CD8+ T cell-epitope DNA vaccine

tanktherapistBiotechnology

Oct 23, 2013 (4 years and 16 days ago)

101 views

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

DNA vaccine


BAZHAN Sergei
,
bazhan@vector.nsc.ru


KARPENKO L
arisa
.
,

ILYICHEVA T
atyana
,


BELAVIN P
avel
,


SEREGIN, Sergei

ANTONETS D
enis
,

ILYICHEV A
lexander





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, Novosibirsk

AIDS


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

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

AIDS


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

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

CD
8
+

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.

BGRS’2010
,
SATELLITE MICROSYMPOSIUM

ISTC,

June 22, 2010, Novosibirsk

AIDS


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

United States

Russia

Workshop on HIV

Prevention
,

EECAAC
, October 28
-
30, 2009, Moscow

Design of poly
-
CD8+ T cell
-
epitope

based
immunogens

E1

E2

E3

E4

E5

E6

E7

E8

E9

E10

H

Construct C1
:

Epitopes

are linked together without flanking residues

Pr

E1

Pr

E2

Pr

E3

Pr

E4

Pr

E5

H

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
+TAP

Pr
+TAP

E1

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

E9

Pr
+TAP

E2

E3

E4

E5

E6

E7

E8

H

E
10

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

United States

Russia

Workshop on HIV

Prevention
,

EECAAC
, October 28
-
30, 2009, Moscow



HindIII


Acc
65

I

BstE II



XhoI

Vector plasmid pV1
==========
=
AAGCTT
=
Kozak
=
GGTACC
====
GGTGACC
=
stop
-
codon
=
CTCGAG
=
===
=



HindIII




Acc
65
I

BstE II

XhoI

Vector plasmid pV2
==
AAGCTT
=
Kozak
=
Ub
-
V(76)
=
GGTACC
====
GGTGACC
=
stop
-
codon
=
CTCGAG
=
===
=




HindIII


Acc
65
I


BstE II

XhoI

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;



Acc
65 I and
Bst
E 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

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


Vectors


Gene products

1

pV

C1

pV1

Polyepitope 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

BGRS’2010
,
SATELLITE MICROSYMPOSIUM

ISTC,

June 22, 2010, Novosibirsk

AIDS


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

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

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

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

BGRS’2010
,
SATELLITE MICROSYMPOSIUM

ISTC,

June 22, 2010, Novosibirsk

AIDS


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

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

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

Pr
+TAP

E1

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

E9

Pr
+TAP

E2

E3

E4

E5

E6

E7

E8

H

E
10

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

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

Pr
+TAP

E1

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

Pr
+TAP

E9

Pr
+TAP

E2

E3

E4

E5

E6

E7

E8

H

E
10

Ub

BGRS’2010
,
SATELLITE MICROSYMPOSIUM

ISTC,

June 22, 2010, Novosibirsk

AIDS


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

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

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