Group B: Ana Rita Domingues, Nicholas Gauthier, Ilka Hoof,

1
Possible HIV Knockout?
Group B Ana Rita Domingues, Nicholas Gauthier,
Ilka Hoof, Eleonora Kulberkyte, and Nicolas
Rapin Immunological Bioinformatics, June 2006
Introduction
The C-terminal cleavage probability is predicted
to be high (see Fig. 3 below) for all MHC class I
epitopes, with only one of them containing a
strong internal cleavage site.
Acquired immunodeficiency syndrome (AIDS) is one
of the worst diseases affecting mankind today.
Since it was discovered in 1981, AIDS has become
a major pandemic, and has killed approximately
16.3 million people. AIDS is caused by HIV
(human immunodeficiency virus) and is spread by
sexual contact, infected blood, and can be passed
from mother to infant. It is estimated that more
than 34 million people are infected worldwide,
most of which are in Sub-Saharan Africa and
South/Southeast Asia 1,2.
Figure 1 Diagram of HIV with proteins 3.
HIV is a retrovirus, which means it carries its
genetic information in the form of RNA and
replicates via a DNA intermediate. HIV infects
the components of the human immune system such as
CD4 T-cells, dendritic cells and
macrophages. Much scientific research has been
done to find a treatment/vaccine for HIV. The
most common therapies used today are
antiretroviral drugs that either interfere with
reverse transcription or inhibit the viral
protease 2. Finding a vaccine is a difficult
task as HIV mutates frequently and the infection
can remain latent for long periods before causing
AIDS. This poster aims to present an in silico
approach to developing a vaccine for HIV. We
have chosen several epitopes from HIV-1 that are
predicted to trigger B-cell and T-cell responses.
The proteins we focused on were gp120, a surface
protein that is involved in the binding of virus
to the host cell, and gag, a structural protein
from the virus core. The polyprotein gag is
cleaved into several sub-proteins of which p24
and p17 are the largest in size.
Figure 3 Epitope Atlas of the polytope
construct. Generated from the four chosen MHC
class I and one MHC class II epitopes.
Since proteasomal cleavage is a stochastic
process, the epitopes are expected to be
frequently processed correctly, presented by MHC
class I molecules, and trigger a CD8 T-cell
response. The MHC class II epitope was added to
the polytope as there is evidence that even
endogenous antigens can be presented by MHC class
II molecules in antigen presenting cells.
Methods
All HIV-1 proteins were processed using NetCTL
4 in order to find putative MHC class I
epitopes. NetCTL combines the prediction of the
main stages in antigen processing probability
for proteasomal cleavage, TAP transport, and MHC
class I binding affinity. Predictions were run
on HLA supertypes A1, A2, A3, and B7. These HLA
supertypes were used because they cover over 95
of the human population 5. After extensive
screening, gag was chosen because it produced the
largest number of high scoring epitopes in NetCTL
prediction throughout the selected supertypes.
The best epitope for each supertype was chosen
based on its NetCTL score and the degree of
sequence conservation. The sequence conservation
was derived from a multiple sequence alignment
of 614 gag sequences provided by the Los Alamos
HIV Sequence Database 6. To maximize the
activation of the immune system, MHC class II
presentation of gag epitopes was
also investigated using the EasyGibbs sampler
program 7. The dataset used to train the Gibbs
sampler consisted of known HLA-DR4 epitopes
(provided by EasyGibbs). A polytope containing
both MHC class I and II epitopes was generated
using a polytope optimization program in order to
minimize internal proteasomal cleavage and
maximize C-terminal cleavage. B-cell epitopes
were predicted using the DiscoTope 1.0 server
8. A suitable candidate was chosen according
to conservation score and visual evaluation of
accessibility in the 3D-structure of gp120. Both
the B-cell epitope and the highest scoring MHC
class II epitope were included into the protein
vaccine construct. The 3D-structure of the
construct was predicted using the CPHmodels 2.0
server 9.
Figure 4 3D-structure of the protein-based
vaccine predicted by CPHmodels 9. The B-cell
epitope is highlighted in red and the MHC class
II epitope is highlighted in blue.
As a suitable target to boost B-cell response
the surface protein gp120 was chosen. Most
putative epitopes which were predicted by
DiscoTope were located in loop regions, and as
these are highly variable in gp120, the conserved
helix ?1 was chosen as a qualified B-cell
epitope. The peptide vaccine construct consists
of the complete gp120 structure 1RZJ (from the
Protein Data Bank) into which the predicted MHC
class II epitope FYKTLRAEQASQEVKNWM was inserted
in the loop region after position 292 (blue in
Fig. 4). The 3D-structure appears stable and the
B-cell epitope is still accessible (red in Fig.
4).
Discussion
Throughout the course project we have identified
several promising MHC class I, MHC class II, and
B-cell epitopes that can be used as possible HIV
vaccines. Our final vaccine strategy contains
both a polytope that can be used for a DNA
vaccine and a peptide that can be used as a
protein vaccine. Our strategy is to target and
activate both the humoral and cell-mediated
immune response in the same vaccine. This
strategy should make it very difficult for HIV to
survive within a vaccinated individual. Due to
time constraints we were unable to consider all
aspects needed for a trial vaccine. Autoimmunity
could be a problem if antigen in our epitopes
closely matches sequestered self antigen. In
addition RNA splicing could destroy our polytope
and result in lower epitope synthesis.
Furthermore it may be beneficial to add
ubiquitination signals into the polytope to help
target it to be degraded by the proteasome.
Placing a signal peptide on the MHC class II
epitope which targets it to lysosomes and
endosomes can further enhance MHC class II
presentation.
Results
The final polytope construct has a length of 70
amino acids and contains four MHC class I
epitopes along with the highest scoring MHC class
II epitope. All MHC class I epitopes are
positioned in highly conserved a-helical
structures (see Fig. 2 below) .
References
1 - Immunobiology. Janeway, C. et al, Garland
Publishing, 2001 2 - Immunology. Goldsby, R.
et al, W.H.Freeman and Company, 2003 3 -
http//en.wikipedia.org/wiki/HIV 4 - An
integrative approach to CTL epitope prediction. A
combined algorithm integrating MHC-I binding, TAP
transport efficiency, and proteasomal cleavage
predictions. Larsen M.V., Lundegaard C., Kasper
Lamberth, Buus S,. Brunak S., Lund O., and
Nielsen M. European Journal of Immunology. 35(8)
2295-303. 2005 5 - Nine major HLA class I
supertypes account for the vast preponderance of
HLA-A and -B polymorphism. Sette, A., and J.
Sidney. 1999. Immunogenetics 50201-212. 6 -
http//hiv-web.lanl.gov 7 - Improved prediction
of MHC class I and class II epitopes using a
novel Gibbs sampling approach. Nielsen M,
Lundegaard C, Worning P, Hvid CS, Lamberth K,
Buus S, Brunak S, Lund O. Bioinformatics. 2004
201388-97 8 - Prediction of discontinuous
antibody binding epitopes in proteins. Pernille
H. Andersen, Morten Nielsen and Ole Lund 2006,
submitted 9 - CPHmodels 2.0 X3M a Computer
Program to Extract 3D Models. O. Lund, M.
Nielsen, C. Lundegaard, P. Worning.. Abstract at
the CASP5 conferenceA102, 2002.
Figure 2 3D-structure of HIV-1 p24 and p17
structural proteins. The four predicted MHC
class I epitopes are highlighted by overall
conservation. Note that the two epitopes in p17
overlap.