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

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Presentation on theme: "New Strategies Towards Vaccine Design Challenging HIV Ralf Wagner Univ. Prof. for Medical Microbiology and Gene Therapy, University of Regensburg."— Presentation transcript:

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

2 immunogen design molecular virology ex vivo analysis basic in vivo / mice preclinic / rhesus macaques clinic / phase 1/2/3 envelope proteins: Env structural proteins: Gag, Pol regulatory proteins: Tat, Rev, Nef accessory proteins: Vpr Targets of immune response ++ Peptides DNA Viral Vectors Bacterial Vectors Pseudovirions Adjuvants ( CpG, PEI, MF59 ) Proteins Delivery / Combination of Multivalent HIV/AIDS Vaccine Strategies ? rAd, NYVAC 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 Immune response A (Africa) B (Europe, N & S-America) C (Asia, Africa …) E (Asia) F (Africa) G (South America, Africa) HIV Clades Starting point

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

4 Knowledge on specific targets/ epitopes - Control of virus replication (35)(1)(22)(8) HLA-B14 CTL-Epitope conservative non- conservative Infectivity % spec. Lysis D R F Y K T L R A E K Y F R S/ A V K G W Y R Q I F H Y F Focus IR to conserved proteins and epitopes limits immunological escape True for both T and B cell responses DFG

5 CRM1 ran REV RRE degradation wt - gag cis active repressor sequenzes UTR SD  REV RRE splicing- machinery ? wt-gag AA Human HIV-1 AlaGCC GCA ArgAGG AGA AsnAAC AAT CysTGC TGT GlnCAG CAA GluGAG GAA GlyGGC GGA UTR-wtgag-RRE gag-RRE UTR-wtgag-RRE CCCCNNNN Northern constitutive export syn-gag syngag Knowledge on virus replication (I) BMBF

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

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

8 Sequence analysis of C (CN54) LTR gag pol vif vpr vpu env rev tat tev nef CladeChassisStatusDeveloper / Manufacturer GagPolNefC (CN54)DNA-CGMP, B, T, I, Fill.UREG / Cobra EnvC (CN54) DNA-CGMP, B, T, I, Fill. UREG / Cobra GagPolNef / EnvC (CN54)NYVAC-CGMP, B, T, I, Fill.Sanofi Pasteur GagPolNef / EnvC (CN54)MVA-CGMP, B, T, I, FillEsteban GagPolNef / EnvC (CN54)vTT-CGMP, B, T, I, FillY. Shao / S-CDC GagPolNef / EnvB NYVAC-BGMP, B, T, I, Fill.Sanofi Pasteur GagPolNef / EnvB MVA-BGMP, B, T, I, Fill. Esteban Gene Design, Protein Design and Vectors PrRT-NCNRT-CRT Myr- (A)ΔFSPr - (D  N)scNef Gag Env gp kDa readthrough polyprotein, cytoplasmic, not processed soluble secreted monomeric gp120 RNA and codon optimized

9 Clinical Analysis - Responders (  IFN + T cells) 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 Percentage of responders DNA C + NYVAC C NYVAC C alone P < P = P = P < DNA-C NYVAC-C Study design: 2 x 20 HIV negative volunteers London (MRC; J.Weber, S. McCormack), Lausanne (CHUV; G. Pantaleo)

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

11 #SFC/10e6 PBMC EnvGag PolNef Human EnvGag PolNef Rhesus Magnitude of Env >> Gag Specific T Cell Responses Clinical Analysis - Responders (  IFN + T cells)

12 Re-Design I: T Cell Immunogens Immunogen Formulation Poxviral Vector (Chassis) TVDC

13 NYVAC-C DNA-C ? MHC-II MHC-I CTL T No co-stimulatory signals No MHC II presentation No T cell help NYVAC-C DNA-C ? MHC-II MHC-I CTL THTH DC CTL THTH ? 100 nm Pr55 Gag cell membrane translation VLP particle release membrane targeting Pr55 Gag 200 nm Myr+ (A  G)+FS PrRT-NCNRT-CRT scNef Gag Pr - (D  N) Re-Design – T cell Immunogens

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

15 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 Re-Design – T Cell Immunogens & Plasmid

16 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 Re-Design – Immunogen Formulation

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

18  Re-introduction of -1 ribosomal frameshift leads to ~ 20x increased Gag expression, NOT on cost of PolNef  Gag alone expresses equally well  Level of Gag expression and VLP formation crucial to level of induced T cell responses  Co-administration of Env suppresses Gag specific T cell responses Recommendations:  DNA: 3 plasmids encoding Gag, PolNef and Env optimize molar ratios (trivalent)  NYVAC: 2 viruses expressing GagPolNef and Env (bivalent) Conclusions Immunogens - Formulation

19 ABC N M K FE P DHJ L G OI aprox. 15Kb Fragmented genesIntact genes NYVAC aprox. 10Kb NYVACKC Re-Design – Poxviral Vector Design  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 Myr+ (A  G)+FS PrRT-NCNRT-CRT scNef Gag Pr - (D  N) Env gp140 bivalent 2 viruses Collaboration Bert Jacobs, Karen Kibler

20 Re-Design II: Assess immunogenicity of next generation immunogens in NHP „Optimal“ immunization schedule TVDC

21 NHP Study Objectives: TVDC  To evaluate the immunogenicity of:  2 nd generation immunogens, DNA and NYVAC  replication competent NYVAC-KC and replication deficient 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 antibody responses

22 2 nd 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 NYVAC-C-KC (IM) NYVAC-C (IM) NYVAC-C + Protein (IM ) DNA-C (IM) NYVAC-C-KC (Scar) Protein NYVAC-C-KC (IM) NYVAC-C (IM) ICS ELISpot 48 Ab Re-Design – Opt Clinical Study Protocol (Rhesus) Protein NYVAC-C + Protein (IM ) N G DNA prime groups No DNA groups

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 Group 1 – NYVAC-C-KC 2 wks Post 1st NYVAC Week 22 NYVAC-C KC: replication competent in human cells SFUs per 10 6 Blood Mononuclear Cells IFN-  ELISpot Week 50Week 57 Week 22 Week 50 Week 57 P540P566P537P555P532P534P547P549 P568P544P536P552P546P558P562P564 P539P554P567P545P557P559P563P wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost Week 48 Week 50 Week 57 Group 2 – NYVAC-C-KC IM Group 3 – NYVAC-C IM NYVAC-C KC: replication defective in human cells 2 wks Post 1st NYVAC 16 wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost 7wks Post 3rd Protein Boost 16 wks Post 2nd Protein Boost 2 wks Post 3rd Protein Boost P539P554P567P545P557P559P563P548 P539P554P567P545P557P559P563P548 P539P554P567P545P557P559P563P548 Week 22 2 wks Post 1st NYVAC P568P544P536P552P546P558P562P564 P568P544P536P552P546P558P562P564 P568P544P536P552P546P558P562P564 Week 48 7 wks Post 3rd Protein Boost P540P566P537P555P532P534P547P549 P540P566P537P555P532P534P547P549 P540P566P537P555P532P534P547P549 7 wks Post 3rd Protein Boost Week D3/N-KC(im)/3P D3/N(im)/3P D3/N-KC(scar)/3P

27 T-Cell Responses After NYVAC+gp120 Immunization Group 4 – NYVAC-C-KC 7 wks Post 2 nd NYVAC-C-KC (IM) + 3 NYVAC-C-KC/Protein Boost Week 28 Group 5 – NYVAC-C NYVAC-C KC: replication competent in human cells SFUs per 10 6 Blood Mononuclear Cells NYVAC-C: replication defective in human cells IFN-  ELISpot P538P543P530P565P541P531P535P533 P560P569P553P542P561P556P550P wks Post 2 nd NYVAC-C-KC (IM) + 2 NYVAC-C-KC/Protein Boost Week 50 2 wks Post 2 nd NYVAC-C-KC (IM) + 3 NYVAC-C-KC/Protein Boost Week 57 7 wks Post 2 nd NYVAC-C (IM) + 3 NYVAC-C/Protein Boost Week wks Post 2 nd NYVAC-C (IM) + 2 NYVAC-C/Protein Boost Week 50 2 wks Post 2 nd NYVAC-C (IM) + 3 NYVAC-C/Protein Boost Week 57 P560P569P553P542P561P556P550P551P560P569P553P542P561P556P550P551 P560P569P553P542P561P556P550P551 4 wks Post 2 nd NYVAC-C (IM) + 2 NYVAC-C/Protein Boost Week 48 P538P543P530P565P541P531P535P533P538P543P530P565P541P531P535P533P538P543P530P565P541P531P535P533 4 wks Post 2 nd NYVAC-C-KC (IM) + 2 NYVAC-C-KC/Protein Boost Week 48 Nkc2NkcP3 Primary Immunization Boost N2NP3 Primary Immunization Boost

28 Key time points:  Groups #1-3 (DNA prime) at week 22  Groups #4,5 (no DNA) at week 28 ValueExact P 1.Group 1 vs. Group 2 vs. Group Group 4 vs. Group Group 4 vs. Groups1-3 <0.001 Group Comparison of IFN-  ELISpot Responses by Wilcoxon Rank Sum Test

29 CD4+ and CD8+ T Cell Function Groups G 2 T G 1-3 G 4, 5 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 Immunization Regimen (DNA Prime vs. No DNA) Associated to Better Antibody Response?

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 cells Kruskal-Wallis Rank Sum Test HIV Isolate BaL Log10 Titer MW965.26SF162.LSSS1196.1Bx08.16MN.3TV Groups 1-3-DNA Prime Week 36 (after 2 nd protein) Groups 4,5-No DNA Week 26 (after 2 nd NYVAC/protein)

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

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

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

38  NYVAC-KC and NYVAC plus gp120 groups compared to DNA prime groups showed:  Greater NAb activity against Tier 1 viruses and SHIVs (TZM.bl assay)  Greater ADCC activity  Earlier and greater cross-clade IgG antibody titers (more than 6 Mo after 1st immunization)  Earlier, but comparable plasma Env IgA and IgG V1/V2 responses  Greater magnitude in T-cell responses do not necessarily translate in better NAb responses Conclusions II – Antibody Response (prior to week 48 boost)

39 What Are the Changes in the Profile of Antibody Responses After - Protein (Grs. #1-3) and - NYVAC+Protein (Grs. #4/5) Boost at Week 48 ?

40 Neutralizing Antibody Titers All for TZM.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: -Substantial transient increase in T-cell response -Stably elevated but no increase in antibody response  DNA groups (only gp120 boost) showed: - Slight transient increase in T-cell response - Substantial increase in antibody response Conclusions III – Vaccine-Induced Responses at Week 48 boost

45 DNANYVAC T-cell Antibody Strong T-cell Polarization Phase Protein Weak Induction of Antibody Protein Late Increase of Antibody Response Months T-Cell Polarizing Vaccine Regimen Magnitude of Response

46 NYVAC + Protein Strong Antibody Polarization Phase NYVAC + Protein Late Increase of T-cell Response NYVAC + Protein T-cell Antibody Weak T-cell Polarization Phase Months Antibody Polarizing Vaccine Regimen Magnitude of Response

47 Future Plans – Optimization of vaccine regimens  Acceleration of the induction of the antibody response  Acceleration of prime/boost immunization schedule of T- cell polarizing vaccine regimens  Circumvent the interference of potent vaccine-induced T- cell response with the induction of potent antibody response  Development of immunization regimens resulting in a synergistic effect between T-cell and antibody polarizing vaccine regimens  Increase breadth of T and B cell responses

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

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

50 Synthetic biology techniques to generate different types of Env libraries (randomized, SeqPer) and … to efficiently subclone libs into pQL13 enabling inducible expression of Env; cotranslational coupling of selection marker; coupling of phenotype and genotype HT generation of stable cell- lines (<10E5) by bulk transfection of Env libraries cloned into pQL13 FACS based screening procedure bNMAbs, germ line versions Genotype Phenotype Single integrations Flp-In stable cell-lines Antibiotic selection for 25 days Analysis by deep sequencing Env library pQL13 ⑥ QL cloning ① Transfection: pQL13 + helperplasmid ② „Linked“ cellular Env-library ⑤ Nested PCR Laser Cells ④ FACS-sorting ③ bnMAB binding Re-Design – B Cell Responses (IAVI, Replivax) Objective: Exploit bnMAbs / germ line versions to select Env‘s with new properties and the potential to induce broad neutralization

51 Applying Synthetic Biology Principles for Better Vaccines: Challenging HIV Construction Cycle(s) Knowledge Gene Design - Modelling GeneOptimizer TM Protein Design - Modeling Initialisation Immunogen Design Master Los Alamos Datenbank s(x) Epitop-Scoring Optimization Immunogen CD4 Env CFSE Clinical trial: Phase IIa Specification Parts, devices Chassis - Delivery plasmids, bacteria, viruses Screen ex vivo / in vivo testing Molecular Epidemiology Pathogenesis Viral Replication „Master Gene“

52 Benedikt Asbach Alex Kliche Josef Köstler Kathi Böckl Jens Wild K. Kindsmüller P. Rosenstock

53 Funding EU 7th Framework CutHiVac Gates Foundation PTVDC, VDC Gates Foundation RepliVax IAVI Stabilized new trimers NIH HIVRAD BMBF HIVCOMPNET, BioChance DFG, BFS, Hektor Stiftung Industry Sanofi Pasteur Jim Tartaglia Novartis Susan Barnett Polymun Dietmar Katinger GeneArt AG Univ. Partners CHUV, Lausanne Guiseppe Pantaleo UCL, London Robin Weiss Univ. Cambridge UK Jon Heeney DPZ Göttingen C. Stahl Hennig Univ. Leiden Cornelius Melief CDC Beijing Shao Yiming Univ. Oxford, UK Quentin Sattentau Acknowledgement Wagner Group: Molecular epidemiology, immunogen development, biochemistry, gene design, DNA vaccine, NYVAC / vector prototypes, ex vivo analysis / preclinic & immunization schedule

54 Acknowledgements (PTVDC) PTVDC Participating OrganizationKey Scientific Personnel Centre Hospitalier Universitaire Vaudois (SponsoringOrganization) G. Pantaleo (PI) T. Calandra Fred Hutchinson Cancer Research CenterL. Corey Consejo Superior de Investigaciones CientificasM. Esteban Biomedical Primate Research Centre /University of CambridgeJ. Heeney Arizona State UniversityB. Jacobs Institute of Research in BiomedicineA. Lanzavecchia University of WashingtonD. Koelle University Paris 12, CHU Henri MondorY. Levy Murdoch UniversityS. Mallal/M. John Leiden University Medical CentreC. Melief University of MontrealR-P. Sekaly Imperial College of Science, Technology and MedicineJ. Weber Sanofi PasteurJ. Tartaglia University of RegensburgR. Wagner Project Management Song Ding

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56 Thanks To Bill & Melinda Gates Foundation for its Support in the Development of the DNA/NYVAC Platform Special thanks to: Nina Russell, Pervin Anklesaria and Jose Esparza for their support in the development of the PTVDC, PVRD and more recently RepliVax projects

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58 Knowledge to specify target genes / epitopes - Control o virus replication Focus IR to conserved proteins and epitopes limits immunological escape True for both T and B cell responses DFG


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