SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED
SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES
PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM
HIV Cohort Study
Roberts SG1, Almeida CM1, Bronke C1, Ahmad I1, Al Damuk A1,Cooper D1, Corkery M1, Keane N1,
Heckerman D3, Chopra A1, Mallal S1,2, John M1,2
Centre for Clinical Immunology and Biomedical Statistics, Institute of Immunology and Infectious Diseases, Murdoch
University, Perth, Western Australia, 2Department of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Perth,
Western Australia, 3Microsoft Research, Microsoft Inc, Redmond, USA
HIV escapes immune recognition by mutating critical amino
acid residues in known HLA-restricted epitopes. Such changes
may limit the ability of ex-vivo assays to detect and map the
epitopes subject to such selection in-vivo. We used HLAassociated polymorphisms in HIV generated from an analysis of
a combined cohort of 800 anti-retroviral nave, HLA-diverse,
predominantly subtype B-infected individuals from the US and
Western Australia to predict and map HIV-specific T cell
responses(see figure 1). A novel epitope prediction program
Epi-pred was used to predict optimal length epitopes around
sites of HLA-allele specific polymorphism and these were tested
in IFN- ELISpot assays, taking into account the autologous
HLA genotype and the autologous viral sequence of 200 US
cohort individuals. The ELISpot assay was optimised and
automated for high-throughput testing of multiple HLAcustomised plates1 (see figure 2).
On average, 11 epitopes were
tested for each patient and of
these 18% elicited an IFN-
In an analysis of
the first 29 individuals tested
in this system, seven putative
novel epitopes were detected
(see table 1).
instances, the HLA-driven
change led to loss of reactivity
as predicted for classical CD8+
T-cell escape, however more
complex patterns of reactivity
were seen, particularly in Nef
epitopes (see figure 3).
Table 1. Seven novel epitopes were seen in the 29
Figure 3. Responses to adapted and non-adapted epitopes detected in ELISpot assays. (3A) Classical
escape the adapted epitope elicits a lower response compared to the non-adapted epitope. (3B) Nonclassical escape the adapted epitope elicits a higher response than the non-adapted epitope. (3C) No
difference in responses seen.
Method used to
690 SFU/ Nonadapted
Novel epitopes/escape variants:
High priority in
Known epitopes/novel escape
variants: Medium priority in
Test non-adapted and adapted epitopes.
Pollyallelic peptide panel
variants: Low priority in
Tat 42-50 HLAA*0301
No difference in
All associations/epitopes classified as individual cellular hypotheses to test with PBMCs.
CD8+ T-cell epitopes (8 to 11-mer) predicted by Epi-pred
Known epitopes from LANL
HIV mutates residues that affect HLA binding , TCR
recognition or intracellular processing allowing the virus to
escape detection by the host immune system (see figure 4 & 5).
If escape is extra-epitopic, then test non-adapted
Nef 71-81 and HLA-B*3501
Nef 90-97 and 83-91 and HLA-B*0801
Coat with INF-
Figure 2. Overview of the ELISpot
method. Each of the steps illustrated
can be set up as a separate method
using the Biomek FX.
Add peptide &
Figure 4. Amino acid change in anchor
Study population was drawn from ACTG 5142/5128, Beckman
Coulter, Bill & Melinda Gates Foundation, National Institutes
of Health, National Health & Medical Research Council and
CCIBS staff [email protected] [email protected]
900 SFU/106 PBMCs
Accredited for compliance with ISO/IEC 17025 interpreted
for research using CITAC Guide CG2, for HLA sequence
based typing, viral sequencing and ELISpot analysis.
Accreditation number 15785
720 SFU/106 PBMCs Neo-epitope
Figure 5. Mutations affecting TCR recognition and
The screening strategy was based on a genetics directed
approach, in which specific sites and epitopes were tested
based on their in-vivo polymorphism. The results have enabled
us to identify possible novel epitopes that warrant further
investigation using confirmatory assays. At a population level,
such screening takes into account the most prevalent HLA
genotypes and HLA-restricted responses in that population
efficiently. These results provide further insights into CD8+ Tcell responses against HIV and have implications for HIV
1. Almeida et al. 2009. Automation of the ELISpot assay for high-throughput
detection of antigen-specific T-cell responses. J Immunol Methods; 344:1-5
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