Tomáš Hanke Weatherall Institute of Molecular Medicine University of Oxford United Kingdom

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1 Immunogen Designs to Elicit Broad and Robust Cell Mediated Immune Responses: Introduction to the Problem and Design of Immunogenic Inserts for Inclusion in HIV-1 Vaccines Tomáš Hanke Weatherall Institute of Molecular Medicine University of Oxford United Kingdom

2 Effective HIV-1 vaccine may have to stimulate both Neutralizing Antibody & Cell-Mediated Immunity The challenges for induction of broadly NAb and T cells are Substantial Very different The rational, scientific approach is to tackle and solve these two problems separately.

3 Early Window of Opportunity 8% of new infections are seeded by a single virus Window of opportunity AT Haase Nature 464, (21)

4 Prophylactic CD8 + T Cells Have to Win Early Numbers reach protective threshold may require boosts CTL (+Ab) * Specificity transmitting virus early vs. late epitopes conserved vs. variable epitopes efficiently presented on HIV-1-infected cells * Breadth recognize multiple epitopes / epitope variants * Functionality proliferate, express effector functions killing / production of soluble fators (functional correlates of protection are not known) * Homing be at the right time at the right place * Good CD4 + T cell help/co-stimulation

5 T cell VACCINE design: HIV-1-derived immunogens/transgene products Vectors/formulations (attenuated viruses, liposome, emulsions etc.) Immunomodulators (stimulation of innate/adaptive immunity, recruitment of DC or effector cells, blockade of suppressive factors) Different viruses induce different qualities of T cells Different vaccine vectors will induce different qualities of T cells Different qualities of T cells may be required to control different viruses

6 T cell VACCINE design: HIV-1-derived immunogens/transgene products Vectors/formulations (attenuated viruses, liposome, emulsions etc.) Immunomodulators (stimulation of innate/adaptive immunity, recruitment of DC or effector cells, blockade of suppressive factors) T cell VACCINE delivery: Dose/number of deliveries Slow release/persistance Routes (i.m., i.d., s.c., mucosally, orally/multi-site) Devices (biojector, gene gun, microneedles, spray, patch etc.) Homologous or heterologous prime-boost regimens Vaccine design and delivery together affect the vaccine-induced T CELLs: Specificity/Depth Breadth Frequency Compartmentalization (αβ/γδ; CD4/CD8; Th1/Th2/Th17/Treg; E/EM/CM) Proliferative capacity Functionality (killing, production of soluble factors) Homing

7 Correlates of T Cell Protection are multi-parameter Specificity Breadth Vaccine-induced protective T cell responses (Poly)functionality Depth Proliferative capacity Clonotype Functional Avidity Frequency In blood Homing

8 HIV-1-Derived Immunogens Intrinsic immunogenicity contain known and yet unidentified epitopes improving specific epitopes may destroy others HIV-1 protein/region of origin known association with control variability immunodominance early vs late presentation on HIV-1-infected cells Conventional and unconventional [open] reading frames Direct vs. cross priming stability intracellular / secreted / forming particles maximize expression / cell toxicity Targeting to intracellular compartments, professional APC Safety (mimicking self proteins, using self, activating) Antigenicity particularly for subdominant epitopes

9 Antigenicity of Epitopes Key to the success of T cell vaccines CD4 L Y CD8 Is the epitope presented on HIV-1-infected cells = Are infected cells killed? CD4 L CD4 ELISPOT and ICS detection using [overlapping] peptides CD8 L Y Epitope Y L Y Y CD8 CD8 CD8 AP C Y CD8 CD8 T cell response induced during natural infection

10 The Biggest Challenge to Both T and B Cell Vaccine Development Are HIV-1 Diversity and Escape Korber et al. JVI 29

11 Specificity Heterologous vaccine regimens are getting better at inducing high frequencies of HIV-1 specific T cells However, these vaccine-induced T cells have to recognize multiple HIV-1 strains circulating in the population and emerging escape mutants within an infected individual HIV-1 escapes 5-75% of T cell responses generated during primary infection (CHAVI)

12 Ways to Tackle HIV-1 Diversity Mixed/heterologous immunogens Natural sequences with the shortest distance to other isolates in the clade or group Artificial central sequences (ancestral, consensus or centre-of-tree) Focus on the small founder population of transmitted viruses (look for common features) Mix of mosaic proteins (computer generated to maximize coverage of all known T cell epitopes including escape mutants) Conserved regions Fischer et al. Nature Medicine 13, 1-16 (27)

13 HIVconsv Immunogen Based on Conserved Regions of the HIV-1 Proteome The 14 most highly conserved regions of HIV-1 proteins Létourneau S. et al., PLoS ONE 2:e984 (27)

14 The HIVconsv Immunogen Alternating 4 major clades to ensure equal clade representation Clade Consensus to reflect variation within clades Each fragment just once to avoid possible immune interference Simple and relatively small to facilitate insertion into vectors Mutations in these regions are likely associated with fitness loss Altering natural (unprotective) hierarchy of epitope responses

15 HIVconsv Vaccines SLP adjuvanted peptides DREP/VREP Semliki Forest virus replicons Synthetic gene - Humanized codons ChAdV-63 attenuated chimp adenovirus BCG AERAS-41 Plasmid DNA naked DNA MVA attenuated poxvirus HAdV-5 STEP trial

16 Synthetic Long Peptides amino acids No competing antigens derived from a vector Processed by APC vs. direct binding to MHC class I good help No junctional epitopes (grey boxes) Total of 46 peptides divided into six sub-pools delivered to six anatomically separate sites Adjuvantation - Imiquimod topically (Aldara cream) - Montanide ISA-51 emulsion

17 Six Read Out HIVconsv Peptide Pools ~ 35 peptides (15/11 AA) in each pool Peptide Pools

18 SLP.HIVconsv Induce Broader T Cell Responses Compared to Single ORF Genetic Vaccines 2 Mean responses to individual pools 1-6 (purple pool 1 is gag) Ex vivo IFN-γ SFU/1 6 PBMC Mean total S S C M V D D D A M S S C M V D DNA.HIVconsv; A HAdV5.HIVconsv; M MVA.HIVconsv; S SLP.HIVconsv; C ChAdV63.HIVconsv; V VREP.HIVconsv

19 SLP.HIVconsv-Boosted Broad, Polyfunctional CD8 T Cells Broad Median 4 Gag CD8 T cell epitopes Median 12 CD8 T cell epitopes Median 25 identified epitopes total Proliferate Day DDDAMSSCMV ONE OZONE OCTAVIA Pool 1 ONE Polyfunctional 1, 2 and 3 functions d47 d497 d59 CD Ø OZONE CFSE OCTAVIA IFN-γ, TNF-α and IL-2

20 Even Highly Complex Regimen Continues To Recruit New T Cell Clonotypes Mamu-A*1/CM9 tetramer+cd8+cd3+ -sorted cells T cell receptor sequences of individual responding T cell clones DDDAM DDDAMSSCMV Public (coloured) vs. private clonotypes

21 Conserved Fragments Are Not Immunologically Inert Total of 1112 CD8 T cell epitopes smaller than 12 AA in the Los Alamos National Laboratory database (27) Each fragment contains at least one known epitope HIVconsv contains 27 (24%) Of these 27: 192 (71%) perfect match 59 (22%) differ by one AA Similar coverage to the mosaic approach

22 Ex vivo Immune Responses to HIVconsv Peptide (15/11) Pools in Chinese MSM Patients (n=3) Frequency of responders Magnitude of recognition Peptide Pools

23 Conserved regions of HIV-1 proteome are immunogenic in humans How efficiently HIVconsvinduced T cells recognize and kill HIV-1-infected cells?

24 HIV-CORE (Conserved Regions) Clinical Trials in Oxford Using HIVconsv Vaccines HIV-CORE1 PATIENTS ON HAART Jan 21 HIV-CORE2 HEALTHY VOLUNTEERS Oct 21 D DNA.HIVconsv; M MVA.HIVconsv; C ChAdV63.HIVconsv

25 HIV-CORE1 HIV-1-infected patients on HAART Ex vivo IFN-γ ELISPOT using 6 peptide pools 15/11 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 Day Day Day Day SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 SFU/million PBMCs S SP 1 SP 2 SP 3 SP 4 SP 5 SP 6 Day Day Day Day MVA.HIVconsv low dose Peptide Pools

26 HIV-CORE1 HIV-1-infected patients on HAART Ex vivo IFN-γ ELISPOT using individual peptides SFU/million PBMCs SFU/million PBMCs Screen Day 23 Screen Day SFU/million PBMCs SFU/million PBMCs Screen Day Day SFU/million PBMCs Screen Day SFU/million PBMCs Screen Day MVA.HIVconsv low dose

27 HIVconsv Conclusions HIVconsv is unique immunogen, which Was designed to induce cross-clade T cell responses Focuses responses on parts of HIV-1 that cannot be easily mutated without a likely significant fitness cost (Achilles heel of HIV-1) Redirects responses relative to natural infection (unprotective) Is simple in design and delivery SLP.HIVconsv vaccine formulation Boosts broad T cell responses superior in breadth to sorf vaccines The multi-site delivery merits evaluation in humans More complex regimens such as DDDCM Are more immunogenic than simple regimens Simple regimens were not hugely successful in humans so far Require multiple immunizations - other vaccine strategies/haart are more complex These regimens may prove the T cell concept

28 Which vaccine strategy will provide the best control of HIV-1 infection? Definitive answer can only come from efficacy trials in humans

29 ACKNOWLEDGEMENTS UNIVERSITY OF OXFORD MAXIMILLIAN ROSARIO EUNG-JUN IM ANNE BRIDGEMAN SVEN LETOURNEAU NICOLA BORTHWICK HONGBING YANG GEMMA HANCOCK ANDREW MCMICHAEL LUCY DORRELL XIAO-NING XU UNIVERSITY OF LEIDEN ESTHER QUAKKELLAR JAN W. DRIJFHOUT CORNELIS MELIEF KAROLINSKA INSTITUTE MARIA KNUDSEN KARL LJUNGBERG PETER LILJESTROM LOS ALAMOS NATIONAL LABORATORY BETTE KORBER OKAIROS VIRGINIA AMMENDOLA ALFREDO NICOSIA STEFANO COLLOCA VACCINE RESEARCH CENTER MAIRE QUIGLEY BRENNA HILL DANIEL DOUEK CARDIFF UNIVERSITY MEDICAL CENTRE DAVID PRICE