SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED

SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES PREDICTED

SCREENING FOR HIV RESPONSES USING OPTIMAL EPITOPES
PREDICTED BY HLA-VIRAL SEQUENCE POLYMORPHISM
ASSOCIATIONS.

Western Australian
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
1

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-
response.
In an analysis of
the first 29 individuals tested
in this system, seven putative
novel epitopes were detected
(see table 1).
In many
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).

Novel epitopes
Protein

Start
position

HLA

Epitope

Positive
responses
(n)

Env

209

A*0101

SFEPIPSHY

1

Env

310

Gag

406

Nef

9

Nef

178

B*1801

KEVLVWKF

1

Pol

8

Multiple

FPQGKAREF

1

Pol

901

B*2705

KRKGGIGGY

1

A*0101/Cw*0401 GPGPGRAFY
A*0302

1

RAPRKKGC
WK

1

A*0101/A*0302 SVVGWPAVR

1

Table 1. Seven novel epitopes were seen in the 29
patients tested.

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.

Figure 1.
Method used to
select patients
peptides to
perform
ELISpots on.

3A

Classical escape

Pol 901-909

HLA-B*2705
690 SFU/ Nonadapted
106
PBMCs epitope

KRKGGIGGY
SAG-----3

KRKGGIGEY

Novel epitopes/escape variants:
High priority in
screening assays

Known epitopes/novel escape
variants: Medium priority in
screening assays

Test non-adapted and adapted epitopes.

KEVLVWKF
DSR-----3

KEVLMWKF

Env 209-217

HLA-B*1501

5----- EKI

RLRPGGKKKY
KLK -----3

RLRPGGRKKY

Pollyallelic peptide panel
Known epitopes/escape
variants: Low priority in
screening assays

Gag 20-29

560
SFU/106
PBMCs
0
SFU/106
PBMCs

SFEPIPSHY
CAP -----3

SFEPIPSIY

Adapted
epitope

Tat 42-50 HLAA*0301

1900
SFU/106
PBMCs

Nonadapted
epitope

2800
SFU/106
PBMCs

Adapte
d
epitope*

5-----ITK

GLGISYGRK
KRR-----3

840 SFU/106
PBMCs

Non-adapted
epitope

900 SFU/106
PBMCs

Adapted
epitope

HLA-A*0101

5----- PKV

Non-adapted
epitope

No difference in
responses

HLA-B*1801

5-----DPE

10
Adapted
SFU/106 epitope
PBMCs

3C

Non-classical escape

Nef 178-185

5-----HNF

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

3B

20 SFU/
106
PBMCs

Nonadapted
epitope

740
SFU/106
PBMCs

Adapted
epitope

GLGISYGRR

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

ELISpot assay

Nef 90-97 and 83-91 and HLA-B*0801
5-----YKGALDLSHFLKEKGGLEGL-----3

Coat with INF-
capture Ab

Figure 2. Overview of the ELISpot
method. Each of the steps illustrated
can be set up as a separate method
using the Biomek FX.

RPQVPLRPMTF

Add peptide &
cells

570 SFU/10
PBMCs

200 SFU/106
PBMCs

Non-adapted
epitope

Adapted
epitope

Figure 4. Amino acid change in anchor
position

Add
streptavidin
enzyme
Add TMB

Acknowledgements
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]
http://www.ccibs.org

Non-adapted
epitope

FLKEEGGL

900 SFU/106 PBMCs

Adapted
epitope
imputed

FLKEMGGL

2500 SFU/106
PBMCs

Adapted
epitope
imputed

FLKENGGL

420 SFU/106
PBMCs

Adapted
epitope
imputed

FLKEQGGL

1540 SFU/106
PBMCs

Adapted
epitope
imputed

5-----YKGALDLSHFLKE(E/M/N/Q)GGL
EGL-----3

Add
biotinylated
detection Ab

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

RPQVPLRPMTY

6

3540 SFU/106
PBMCs

720 SFU/106 PBMCs Neo-epitope

Figure 5. Mutations affecting TCR recognition and
intracellular processing.

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
vaccine design.
References
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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