization on transgene expression Conclusion: RNA- and codon-optimization recommended if various vector systems shall be compared / combined RNA-and Codon Optimization

9 Reduced Risk for Generating Replication Competent Recombinants...following RNA- and Codon-Optimization 2-Plasmid-system Transfervector gagpol - /Env + packaging construct wt vs syn cotransfection and harvest particles Permissive CD4+ cell culture system Replication post recombination Tat driven SEAP expression CMV BGHpA 1 E CMV U3 R U5 Ψ UTR * Ψ gag gag Δ pol pol Rev Recombination RCR vif rev1 vpr vpx RRE env tat1 tat2 Δ SFFV-Pr Δ Δ rev2 U3 R U5 * BGHpAd GFP SEAP non-replication competent HIV-reporter SEAP Aktivität wt syn syngp Sgp Δ 2 Mock days post transfection Safety Thema Profiles

Reduced Replicative Capacity of Engineered Recombinant HIVs chimeric viruses HIV-1 LTR gag codon-modified sequence pol

HXCS 5 gag (bp 154-83) HXNS 5 gag (bp 337-83) HXSA 3 gag (bp 83-1333) HX1 ----- Hxsyn gag/pol (bp 154-1333.

c HXCS HXNS HXSA HX1 HXsyn 12 h p.t. 9 kb 4 kb 2 kb 9 kb 4 kb 2 kb (Wagner et al., 2; Steck et al., in prep.

10 Reduced Replicative Capacity of Engineered Recombinant HIVs chimeric viruses HIV-1 LTR gag codon-modified sequence pol Cla I Nsp V Spe I Apa I Bcl I frameshift en v wild-type sequence LTR Mutant: Codon-optimized region (gag ATG=112): HXCS 5 gag (bp ) HXNS 5 gag (bp ) HXSA 3 gag (bp ) HX Hxsyn gag/pol (bp ) Processing & maturation cells viral particles HXCS HXNS HXSA HX1 HXsyn Gag p24 Gag p24 env RNA splicing & export n c HXCS HXNS HXSA HX1 HXsyn 12 h p.t. 9 kb 4 kb 2 kb 9 kb 4 kb 2 kb (Wagner et al., 2; Steck et al., in prep.) Viral infectivity p24 [ng/ml] Replication kinetics (MT4) HXSA HX days Safety Thema Profiles

11 RNA and Codon Optimization DNA Vaccines Increased immunogenicity in BALB/c mice isotype titers IgG1 IgG2a pg pg im im wt-gag syngag wt-gag syngag cytokine secretion (pg/ml) pg IL-5 IFN-γ pg im im wt-gag syngag wt-gag syngag NET specific lysis (%) wt- gag (p.g.) syngag (p.g.) wt- gag (i.m.) syngag (i.m.) mock Wt-Gag: No Gag specific antibodies, IFN-g or CTLs detected Syn Gag: High levels of gag specific antibodies and IFN-g; CTL induction 1 Im (Th1, CTL) >>> pg (Th2) E/T Immunogenicity Thema

Molecular Epidemiology: Prerequisite for Immunogen Design India China C B Asia C C B B C B C C B B Prevalence Incidence Virus

pol vpr gag env vpu Graf et al., 1998, Su et al.

Wagner / Wolf / Cobra Env C (CN54) DNA-C GMP, B, T, I, Fill.

12 Molecular Epidemiology: Prerequisite for Immunogen Design India China C B Asia C C B B C B C C B B Prevalence Incidence Virus population Regional clustering Host genetic background Subcloning pcr-script Sequence analysis of C (CN54) tev vif tat rev LTR LTR pol vpr gag env vpu Graf et al., 1998, Su et al., 2 Clade Delivery Status Developer / Manufacturer GagPolNef C (CN54) DNA-C GMP, B, T, I, Fill. Wagner / Wolf / Cobra Env C (CN54) DNA-C GMP, B, T, I, Fill. Wagner / Wolf / Cobra GagPolNef / Env C (CN54) NYVAC-C GMP, B, T, I, Fill. Sanofi Pasteur GagPolNef / Env C (CN54) MVA-C GMP in progress Esteban GagPolNef / Env C (CN54) vtt-c in progress 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 cgmp Immunogens Thema

13 In vivo Studies in Rhesus Monkeys Mimicking a Human Phase I Trial CMI CMI CMI CMI CMI CMI NYVAC/C i.m. NYVAC/C i.m. CMI CMI CMI CMI CMI CMI CMI CMI CMI monkeys per group DNA/C DNA/C NYVAC/C i.m. NYVAC/C i.m. Preclinical Trials

14 In vivo Studies in Rhesus Monkeys Mimicking a Human Phase I Trial Antigen-specific IFNγ release at week 24 (4 weeks after 1st NYVAC) number of peptide pools Rl187 Rl432 Rl / NYVAC-C DNA-C / NYVAC-C C54 Rl313 Rl457 C24 Rl346 Rl1147 C28 2/1 respond 8/1 respond several pools recognised Rl468 Rl543 C136 Rl45 Rl474 Rl12165 C145 Rl467 Rl58 C86 gag1 gag2 pol1 pol2 pol3 env1 env2 Preclinical Trials P. Mooij, J. Heeney et al.

15 In vivo Studies in Rhesus Monkeys / NYVAC-C DNA-C / NYVAC-C Ri346 C24 Ri Ri12165 C145 Ri58 Ri474 Ri45 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 number of responders week IFNγ IL-2 IL-4 C28 Ri1147 Ri346 C24 Ri12133 Ri IFNγ IL-2 IL-4 Ri12165 C145 Ri58 Ri474 Ri45 Ri543 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 gag 1 gag 2 pol 1 pol 2 pol 3 env 1 env 2 number of responders week 26 IFNγ IL-2 IL-4 IFNγ IL-2 IL-4 Preclinical Trials P. Mooij, J. Heeney et al.

16 EV2: Study Design HIV Negative Volunteers Objective: Determine the impact of DNA-C versus NYVAC-C alone on priming T-cell responses Study design: 2 x 2 HIV negative volunteers A. DNA-C prime (W, W4; 4mg im), NYVAC-C boost (W2, W24; 2x1E7 im) B. NYVAC-C (W2, W24; 2x1E7 im) London (MRC; J.Weber, S. McCormack), Lausanne (CHUV; G. Pantaleo) Readout (Lausanne): Several time points ELISPOT analysis (γifn, IL2) FACS analysis (CD4, CD8, CCR7, IL2, γifn) T cell phenotype / function Epitope mapping Phase I Clinical Trial

17 EV2: Percentage of Responders (γifn+ T cells) A Percentage of responder NYVAC C Alone (n=15) P =.3 DNA C + NYVAC C (n=2) B Percentage of responders P <.1 P =.1 P <.1 P =.19 NYVAC C alone DNA C + NYVAC C W W5 W2 W24 W26 W28 W48 DNA-C NYVAC-C Higher percentage of responders in DNA-C prime / NYVAC-C boost group (>9%) compared to NYVAC-C group (<4%) Durability DNA-C / NYVAC-C >> NYVAC-C Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

18 EV2: Magnitude of T cell Responses (γifn) A DNA C + NYVAC C SFU / 1 6 cells ES1 W W5 W2 W24 W26 W28 W ES2 W W5 W2 W24 W26 W28 W ES8 W W5 W2 W24 W26 W28 W ES1 W W5 W2 W24 W26 W28 W ES11 W W5 W2 W24 W26 W28 W48 SFU / 1 6 cells ES23 W W5 W2 W24 W26 W28 W W ES24 W5 W2 W24 W26 W28 W ES26 W W5 W2 W24 W26 W28 W EU3 W W5 W2 W24 W26 W28 W EU5 W W5 W2 W24 W26 W28 W48 SFU / 1 6 cells EU9 W W5 W2 W24 W26 W28 W EU11 W W5 W2 W24 W26 W28 W EU12 W W5 W2 W24 W26 W28 W48 DNA C (W and W4) NYVAC C (W2 and W24) Neg GAG 1 GAG 2 GAG/POL POL1 POL2 NEF ENV NYVAC C Alone SFU / 1 6 cells ES3 W W5 W2 W24 W26 W28 W ES4 W W5 W2 W24 W26 W28 W ES6 W W5 W2 W24 W26 W28 W ES2 W W5 W2 W24 W26 W28 W EU7 W W5 W2 W24 W26 W28 W48 Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

19 EV2: Magnitude of T cell Responses (γifn) B Env Responses C Gag/Pol/Nef Responses 1 DNA C + NYVAC C 1 NYVAC C alone 4 SFU/1 6 cells SFU/1 6 cells Week Week Week 26 Week 28 Week 26 Week 28 DNA C + NYVAC C NYVAC C alone D Percentage NYVAC C DNA C + NYVAC C Higher magnitude of T cell reponses in DNA-C prime / NYVAC-C boost group (>9%) compared to NYVAC-C group (<4%) Env >> GagPolNef SFU/1 6 cells Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

20 EV2: Functional Analysis of T cell Responses A Volunteer # EU11, DNA C + NYVAC C Gated on CD4 T cells IL-2 % % % TNF-α.2% % % CD4.34% Neg CD4 and CD8 responses are polyfunctional.4%.7%.11%.9 6.6% Env (IL-2, γifn, TNFα) IFN-γ.3% IFN-γ.1% CFSE B Volunteer # EU11, DNA C + NYVAC C Gated on CD8 T cells %.1% % 1.1% %.1% IL-2 % TNF-α % CD17a % CD8 Neg.7%.3% %.8% 1.94% Env.22.3%.17 IFN-γ IFN-γ IFN-γ CFSE Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

EV2: Epitope specific T cell Responses A 8 number of epitopes C Gated on CD8 T cells #ES26 #ES2 Number of ENV epitopes 6 4 2 ES2 ES8

14.14.2.3 -----YSENSSEYY- ------SENSSEYYR.1.1 IFN-γ IL-2.1.1 FYRLDIVPLTKKNYS ENVTENFNMWKNEMV.2.4.3.15.2.5 IFN-γ D pentamer #EU16 HLA-A*11-YSENSSEYY.

21 EV2: Epitope specific T cell Responses A 8 number of epitopes C Gated on CD8 T cells #ES26 #ES2 Number of ENV epitopes ES2 ES8 ES16 ES2 ES21 ES23 ES26 EU3 EU11 IL Unstimulated LTKKNYSENSSEYYR ----NYSENSSEY-- B Gated on CD4 T cells #ES26 #ES23 NEG NEG YSENSSEYY SENSSEYYR.1.1 IFN-γ IL FYRLDIVPLTKKNYS ENVTENFNMWKNEMV IFN-γ D pentamer #EU16 HLA-A*11-YSENSSEYY.9 CD8 T cell responses are broad (mean 4,2 epitopes) and polyfunctional Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

22 EV2: Longevity of T cell Responses (γifn, IL2) A NYVAC C Alone DNA C + NYVAC C SFU / 1 6 cells # ES4 * * * W28 W48 W # ES2 # ES8 #ES * * 45 * * * W28 W48 W72 W28 W48 W72 W28 W48 W72 6 * * * Neg GAG 1 GAG 2 GAG/POL POL1 POL2 NEF ENV B Responses are durable SFU / 1 6 cells (mean±se) DNA C + NYVAC C NYVAC C alone W28 W48 W72 7% positivity at W72 (DNA-C / NYVAC-C) ~3 SFU / 1E6 cells at W72 Phase I Clinical Trial A. Harrari, G.Pantaleo et al.

23 Dominant Env- / Mitigated Gag Responses Possible explanations: Intracellular trafficking / localisation: gp12 secreted, GagPolNef not Gp12: 55 kda + glycosylation; GPN: 16 kda non glycosylated Gag expression >>> GagPolNef; SIV-Gag >> HIV-Gag Env vs GagPolNef: competition for HLA epitope presentation Modifications: Altered immunization regimen: 3 x DNA-C (mixture DNA-GPN and DNA- Env) followed by 1 x NYVAC-C (EV3) instead 2 x DNA-C followed 2 x NYVAC-C (EV2) At least 2 different NYVAC viruses: Env + GagPolNef instead Env / GagPolNef; different inoculation sites Increase immunogen processing and epitope presentation DRIP Improve cross-presentation: VLP / secreted Gag derivatives Targeting to DCs (e.g. Dec25 targeting, FcR targeting) Incorporate molecular adjuvants (CD4L, CTB...) Immunogen Design

24 Outlook: BMGF-PTVDC Discovery Group Myr- (A) ΔFS Pr - (D N) scnef Gag Pr RT-N C N RT-C RT Myr- (A) Myr+ (A G) Myr- (A) Myr+ (A G) Myr+ (A G) Signal peptide Gag Gag +FS Gag Pr - (D N) ΔNef Gag Gag Gag +FS +FS +FS +FS Pr - (D N) scnef Pr RT-N C N RT-C RT Pr - (D N) scnef Pr RT-N C N RT-C RT Pr RT-N RT-C RT Pr - (D N) ΔNef Pr RT-N RT-C RT Pr - (D N) Pr RT-N RT-C RT scdec25-ab Gag reference construct allow ribosomal frameshifting (Gag 95 / PolNef 5) allow ribosomal frameshifting (Gag 95 / PolNef 5), VLP budding allow ribosomal frameshifting No NEF, (Gag 95 / Pol 5) allow ribosomal frameshifting No NEF, VLP budding 2 distinct ORFs secretion, DC targeting best in class: increase expression & improve access to DRIP pathway Immunogen design

25 What we have achieved Where we need to go Targets of immune response HIV Clades A (Africa) B (Europe, N & S-America) C (Asia, Africa ) E (Asia) F (Africa) G (South America, Africa) + + envelope proteins: Env structural proteins: Gag, Pol regulatory proteins: Tat, Rev, Nef accessory proteins: Vpr immunogen design molecular virology Immune response neutralizing AB mucosal immunity CD4+ T cell response CD8+ CTLs broad, cross clade, high quality, polyfunctional long term memory Delivery / Combination of Multivalent HIV/AIDS Vaccine Strategies Proteins Peptides DNA Viral Vectors Novel Adjuvants (CpG, PEI, MF59)? basic immunology / mice preclinic / rhesus macaques clinic / phase 1/2 Pseudovirions Ad, NYVAC Bacterial Vectors Summary

26 Acknowledgements Universität Regensburg RIMMH, Hans Wolf Christine Ludwig Benedikt Asbach Asli Bauer Andi Alex Kliche Thomas Benen Ruth Kapzan Tim Henrik Bruun Kristina Schilling Jens Wild Veronika Wanninger Denijal Kosovac Helga Hofmann Josef Köstler Yvonne Ködel Katharina Böckl Simon Bredl Nikole Kirner Supported by: EC 6th Framework CHIVAC, EUROVAC, DECVAC (GENEART) BMGF PTVDC, VDAC NIH HIVRAD BMBF HIVCOMPMET Bavarian Res. Foundation Hektor Foundation BPRC (NL) Jonathan Heeney Petra Mooij Sunita Balla Sanofi Pasteur Jim Tartaglia Marie-Joelle Frachette CAPM (China) Shao Yiming, Ling Su CHUV, CH Guiseppe Pantaleo Alexandre Harrari Pierre A. Bart MRC, UK Jonathan Weber Sheena McCormac Thema

Trial Objectives immunogenicity safety

Trial Objectives immunogenicity safety Optimal priming of poxvirus vector (NYVAC)-based HIV vaccine regimens requires 3 DNA injections. Long term Results of the EV03/ANRS Vac20 Phase I/II Trial. Yves Levy, Kim Ellefsen, Wolfgang Stöehr, Pierre-Alexandre