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Promising strategies for designing poly- CD8+ T cell-epitope DNA vaccine BAZHAN Sergei, KARPENKO Larisa., ILYICHEVA Tatyana, BELAVIN.

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Presentation on theme: "Promising strategies for designing poly- CD8+ T cell-epitope DNA vaccine BAZHAN Sergei, KARPENKO Larisa., ILYICHEVA Tatyana, BELAVIN."— Presentation transcript:

1 Promising strategies for designing poly- CD8+ T cell-epitope DNA vaccine BAZHAN Sergei, bazhan@vector.nsc.ru KARPENKO Larisa., ILYICHEVA Tatyana, BELAVIN Pavel, SEREGIN, Sergei ANTONETS Denis, ILYICHEV Alexander State Research Center of Virology and Biotechnology "Vector", Novosibirsk region http://www.vector.nsc.ru AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

2 Design of the artificial polyepitope immunogens capable of eliciting high levels of the CD8+ CTL responses to all the contained epitopes is a promising approach in creation of an efficient vaccines. When designing such immunogens, it is necessary to optimize the processing and presentation of contained epitopes taking into account major steps of MHC class I- dependent antigen processing. Introduction AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

3 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow As is known, CD8+ CTLs recognize the viral protein antigens synthesized in the cell as short peptides (8–12 amino acid residues) associated with specific MHC class I molecules rather than full-sized proteins. These short antigenic epitopes are produced from endogenously expressed protein antigens by the proteasome-mediated processing with subsequent transportation to the ER lumen by TAP1/TAP2 heterodimers (TAP – Transporters Associated with antigen Processing) where they bind to the MHC class I molecules. Obtained complexes [peptides-MHC class I molecules] are transported through the trans-Golgi network to the cell surface where they are presented to CD8+ CTLs. MHC class I-dependent antigen presentation pathway Overview of the MHC I antigen-processing pathway (B. Lankat-Buttgereit and R. Tampe, 2002) BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

4 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow The objective of this study was optimization of polytope sequence for inducing high level of CD8+ CTL responses, notably: TAP-dependent transport of generated peptidic fragments into endoplasmic reticulum where they bind to MHC class I molecules. For this purpose we carried out the following studies: designing a set of artificial immunogens encoding different strategies of antigen processing and presentation; and comparison of immunogenicity of experimental DNA vaccines encoding obtained vaccine constructs. proteasomal/immunoproteasomal cleavage of antigen; The objective of this study BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

5 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Choice of the CD8+ T cell epitopes for polyepitope design HLA A*0201-restricted CD8+ T cell epitopes chosen for polyepitope design (retrieved from the Los Alamos HIV Molecular Immunology Database Epitope (peptide) sequences Short notation Protein (amino acid residues) Score(*)Epitope variation among HIV-1 subtypes 1SLYNTVATLSLYp17 (77–85)157.227Conserved (A, B, and C) 2ALVEICTEMALVRT (33–41)20.369Conserved (B) 3VIYQYMDDLVIYRT (179–187)18.008Conserved (A, B, and C) 4ILKEPVHGVILKRT (309–317)39.025Conserved (A, B, and C) 5QMHEDIISLQMHgp160 (103–111)145.490Conserved (A, B, and C) 6KLTPLCVTLKLTgp160 (121–129)74.768Conserved (A, B, and C) 7RLRDLLLIVRLRgp160 (770–778)20.437Conserved (A and B) 8VLEWRFDSRLVLENef (180–189)8.832Conserved (B) 9RILQQLLFIRILVpr (62–70)67.142Conserved (A, B, and C) 10RGPGRAFVTIRGPgp160 (311–320)0.129Weakly conserved (*) Predicted estimate of the disassociation half-time of the molecule containing this subsequence. BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

6 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Design of poly-CD8+ T cell-epitope–based immunogens E1E2E3E4E5E6E7E8E9E10H Construct C1 Construct C1: Epitopes are linked together without flanking residues PrE1PrE2PrE3PrE4PrE5HPrE6PrE7PrE8PrE9PrE10 Construct C2 Construct C2: Epitopes are flanked with spacer residues “Pr” to optimize liberation of determinants by standard and immuno proteasomes Each of the polypeptide construct contains the universal PADRE peptide (AKFVAAWTLKAAA) that is highly immunogenic CD4+ T cells epitope restricted by numerous class II allomorphs for mouse and human. Construct C3 Construct C3: Epitopes are flanked with spacer residues to optimize proteasome liberation and TAP transport Pr+TAP E1Pr+TAP E9Pr+TAPE2E3E4E5E6E7E8 HE10 Additionally, each construct contains the ovalbumin derived C-terminal SIINFEKL epitope for monitoring expression and immunogenicity using the 25-D1.16 antibody specific for Kb-SIINFEKL complexes. BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

7 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow HindIII Acc65 I BstE II XhoI Vector plasmid pV1 Vector plasmid pV1===========AAGCTT=Kozak=GGTACC====GGTGACC=stop-codon=CTCGAG===== HindIII Acc65 I BstE II XhoI Vector plasmid pV2 Vector plasmid pV2==AAGCTT=Kozak=Ub-V(76)=GGTACC====GGTGACC=stop-codon=CTCGAG===== HindIII Acc65 I BstE II XhoI Vector plasmid pV3 Vector plasmid pV3===========AAGCTT=Kozak=GGTACC====GGTGACC=Ub=stop-codon=CTCGAG== The structures of vector plasmids for cloning genes encoding target polyepitope constructs Kozak – Kozak motif; Ub – ubiquitin; Acc65 I and BstE II – sites for embedding target genes in vector plasmids pV1, pV2 and pV3 Construct C1 Construct C2 Construct C3 Experimental Design BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

8 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow The list of recombinant plasmids encoding the target immunogens No.Recombinant plasmids VectorsGene products 1pV–C1pV1 Polyepitope constructs C1, C2, and C3 2pV–C2pV1 3pV–C3pV1 4pV–UbC1pV2 Polyepitope constructs C1, C2, and C3 with Ub genetically appended to their N-terminus 5pV–UbC2pV2 6pV–UbC3pV2 7pV–C1UbpV3 Polyepitope constructs C1, C2, and C3 with Ub genetically appended to their C-terminus 8pV–C2UbpV3 9pV–C3UbpV3 Experimental Design BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

9 Results Mean fluorescent intensity (MFI) of [SIINFEKL–H-2 Kb] complexes in the 293- Kb cells transfected with the studied recombinant plasmids United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow All data are expressed as the mean ± S.E.M. A.U., arbitrary unit. Untr, untransfected cell (negative control 1); vector plasmid pV1 (negative control 2); a, statistically significant differences in comparison with negative control 1; b, statistically significant differences in comparison with negative control 2; A – MFI of [SIINFEKL–H-2 Kb] in the cells transfected with the recombinant plasmids encoding constructs C1, C2, C3, UbC1, UbC2, UbC3, C1Ub, C2Ub, C2U3; B – MFI of [SIINFEKL–H-2 Kb] in the cells transfected with three groups recombinant plasmids {namely pC =[pV-C1, pV-C2, pV-C3], pUbC=[pV- UbC1, pV-UbC2, pV-UbC3] and pCUb=[pV-C1Ub, pV-C2Ub, pV-C3Ub]} to assess the contribution of the ubiquitin to produce [Kb-SIINFEKL] complexes; C – MFI of [SIINFEKL–H-2 Kb] in the cells transfected with three groups recombinant plasmids {namely pC1(Ub)=[pV-C1, pV-UbC1, pV−C1Ub], pC2(Ub)=[pV-C2, pV-UbC2, pV-C2Ub] and pC3(Ub)= [pV-C3, pV-UbC3, pV-C3Ub]} to assess the contribution of the constructs C1, C2, and C3 to produce [Kb-SIINFEKL] complexes. BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

10 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Results Protocol of HLA-transgenic mice immunization Prime with rDNA 14 days Boost with rDNA Boost with rVV-UbC1 Splenocytes were harvested and stained for IFN-γ positive T CD8 cells in response to panel of peptides 15 days 6 days Immunization groups Plasmids 1pV–C1 2pV–C2 3pV–C3 4pV–UbC1 5pV–UbC2 6pV–UbC3 7pV–C1Ub 8pV–C2Ub 9pV–C3Ub 10 (control)pV1 (vector) HLA-A2 BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

11 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Responses of IFN-γ–containing cells (CD8+ T cell) to specific peptides in groups of the HLA-A2 transgenic mice immunized with naked recombinant plasmids encoding target immunogens. Gray bars - immunization with the recombinant plasmids encoding target immunogens. White bars - immunization with the control vector plasmid pV1. Results. The immunogenicity of the vaccine constructs Ratio of the level of IFN-γ–containing cells stimulated with specific peptides to the level of the cells stimulated with control peptide BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

12 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Our results from in vitro and in vivo experiments demonstrate that the most promising vaccine candidate is construct pV-UbC3 Conclusion Construct UbC3: Epitopes are flanked with spacer residues to optimize proteasome liberation and TAP transport Pr+TAP E1Pr+TAP E9Pr+TAPE2E3E4E5E6E7E8 HE10 Ub a) based on genetic attachment of ubiquitin sequence to the N-terminus of polyepitope constructs to target them to the proteasome b) uses the amino acid residues flanking the determinants to provide a proteasomal processing of the polyepitope construct or the motifs for TAP proteins, necessary for transporting the proteasome generated peptides into the ER c) exhibits the greatest antigenicity (for SIINFEKL at least) and immunogenicity BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

13 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Conclusion Because UbC3 was optimized according to a)proteasome degradation, b)peptide liberation, and c)TAP transport, obtained results supports the concept of rational vaccine design based on available knowledge of the MHC class antigen processing pathway. Construct UbC3 Pr+TAP E1Pr+TAP E9Pr+TAPE2E3E4E5E6E7E8 HE10 Ub BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

14 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow PolyCD8+ T cellDesigner allows to select the minimal set of epitopes with the known (or predicted) specificity towards various allelic variants of MHC class I molecules covering the overall repertoire with a specified redundancy. This program makes it possible to select the flanking sequences for optimizing the binding of selected peptides with TAP and then to join the obtained peptide fragments into a polyepitope construct to provide the proteasomal processing and liberation of epitopes. The developed software can be used for rational designing new candidate polyepitope vaccines. More detailed information about PolyCTLDesigner is available at http://tepredict.sourceforge.net/PolyCTLDesigner.html PolyCTLDesigner software BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria

15 United States–Russia Workshop on HIV Prevention, EECAAC, October 28-30, 2009, Moscow Acknowledgements SRC Virology and Biotechnology Vector, Russia Karpenko L.I. Ilyicheva T.N. Seregin S.V. Danilyuk N.K. Belavin P.A. Antonec D.V. Ilyichev A.A. Ignatyev G.M. Laboratory of Viral Diseases, NIAID, NIH, USA Yewdell J.W. Bennink J.R. Irvine K. Gibbs J. State Research Center of Virology and Biotechnology "Vector", Novosibirsk region http://www.vector.nsc.ru http://www3.niaid.nih.gov BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’ 2010, 18-23 July, 2010, Vienna, Austria


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