Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity.
BACKGROUND
Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity.


METHODS
We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response.


RESULTS
The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05).


CONCLUSIONS
These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.
Amicosante
Massimo
M
Laboratory of Clinical Pathology, National InstiTute for Infectious Disease, Lazzaro Spallanzani, Rome, Italy. amicosante@inmi.it
Gioia
Cristiana
C
Montesano
Carla
C
Casetti
Rita
R
Topino
Simone
S
D'Offizi
Gianpiero
G
Cappelli
Giulia
G
Ippolito
Giuseppe
G
Colizzi
Vittorio
V
Poccia
Fabrizio
F
Pucillo
Leopoldo P
LP
D000328:Adult; D000595:Amino Acid Sequence; D001681:Biological Assay; D005260:Female; D015683:Gene Products, gag / Q000276:immunology; D006678:HIV / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism
D015683:Gene Products, gag; D006680:HLA Antigens; D010455:Peptides; D007371:Interferon-gamma