Rationally designed DNA construct encoding MHC class I restricted epitopes derived from Leishmania major proteins successfully provokes cytotoxic CD8 T cell responses in Balb/c mice
Negar
Seyed1,
Tahereh
Taheri1,
Charline
Vauchy2, 3, 4,
Magalie
Dosset2, 3, 4,
Iraj
Sharifi5,
Pierre
S.
Rohrlich2, 3, 4, 6 and
Sima
Rafati1*
-
1
Pasteur Institute of Iran, Iran
-
2
Université de Franche-Comté, France
-
3
INSERM U1098; Unité Mixte de Recherche;, France
-
4
Etablissement Français du Sang de Bourgogne Franche-Comté, France
-
5
Leishmaniasis Research Center, Kerman University of Medical Sciences, Iran
-
6
CHRU de Besançon, Service de pédiatrie, France
Today protective and therapeutic peptide-based vaccine concept has drawn attraction in the field of intracellular infections where CD8 cytotoxic T cell responses are crucial. In this study we used a rationally designed DNA construct encoding different in silico predicted HLA class I restricted peptides from several Leishmania major proteins plus 4 H2-Kd restricted ones. We immunized Balb/c mice with the construct to verify that the plasmid could induce CD8 T cell responses in vivo.
Different MHC class one restricted peptides were lined up together to satisfy two main criteria: least junctional peptides and most cleaved peptides of interest out of the poly-epitope sequence using NetCTL and nHLApred software. All different possibilities of arrangements without spacer and with “AAA”, “AAY”, “K” and “AD” were individually analyzed. Beside spacers, H2-kd restricted peptides (Kd1-Kd4) were additionally separated using N-terminal “ARY” sequence (TAP binding motifs). Ubiquitin sequence was joined N-terminal to final arrangement with a G76A substitution to protect deubiquitination by hydrolases. Universal Th1 epitope (TT-830) was integrated in C-terminal position to the polytope sequence to meet the prerequisite of naïve CD8 T cell response activation. The final arrangement was cloned in pCDNA-3.1. 8 week-old male BALB/c mice primarily injected with cardiotoxin 4 days before DNA immunization, received 100µg DNA intramuscularly followed by 2 boosters at one week interval. 10 days after the last booster, mice were sacrificed and splenocytes were subject to ex-vivo IFN-γ production and in-vitro cytotoxicity against individual Kd1-Kd4 peptides by ELISpot and standard chromium-51(51Cr) release assay, respectively.
4 out of 4 H2-Kd restricted peptides in the polytope sequence were immunogenic with high IFN-γ production detected by ELISpot assay. These peptides had been previously characterized from L. major proteome by a reverse approach and detected as potent IFN-γ inducers by CD8 T cells in Kd2>Kd3>kd1>kd4 order. The same peptides in our experiment appeared highly immunogenic with the same order. 3 out of 4 peptides elicited a significant cytotoxic response of spleen T cell against peptide loaded CT26 cells. There was a considerable correlation between the ex vivo Elispot assay and the cytotoxic activity.
In vivo studies confirmed that a rational design of epitopes respecting N-terminal tight ubiqitination with G76A substitution, “AD” flanking spacers for adequate proteosomal degradation, TAP binding motifs and Th1 epitope, effectively raised immune response against constituent epitopes from a multi-epitope DNA construct. These results are of importance for polytope vaccine design regarding DNA strategy which stands higher in stability and cost effectiveness compared to peptide vaccination.
Acknowledgements
Authors from Pasteur Institute of Iran deeply appreciate colleagues at INSERM UMR1098, Besancon, France for their concerns and collaboration in this study.
Keywords:
Leishmania major,
CD8 T cell,
epitope,
Vaccine,
Transgenic mice
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Adaptive Immunity
Citation:
Seyed
N,
Taheri
T,
Vauchy
C,
Dosset
M,
Sharifi
I,
Rohrlich
PS and
Rafati
S
(2013). Rationally designed DNA construct encoding MHC class I restricted epitopes derived from Leishmania major proteins successfully provokes cytotoxic CD8 T cell responses in Balb/c mice.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00987
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Received:
27 Jun 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Prof. Sima Rafati, Pasteur Institute of Iran, Tehran, Iran, s_rafati@yahoo.com