Transcript Methods MHC class-I T cell epitope prediction for Nef

In pursuit of a combined immunogenic vaccine
Bent Petersen, Boumediene Soufi, Francisco Roque, Wan Peng
27685 Immunological Bioinformatics
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 NGlycosite (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.
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.
Introduction
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).
Alelle
supertype
A1
A2
A3
A24
B7
B44
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.
Predicted
Epitope
QVPLRPMTY
LTFGWCFKL
QVPLRPMTY
GWCFKLVPV
FPVRPQVPL
QEILDLWVY
Match
Combined
percentage score
84%
0,7625
72%
1,2634
84%
0,9101
72%
0,7999
100%
1,7828
76%
1,8666
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.
MHC class-II
HLA
supertype
DR0401
DR0402
DR0404
DR0405
DR0408
DR0410
DR0421
DR0423
DR0426
Predicted
Epitope
VRPQVPLRP
IRYPLTFGW
IRYPLTFGW
FKLVPVDPE
IRYPLTFGW
IVGWPAVRE
VRPQVPLRP
IRYPLTFGW
VRPQVPLRP
Match
percentage Score
100%
1,70
44%
3,10
44%
2,55
20%
3,60
44%
1,55
24%
3,45
100%
2,70
44%
2,55
100%
1,70
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
Epitope
CTRPNNNTRK
QSSGGDPEI
Percentage
64%
67%
Table 2 -The two predicted epitopes are shown together with
the percentage of occurrence in the total number os sequences
considered.
-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 classII 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.
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.
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