Methods MHC class-I T cell epitope prediction for Nef

1. In pursuit of a combined immunogenic vaccine Bent Petersen, Boumediene Soufi, Francisco Roque, Wan Peng 27685 Immunological Bioinformatics Introduction Methods MHC class-I T cell epitope prediction for Nef Consensus and ancestral sequences of the Nef protein for the different HIV-1 subtypes were obtained from the Los Alamos HIV sequence database (1). Epitope predictions for MHC HLA supertypes A1, A2, A3, A24, B7 and B44 were made using the NetCTL server (2), by submitting the global consensus. This server combines MHC peptide binding prediction and proteosomal cleavage using NN, and TAP transport efficiency using a weight matrix method. N-Glycosylation sites were identified using N-Glycosite (3). MHC class-II T cell epitope prediction for Nef A consensus sequence for the NEF protein of the HIV-1 was obtained from the Los Alamos HIV sequence database (1). Epitope predictions for all available MHC HLA-DR04 subtypes were made using ProPred (4), a graphical web tool for predicting MHC class II binding regions in antigenic protein sequences. The server implements a matrix based prediction algorithm, employing amino-acid/position coefficient tables deduced from literature. B-cell epitope prediction for gp120 Using Consensus Maker (1), a consensus of 36 sequences from a region surrounding the 3rd variable region in the gene encoding gp120 from the HIV-1, subtype B virus, was made. All of the sequences were aligned using Jalview (5) and submitted to BepiPred (6), predicting the location of linear B-cell epitopes using HMM and propensity scale methods. Polytope prediction The entire HIV1 sequence was adopted from the NCBI (9) website, and was used for CTL epitope prediction using the NetCTL server (1) for all 10 supertypes. For integrating the DR4 class-II epitope, we used the SYFPEITHI server (10). 3D structure construction The consensus sequences for the NEF and gp120 were submitted to FeatureMap3D (11), aligned with the best hit in PDB, while mapping interesting features in the structure. Pymol was used to visualize and prepare the figures for presentation, coloring and annotation. Results MHC class-I HIV Human Immunodeficiency Virus (HIV) is a retrovirus that primarily infects vital components of the human immune system such as CD4+ T cells, macrophages and dendritic cells leading to the syndrome known as AIDS (7). Yet designing a vaccine able to protect against it remains as much of a challenge today, as it was when first discovered. One of the primary reasons for this has to do with the virus’s high capability and susceptibility to mutation leading to multiple strains of the HIV virus (7). Table 1 - The predicted epitopes for each MHC class-I allele supertypes, the percentage of sequences in which they occurred and the combined score of the prediction. The selection of the best epitopes was based both on high score and how often they appeared in the ancestral and consensus sequences for each HIV subtype. Figure 1 - Overall life cycle of HIV, including the formation of the structural and regulatory proteins (8). • Vaccine Design • - Effective vaccine would be able to stimulate both branches of the immune system: Humoral and Cell Mediated. • - This methodology if successful could better prepare the immune system in combating HIV if encountered. • - The epitopes will be chosen from the gp120 protein and the Nef protein of HIV. • - Epitopes predicted using various immunological in silico methods • gp120 B cell epitopes: To induce the Humoral immune system response • - The GP120 viral protein of HIV is absolutely crucial in order for the HIV virus to successfully bind to and infect target cells via the CD4 receptors (7). • Therefore we feel it is a good candidate to induce the humoral branch of the Immune response since it is expressed on the infected cell surface and therefore the human body should respond by stimulating the production of antibodies on B cells that will bind the viral protein and target it for destruction. • Nef Protein epitopes: Used to generate Cell Mediated Immune response • - The Nef protein is one of the 3 major regulatory proteins made by HIV (13). • - Interacts with the host cell signal transduction proteins to provide survival over a long period of time of infected T cells and for destruction of non-infected T cells through apoptosis (13). • - Induce cell mediated immune response via production of cytotoxic T cells. • Delivery of gp120 and Nef protein epitopes into Human system • One possible method of introducing these epitopes to the patient so that the immune system can develop a memory response to these viral agents would be to insert the DNA that encodes for these peptides into a virus that can replicate in a human but will not cause disease. In this way the protein encoding the epitopes will be produced and fold in its correct 3 dimensional conformation since it would be doing so in the actual invivo system. One virus that is known to be non pathogenic in humans is the canarypox virus (14). So in theory this virus could be used to deliver the epitopes of gp120 and the Nef proteins into a human patient inducing both major type of mechanisms of the immune system and potentially be strong enough to immunize a human being against the HIV virus. Figure 2 - PDB entry 2NEF was the highest blast scoring hit. Epitopes are marked according to the degree of overlapping: yellow for a single, orange for two and red for three epitopes in the same region. The glycosylation site is marked as a red ball. MHC class-II Table 2 - The predicted epitopes for each MHC class-II DR04 allele supertypes, the percentage of sequences in which they occurred and the score of the prediction. The selection of the best epitopes was based on score. The percentage shows how often they appeared in the analyzed sequences. Figure 3 - PDB entry 2NEF was the highest blast scoring hit. Epitopes are colored according to the number of matching HLA supertypes. Yellow for 1, orange for 3 and red for 4 types. B-cell epitope • Discussion/Conclusion • 6 supertypes of the MHC class-I alleles were chosen to search for a common epitope since these alleles are found in 99% of the Human population. All epitopes were found in helix and beta sheets, which could suggest importance in epitope recognition by the MHC. Interesting to note the overlap of the A1 and A3 epitopes and the 100% match of the B7 epitope. • With regards to MHC class-II, epitopes were also found in similar motifs as class I. A higher binding affinity could be resultant of a higher structural stability from these regions. • gp120 sequences containing the V3 region were chosen because of it’s immunodominance. Finding conserved epitopes within this hypervariable region suggests importance in viral function. Literature (15) states that the V3 base and neighboring residues and the bridging sheet are important for coreceptor binding. One epitope was predicted to fall precisely onto this region. • After running the entire genome through NetCTL, it was found that all top scoring predicted epitopes were from the Gag-Pol sequence of HIV. With this into consideration, the top scoring epitope of class-II DR04 (14mer) was inserted and fixed in the last position. • - Upon analyzing polytope construct we see strong C-terminal cleavage signal and no aparent optimization, which can be due to prediction of A2 supertype only. • In conclusion, we believe that from the results obtained, our target vaccine could have a potential in providing immunity to the HIVI virus, especially in those individuals with the B7, A1 and A3 alleles for MHC class I, and the DR0401, DR0421 and DR0426 alleles for MHC class II. Of course these predictions would have to be confirmed through experimental means, however we do see potential in studying this further. Table 2 -The two predicted epitopes are shown together with the percentage of occurrence in the total number os sequences considered. Figure 4 - PDB entry 2B4C was the highest blast scoring hit. The 10mer is shown in red, in the V3 base, and the 9mer is shown in yellow. Note that the sequence used was only partial for the gp120 gene, and the unmatched regions are colored grey. References [1] Korber, B. Et al. (2005), HIV Molecular Immunology, Los Alamos National Laboratory,Theoretical Biology and Biophysics, Los Alamos, New Mexico, LA-UR 06-0036 [2] Larsen, M. Et al. (2005), An integrative approach to CTL epitope prediction. A combined algorithm integrating MHC-I binding, TAP transport efficiency, and proteasomal cleavage predictions, European Journal of Immunology, 35(8), 2295-303 [3] Zhang M et al. (2004), Glycobiology. 14(12), 1229-46 [4] Singh,H. and Raghava,G.(2001) ProPred: Prediction of HLA-DR binding sites. Bioinformatics, 17(12), 1236-37. [5] Clamp, M. et al. (2004), The Jalview Java Alignment Editor, Bioinformatics, 20, 426-7. [6] Larsen, J. et al (2006), Improved method for predicting linear B-cell epitopes, Immunome Research, 2, 2 [7] http://medlib.med.utah.edu/WebPath/TUTORIAL/AIDS/HIV.html [8] http://www.pharmacy.umaryland.edu/courses/PHAR531/lectures_old/fig/HIV-Lifecycle.15May02.gif [9] http://www.ncbi.nih.gov/Database/ [10] Rammensee, H. et al. (1999), SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, 50, 213-219 [11] Wernesson, R. et al (2006), FeatureMap3D - a tool to map protein features and sequence conservation onto homologous structures in the PDB, Unpublished. [12] http://www.infekt.ch/index.php?artID=384 [13] http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/hiv1nef/molmast.htm [14] http://www.iavi.org/viewpage.cfm?aid=223 [15] Huang, C. et al.(2005),Structure of a V3 containing HIV-1 gp120 core, Science, 310, 1025-1028.