promising strategies for designing poly- cd8+ t cell-epitope dna vaccine bazhan sergei,...

Promising strategies for designing poly-CD8+ T cell-epitope DNA vaccine

BAZHAN Sergei, [email protected]

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

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

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, NovosibirskAIDS’ 2010, 18-23 July, 2010, Vienna, Austria


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, NovosibirskAIDS’ 2010, 18-23 July, 2010, Vienna, AustriaAIDS’ 2010, 18-23 July, 2010, Vienna, Austria


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

1 SLYNTVATL SLY p17 (77–85) 157.227 Conserved (A, B, and C)

2 ALVEICTEM ALV RT (33–41) 20.369 Conserved (B)

3 VIYQYMDDL VIY RT (179–187) 18.008 Conserved (A, B, and C)

4 ILKEPVHGV ILK RT (309–317) 39.025 Conserved (A, B, and C)

5 QMHEDIISL QMH gp160 (103–111) 145.490 Conserved (A, B, and C)

6 KLTPLCVTL KLT gp160 (121–129) 74.768 Conserved (A, B, and C)

7 RLRDLLLIV RLR gp160 (770–778) 20.437 Conserved (A and B)

8 VLEWRFDSRL VLE Nef (180–189) 8.832 Conserved (B)

9 RILQQLLFI RIL Vpr (62–70) 67.142 Conserved (A, B, and C)

10 RGPGRAFVTI RGP gp160 (311–320) 0.129 Weakly conserved

(*) Predicted estimate of the disassociation half-time of the molecule containing this subsequence.



Design of poly-CD8+ T cell-epitope–based immunogens

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10H

Construct C1: Epitopes are linked together without flanking residues

Pr E1 Pr E2 Pr E3 Pr E4 Pr E5H Pr E6 Pr E7 Pr E8 Pr E9 Pr E10

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: Epitopes are flanked with spacer residues to optimize proteasome liberation and TAP transport

Pr+TAPPr+TAP E1 Pr+TAPPr+TAP Pr+TAPPr+TAP Pr+TAP Pr+TAP Pr+TAP E9Pr+TAPE2 E3 E4 E5 E6 E7 E8H E10

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.



HindIII Acc65 I BstE II XhoIVector plasmid pV1===========AAGCTT=Kozak=GGTACC====GGTGACC=stop-codon=CTCGAG=====

HindIII Acc65 I BstE II XhoIVector plasmid pV2==AAGCTT=Kozak=Ub-V(76)=GGTACC====GGTGACC=stop-codon=CTCGAG=====

HindIII Acc65 I BstE II XhoIVector plasmid pV3===========AAGCTT=Kozak=GGTACC====GGTGACC=Ub=stop-codon=CTCGAG==

The structures of vector plasmids for cloning genes encoding target polyepitope constructsKozak – Kozak motif; Ub – ubiquitin; Acc65 I and BstE II – sites for embedding target genes in vector plasmids pV1, pV2 and pV3

Construct C1Construct C2Construct C3

Experimental Design



The list of recombinant plasmids encoding the target immunogens

No. Recombinant plasmids

Vectors Gene products

1 pV–C1 pV1Polyepitope constructs C1, C2, and C3

2 pV–C2 pV1

3 pV–C3 pV1

4 pV–UbC1 pV2 Polyepitope constructs C1, C2, and C3 with Ub genetically appended to their N-terminus

5 pV–UbC2 pV2

6 pV–UbC3 pV2

7 pV–C1Ub pV3 Polyepitope constructs C1, C2, and C3 with Ub genetically appended to their C-terminus

8 pV–C2Ub pV3

9 pV–C3Ub pV3

Experimental Design


ResultsMean 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.



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 TCD8 cells in response to panel of peptides

15 days

6 days

Immunization groups

Plasmids

1 pV–C1

2 pV–C2

3 pV–C3

4 pV–UbC1

5 pV–UbC2

6 pV–UbC3

7 pV–C1Ub

8 pV–C2Ub

9 pV–C3Ub

10 (control) pV1 (vector)

HLA-A2 HLA-A2 HLA-A2



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

0

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Control pV-C1

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ЦТ

Л/1

2-1

3

Control pV-C2

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RIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIYRIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIYRIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIY

RIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIYRIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIYRIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIY

RIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIYRIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIY

*

*

RIL AQM QMH SLY ILK KLT RGP VLE ALV RLR VIY

Control pV-C3

ЦТ

Л/1

2-1

3

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Control pV-UbC1

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Control pV-UbC2

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Control pV-UbC3

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Control pV-C1Ub

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Control pV-C2Ub

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Control pV-C3Ub

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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+TAPPr+TAP E1 Pr+TAPPr+TAP Pr+TAPPr+TAP Pr+TAP Pr+TAP Pr+TAP E9Pr+TAPE2 E3 E4 E5 E6 E7 E8H E10Ub

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



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+TAPPr+TAP E1 Pr+TAPPr+TAP Pr+TAPPr+TAP Pr+TAP Pr+TAP Pr+TAP E9Pr+TAPE2 E3 E4 E5 E6 E7 E8H E10Ub



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



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


promising strategies for designing poly- cd8+ t cell-epitope dna vaccine bazhan sergei,...

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