Oral Abstract Session 01 - Global HIV Vaccine Enterprise

Topic 11: T Cell Immunity
P11.01
Evaluation of the Dual IFNγ/IL-2 Fluorospot-Assay
with Flow Cytometry For Detection of HLA-Restricted
HIV-Specific T-Cell Responses in HIV Controllers
N. Keane 1 , C. Almeida 1 , S. Roberts 1 , I. Ahmad 1 , S. Mallal 1 ,
M. John 1
1 Murdoch University, Perth, Australia
Background: The IFNγ ELISpot assay is used widely for high
through-put screening of HIV-specific responses in studies of
HIV infection and vaccine studies. However, dual production of
IFN/IL-2 and increased proliferative capacity may be associated
with better natural control of HIV infection. We evaluated a
novel fluorospot assay enabling the identification of dual IFNγ/
IL-2 producing antigen-specific cells and compared it with
intracellular cytokine staining by standard flow cytometry in
individuals with natural control of HIV-infection.
Methods: PBMC from five untreated HIV-infected individuals
were stimulated overnight with HIV peptides or controls in
dual IFN/IL-2 pre-coated plates. Peptide arrays were specifically
selected based on the individual HLA type. Secreted IFNγ and
IL-2 were detected using fluorescent-conjugated antibodies.
Fluorescent spots were enumerated on the iSpot AID reader.
Responses were considered positive if >50 spots/million cells
SFU after background subtraction.Positive responses were then
evaluated by flow cytometry using the Gallios flow cytometer.
Results: Dual IL-2/IFNγ producing cells were detected to anti-CD3stimulated
PBMC from all patients. IFNγ responses alone were
detected to 35 of 136 HLA-restricted peptides tested (median
=73, 52-4190 SFU) across the 5 patients (1/19, 5/19, 7/28, 9/15
and 13/55 for each patient), while IL-2 responses were either low
grade or undetectable for the majority of HIV peptides tested.
Dual IFNγ/IL-2 producing HIV-specific T cells were not detected
using the fluorospot assay. 24/35 peptides induced CD8 T cell-
IFNγ production by flow cytometry.
Conclusion: HIV-specific mono-IFNγ responses were detected
using the novel fluorospot assay. However limited HIV-specific dual
IFNγ/IL-2 responses were detected in this patient group. A greater
number of epitope-specific positive responses were detected
in the fluorospot compared with flow cytometry suggesting the
fluorospot may be more sensitive in detecting a greater breadth
of epitope-specific T cell responses, and therefore better for
screening purposes than flow cytometric methods.
P11.02
AIDS Vaccine 2012
Posters
Early Changes in the CD8 T Cell Immunodominance
Hierarchy in Primary HIV Infection Prior to
Seroconversion
N. Keane 1 , C. Almeida 1 , A. Chopra 1 , D. Cooper 1 , E. Demaine 1 ,
S. Mallal 1 , M. John 1
1 Murdoch University, Perth, Australia
Background: Identification of the earliest CD8 T-cell responses
against HIV may help select critical viral targets for inclusion in
an HIV vaccine. We describe changes to the earliest detected
CD8 T-cell responses and changes in genetic sequence encoding
targeted epitopes in an individual who presented with Fiebig stage
II, clade C acute HIV-infection, 27 days after sexual transmission.
Methods: We examined HLA-restricted CD8 T-cell responses by
IFNγ ELISpot and HIV Gag, Pol, Nef and Env sequence by 454
deep sequencing over 6 timepoints, from days 27-118 after
HIV-transmission. HIV-specific IFNγ responses were detected
against ten HIV epitopes in Gag, Nef and Env at day 27 post HIVtransmission
when the patient’s CD4 T-cell count was at a nadir
of 224 cells/_l, the plasma HIV RNA >106 copies/ml and prior to
any detectable p24 antibody.
Results: Immunodominant responses were detected to the
HLA-B*07:02 restricted Env IIRRIRQGL (IL9) epitope and
the B*07:02 Gag GPGHKARVL (GL9) epitope (>4000SFU). A
detectable but weaker response was observed to HLA-B*08 Nef
FLKEKGGL (FL8) epitope (1010SFU). These responses declined
over subsequent timepoints to 470 and 1630SFU on day 118
coincident with a 2 log fall in the plasma viral load, a rise in the
CD4 T cell count to 533 cells/µL and antibody seroconversion.
The Nef FL8 became the immunodominant response after day
34. IFN_ responses broadened from 10 responses at day 27 to
23 responses by day 118. Analysis of Gag, Nef and Pol genes by
454 deep sequencing showed no evidence of escape within the
targeted epitopes as a cause of their decline over time.
Conclusion: Early changes in the CD8 T-cell immunodominance
hierarchy are apparent in acute HIV-infection prior to
seroconversion, including early immunodominant targeting of
Env epitopes. Subsequent broadening of the CD8 T-cell response
was not associated with CD8 T-cell escape in this case.
217
POSTERS