An adaptation of recombinant vaccinia-based ELISPOT and intracellular cytokine staining for a comparative measurement of cellular immune responses in HIV-1 and HIV-2 infections in West Africa.

An efficient and quantitative tool for rapid assessment of human immunodeficiency virus (HIV)-induced cellular immune responses is important for resource-limited settings, such as in sub-Saharan Africa. Modifications are required to previously reported methods for evaluating ex-vivo antigen-specific cellular responses based on direct recombinant vaccinia virus (rVV) stimulation of peripheral blood mononuclear cells (PBMCs) by enzyme linked immunosorbent assay (ELISPOT) and by flow cytometry intracellular cytokine assay (ICA). We made such modifications in order to detect specific responses and compared quantitative cellular immune responses in HIV-1 and HIV-2 infected Gambians. The sensitivity of the rVV-based ELISPOT assay was on average 1.25 interferon (IFN)-gamma spot forming cells (SFC) per 50 000 PBMCs specific for either infection, and 5 IFN-gamma-secreting CD8+ T cells/50 000 in the ICA. The level of IFN-gamma SFC detected by ELISPOT and by ICA were correlated (P < 0.02). ICA detected pol-specific responses in 88% and 67% of HIV-1 and HIV-2 subjects, respectively, and gag-specific responses in more than 80% of both infections. Lower proportions of responders were obtained with ELISPOT, for which pol responses were present in 60% of HIV-1 and 46% of HIV-2 infected patients, and gag responses in 55% and 69%, respectively. The assays did not show any significant difference in cellular immune responses between HIV-1 and HIV-2 infected subjects with CD4% >or= 20%. These outcomes are comparable with results obtained using standard techniques and thus this method is a suitable, rapid and less expensive assessment of cellular immunity.

Absolute lymphocyte count as surrogate for CD4+ cell count in monitoring response to antiretroviral therapy.

The objective of the study is to determine whether Absolute Lymphocyte Count (ALC) can serve as a surrogate for CD4 T-lymphocyte Cell Count (CCC) in HIV infected Nigerians on Lamivudine/Zidovudine anti-retroviral therapy. 32 adult Nigerians infected with HIV were recruited into the study. They were assessed clinically and categorised into three clinical stages A, B and C according to CDC criteria. They all received lamivudine 150 mg b.d and Zidovodine 300 mg b.d for six months. Blood specimens were taken on enrollment and at four weekly intervals for paired ALC and CCC determination. ANOVA statistics was used to determine whether ALC and CCC (separately) change significantly with increasing duration of therapy. Paired ALC and CCC values were tested for correlation. Sensitivity and specificity of low ALC values in predicting low CCC values were also calculated. The 32 patients comprised 18 males and 14 females aged between 16 and 49 years. The mean age (SD) was 36.1 (+/-7.85) years. The mean ALC value rose from 2485/l and 2352/microl before commencement of therapy to 3026/microl and 3151/microl four weeks after for males and females respectively. These changes were not significant, P>0.05. No further changes were noted over the next 24 weeks. However, the mean CCC values increased from 233/microl before therapy through 339/microl at four weeks, 362/microl at eight weeks to 398/micro1 at 12 weeks. It then fluctuated between 372/microl and 310/microl for the remaining part of the study. These changes were not significant: F: ratio = 1.28 (df = 6,181), P>0.05. A weak but significant positive correlation was established between ALC and CCC. Correlation coefficient was 0.25, P<0.05. The sensitivity and specificity of ALC 2000/microl as a predictor of CCC 200/microl were 57% and 72% respectively. ALC correlates weakly with CCC in patients undergoing antiretroviral therapy and it may not serve as a perfect surrogate for CCC as a monitor of immunological response to therapy.