Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from <i>Leishmania major</i> Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

<div><p>Background</p><p>There are several reports demonstrating the role of CD8 T cells against <i>Leishmania</i> species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.</p><p>Methods and Findings</p><p>HLA-A*0201 restricted epitopes from LPG-3, <i>Lm</i>STI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to <i>ex-vivo</i> and <i>in-vitro</i> evaluation of specific IFN-γ production and <i>in-vitro</i> cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, <i>Lm</i>STI-1-P3 and <i>Lm</i>STI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, <i>Lm</i>STI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.</p><p>Conclusions</p><p>Here we described a new DNA-polytope construct for <i>Leishmania</i> vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of <i>Leishmania</i>. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.</p></div

Percent of specific lysis of targets loaded by P1, P3 and P6 by T cell clones from individual mice at 30∶1 E/T effector to target ratio.

a<p>Responder to tested mice.</p>b<p>Specific lysis at 30∶1 effector to target ratio.</p><p>Percent of specific lysis of targets loaded by P1, P3 and P6 by T cell clones from individual mice at 30∶1 E/T effector to target ratio.</p

Balb/c response against 4 H-2Kd restricted peptides.

<p>4 mice were immunized with polytope construct three times with one week interval and sacrificed 10 days after the last booster. Splenocytes from individual mice were <i>in-vitro</i> re-stimulated by representative peptides (Kd1-4) of Balb/c and specific IFN-γ production was evaluated by <i>ex-vivo</i> ELISPOT assay. A. Representative of two experiments. Columns, mean of spots from duplicate wells for each mice from one representative experiment; bars, SD. Stimulations resulting in spots two times the negative control (unstimulated cells) and more than 10 were considered positive (stars). B. Statistical analysis of consolidated data from 4 mice against each individual peptide. The response against all 4 peptides appeared statistically significant compared to un-stimulated control cells (<i>p</i><0.05) with an exception for Kd4 which was subdominant in comparison to the rest. Horizontal lines represent the mean value. SFC: Spot Forming Cells.</p

Final arrangement of the polytope construct used for immunization.

<p>13 HLA-A*0201 plus 4 H-2Kd control restricted peptides were arranged in tandem with spacers for accurate proteosomal cleavage (in red). Additional N-terminal ubiquitin sequence (bigger box) for proteosomal degradation, and C-terminal TT₈₃₀ epitope (smaller box) for CD8 T cell response enhancement were also included. Peptides depicted in green are from <i>Lm</i>STI-1, peptides in blue from LPG-3, peptides in gray from CPB/CPC and peptides in yellow are H-2Kd restricted (AYS = Kd1, SYE = Kd2, FYQ = Kd3, SYS = Kd4).</p

<i>Ex-vivo</i> evaluation of the specific response against six peptides (5 µg/ml/peptide) in HLA A2/DR1 mice.

<p>A total of 11 mice in two rounds of experiments were immunized with polytope construct three times with one week interval and sacrificed 10 days after the last booster. Splenocytes from individual mice were <i>in-vitro</i> re-stimulated by representative peptides (P1–P6) of HLA-A2 and specific IFN-γ production was evaluated by <i>ex-vivo</i> ELISPOT assay. Each dot represents mean of duplicate wells for each individual mice response against each peptide. Neg.pept (negative control peptide) represents a 9 mer HLA-A*0201 restricted peptide from human telomerase. Horizontal lines represent the mean value. No.pept =  no peptide stimulation. NS: not-significant.</p

Cytotocic T cell response against RMA/s target cells loaded with individual peptides.

<p>Cytolytic activity was tested in a standard 4-hour ⁵¹Cr release assay. RMA/s target cells loaded with individual peptides at a 10 µg/ml final concentration and labeled with ⁵¹Cr radioactive isotope were co-cultured with short term CTL lines making three different effector-to-target ratios. Results are expressed as the mean of triplicates in % of specific lyses: [(experimental − spontaneous release)/(total – spontaneous release)] ×100. A 9-mer HLA-A0201 restricted peptide from human telomerase was used as negative control.</p

<i>In-vitro</i> evaluation of polytope expression using COS-7 cells.

<p>Recombinant pEFGP-PT was transiently transfected into COS-7 cells by means of linear Polyethylenimine 25 KDa. A1 represents COS-7 cells without any transfection, A2 represents COS-7 cells transfected with pEGFP-N3 as positive control and A3 represents COS-7 cells transfected with pEGFP-PT. Panel B represents the corresponding microscopic feature of each condition, B1 represents positive control and B2 represents COS-7 cells transfected with pEGFP-PT after 24 hours.</p

Characteristics of <i>in silico</i> predicted <i>L. major</i> specific CD8⁺ T cell 9-mer peptides included in the polytope construct.

<p>* Peptides analyzed in HLA transgenic mice (P1–P6) and BALB/c mice (Kd1–Kd4) are in bold.</p>a<p>Moderate binder.</p>b<p>High binder.</p>#<p>Peptide falling over pre-set threshold.</p><p>(1) <a href="http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm" target="_blank">http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm</a>.</p><p>(2) <a href="http://www-bimas.cit.nih.gov/molbio/hla_bind/" target="_blank">http://www-bimas.cit.nih.gov/molbio/hla_bind/</a>.</p><p>(3) <a href="http://www.jenner.ac.uk/EpiJen/" target="_blank">http://www.jenner.ac.uk/EpiJen/</a>.</p><p>(4) <a href="http://immunax.dfci.harvard.edu/Tools/rankpep.html" target="_blank">http://immunax.dfci.harvard.edu/Tools/rankpep.html</a>.</p><p>(5) <a href="http://www.imtech.res.in/raghava/nhlapred/comp.html" target="_blank">http://www.imtech.res.in/raghava/nhlapred/comp.html</a>.</p><p>(6) <a href="http://www.cbs.dtu.dk/services/NetCTL/" target="_blank">http://www.cbs.dtu.dk/services/NetCTL/</a>.</p><p>(7) <a href="http://antigen.i2r.a-star.edu.sg/multipred" target="_blank">http://antigen.i2r.a-star.edu.sg/multipred</a>.</p><p>CPB (Cathepsin L-like Protease or Type I Cysteine Proteinase), CPC (Cathepsin L-like Protease or Type I Cysteine Proteinase), <i>Lm</i>sTI-1 (<i>L.major</i> Stress Indussible Protein), LPG-3 (Lipophosphoglycan Biosynthetic Protein).</p><p>Characteristics of <i>in silico</i> predicted <i>L. major</i> specific CD8⁺ T cell 9-mer peptides included in the polytope construct.</p

Transfected <i>Leishmania</i> parasites before and after treatment with proteasome inhibitor.

<p>Stable transfected parasites harboring polytope sequence were generated and used for evaluation of ubiquitinated polytope expression and degradation. A. Shows the fluorescent microscope patterns in 2 microscopic fields. Pale colored parasites before MG132 treatment turned sharp green in the presence of MG132 inhibitor. Part “a” in each field reflects before and part “b” after treatment with 10 µM MG132 for 3 hours. B. Illustrates fluorescent intensity of 2 different transfected clones (Clone#1 and clone #2) before and after treatment with MG132. Column 1: before treatment, column 2: treatment with 5 µM and column 3: treatment with 10 µM of MG132. Numbers on each plot represent the GFP positive population which drastically increases after MG132 treatment. PI: proteasome inhibitor.</p

<i>In vitro</i> evaluation of the specific response against six peptides in HLA A2/DR1 mice after one week stimulation (with 100 u/ml IL-2).

<p>A. Splenocytes from a total of 5 mice immunized with polytope construct three times with one week interval and sacrificed 10 days after the last booster were re-stimulated by representative peptides (5 µg/ml/peptide) of HLA-A2. Specific IFN-γ production was evaluated by <i>ex-vivo</i> ELISPOT assay. Each dot represents mean of duplicate wells for each individual mice response against each peptide. B. Splenocytes were stimulated with 10 µg/ml/peptide instead of 5 µg/ml/peptide. Neg.pept (negative control peptide) represents a 9 mer HLA-A*0201 restricted peptide from human telomerase. Horizontal lines represent the mean value. No.pept =  no peptide stimulation. NS: not-significant. C. P1 stimulation of splenocytes from B. along with Anti-HLA-DR (L243) blocker antibody. The response was CD8⁺ T cell restricted since CD4-T cell blockade by anti-HLA-DR, did not influence the outcome. L243 definitely lowers a potent CD4 T cell response in human PBMC against a TERT derived universal MHC class II restricted cancer peptide (UCP).</p