title: Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity.
abstract: 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.
authors.affiliation: Laboratory of Clinical Pathology, National InstiTute for Infectious Disease, Lazzaro Spallanzani, Rome, Italy. amicosante@inmi.it
authors.forename: Massimo
authors.initials: M
authors.lastname: Amicosante
authors.forename: Cristiana
authors.initials: C
authors.lastname: Gioia
authors.forename: Carla
authors.initials: C
authors.lastname: Montesano
authors.forename: Rita
authors.initials: R
authors.lastname: Casetti
authors.forename: Simone
authors.initials: S
authors.lastname: Topino
authors.forename: Gianpiero
authors.initials: G
authors.lastname: D'Offizi
authors.forename: Giulia
authors.initials: G
authors.lastname: Cappelli
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ippolito
authors.forename: Vittorio
authors.initials: V
authors.lastname: Colizzi
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Poccia
authors.forename: Leopoldo P
authors.initials: LP
authors.lastname: Pucillo
mesh_terms: 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
chemical_list: D015683:Gene Products, gag; D006680:HLA Antigens; D010455:Peptides; D007371:Interferon-gamma