AEGiS-13IAC: Study of HIV-specific CD8+ T cells in infected children using enzyme-linked immuno spot assay (Elispot).

13th International AIDS Conference

Durban, South Africa - July 9-July 14, 2000

Study of HIV-specific CD8+ T cells in infected children using enzyme-linked immuno spot assay (Elispot).

Int Conf AIDS 2000 Jul 9-14; 13:(abstract no. MoOrA221)

Buseyne F, Porrot F, Fleury Corre B, Blanche S, Rouzioux C, Rivifre Y;;; F. Buseyne, Institut Pasteur, Laboratoire d'Immunopathologie virale, Batiment des Retrovirus, 28, rue du Dr Roux, 75015 Paris, France, Tel.: +33 1 4568 8899, Fax: +33 1 4061 3012, E-mail: florence@pasteur.fr

BACKGROUND: HIV-specific cytotoxic CD8+ T cells play a major role in the control of disease progression. These cells can be detected by their IFN-g production after stimulation with antigen. This technique is much more sensitive than methods based on lysis of target cells. We used Elispot technique to define the HIV-specific CD8+ T cells in perinatally infected children.

METHODS: Freshly isolated peripheral blood mononuclear cells (PBMCs) were infected with recombinant Vaccinia viruses expressing the Env, Gag and Pol proteins of HIV and added to wells precoated with anti-IFN-g antibodies. Spot forming cells, i.e., HIV-specific T cells, were detected 16 hours after by the addition of biotinylated anti-IFN-g specific antibodies, followed by streptavidin bound alkaline phosphatase.

RESULTS: Preliminary study showed that all HIV-infected children (10/10) responded to the Env-Gag-Pol construct (mean: 765 IFN-g spot forming cells (SFC)/106 PBMCs versus 21 SFC/106 for wild-type Vaccinia; range: 129 to 1100 SFC/106 PBMCs). In 51Cr release assay using freshly isolated PBMCs the frequencies of responders were lower: 0/19 were positive for Env and 7/39 were positive for Gag. These results showed that the Elispot technique is much more sensitive than standard techniques to detect HIV-specific CD8+ T lymphocytes. Use of recombinant Vaccinia viruses allowed the detection of CD8+ T cells directed against their major viral targets, without need of previous identification of CTL epitope and their HLA-restriction, and can thus be widely used.

CONCLUSION: HIV-specific CD8+ T cells can be easily detected in peripheral blood of HIV-infected children using Elispot assay to detect IFN-g production in response to HIV antigens. Recombinant vaccinia viruses suited well for stimulation of responding cells in this assay. This technique is much more sensitive than standard techniques and will allow re-evaluation of CD8+ T lymphocytes response against HIV in perinatally infected children.

Keywords: AEGIS, Antigens, CD8, HIV Infections, HIV Antigens, HIV Seropositivity, T-Lymphocytes, Cytotoxic, Aluminum Hydroxide, Enzyme-Linked Immunosorbent Assay, Gene Products, pol, Vaccinia, Vaccinia virus, Link, Child, Human, immunology
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MoOrA221