Promising strategies for designing poly-CD8+ T cell- epitope DNA vaccine BAZHAN Sergei , bazhan@vector.nsc.ru KARPENKO

1. State Research Center of Virology and Biotechnology "Vector", Novosibirsk region http://www.vector.nsc.ru • 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 AIDS’2010, 18-23 July, 2010, Vienna, Austria

2. Introduction 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. AIDS’2010, 18-23 July, 2010, Vienna, Austria

3. MHC class I-dependent antigen presentation pathway 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. Overview of the MHC I antigen-processing pathway(B. Lankat-Buttgereit and R. Tampe, 2002) AIDS’2010, 18-23 July, 2010, Vienna, Austria BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

4. The objective of this study The objective of this study was optimization of polytope sequence for inducing high level of CD8+ CTL responses, notably: • proteasomal/immunoproteasomal cleavage of antigen; • 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. BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

5. Choice of the CD8+ T cell epitopesfor polyepitope design HLA A*0201-restricted CD8+ T cell epitopes chosen for polyepitope design (retrieved from the Los Alamos HIV Molecular Immunology Database (*) 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 United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

6. Construct C1:Epitopes are linked together without flanking residues Construct C2: Epitopes are flanked with spacer residues “Pr” to optimize liberation of determinants by standard and immunoproteasomes H E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 H Pr E1 Pr E2 Pr E3 Pr E4 Pr E5 Pr E6 Pr E7 Pr E8 Pr E9 Pr E10 Construct C3: Epitopes are flanked with spacer residues to optimize proteasome liberation and TAP transport Pr+TAP E1 Pr+TAP E2 Pr+TAP E3 Pr+TAP E4 Pr+TAP E5 Pr+TAP E6 Pr+TAP E7 Pr+TAP E8 Pr+TAP E9 Pr+TAP H E10 Design of poly-CD8+ T cell-epitope–based immunogens 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. 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. AIDS’2010, 18-23 July, 2010, Vienna, Austria BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

7. Construct C1 Construct C2 Construct C3 HindIII Acc65 IBstE IIXhoI Vector plasmid pV1===========AAGCTT=Kozak=GGTACC====GGTGACC=stop-codon=CTCGAG===== HindIII Acc65 IBstE IIXhoI Vector plasmid pV2==AAGCTT=Kozak=Ub-V(76)=GGTACC====GGTGACC=stop-codon=CTCGAG===== HindIII Acc65 I BstE IIXhoI 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 Experimental Design BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

8. Experimental Design The list of recombinant plasmids encoding the target immunogens BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

9. Results Mean fluorescent intensity (MFI) of [SIINFEKL–H-2 Kb] complexes in the 293-Kb cells transfected with the studied recombinant plasmids 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 United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

10. HLA-A2 HLA-A2 HLA-A2 Results Protocol of HLA-transgenic mice immunization Boost with rVV-UbC1 14 days 15 days Prime with rDNA Boost with rDNA 6 days Splenocytes were harvested and stained for IFN-γ positive TCD8 cells in response to panel of peptides BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

11. Results. The immunogenicity of the vaccine constructs 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 encodingtarget immunogens. White bars - immunization with the control vector plasmid pV1. 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 United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

12. Construct UbC3: Epitopes are flanked with spacer residues to optimize proteasome liberation and TAP transport Ub Pr+TAP E1 Pr+TAP E2 Pr+TAP E3 Pr+TAP E4 Pr+TAP E5 Pr+TAP E6 Pr+TAP E7 Pr+TAP E8 Pr+TAP E9 Pr+TAP H E10 Conclusion Our results from in vitro and in vivo experimentsdemonstrate that the most promising vaccine candidate is construct pV-UbC3 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 AIDS’2010, 18-23 July, 2010, Vienna, Austria BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

13. Construct UbC3 Ub Pr+TAP E1 Pr+TAP E2 Pr+TAP E3 Pr+TAP E4 Pr+TAP E5 Pr+TAP E6 Pr+TAP E7 Pr+TAP E8 Pr+TAP E9 Pr+TAP H E10 Conclusion • Because UbC3 was optimized according to • proteasome degradation, • peptide liberation, and • TAP transport, • obtained results supports the concept of rational vaccine design based on available knowledge of the MHC class antigen processing pathway. AIDS’2010, 18-23 July, 2010, Vienna, Austria BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

14. PolyCTLDesigner software 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 andthen 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 BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow

15. http://www3.niaid.nih.gov 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 Acknowledgements BGRS’2010, SATELLITE MICROSYMPOSIUM ISTC, June 22, 2010, Novosibirsk AIDS’2010, 18-23 July, 2010, Vienna, Austria United States–RussiaWorkshop on HIVPrevention,EECAAC, October 28-30, 2009, Moscow