Background
Human immunodeficiency virus (HIV) envelope glycoprotein 120 (gp120) can be shed from the cell surface as soluble gp120 (sgp120) contributing to HIV pathogenesis and immune dysfunction. Accurate measurement of sgp120 and host antibody responses is important for understanding its role in HIV disease progression. Conventional enzyme-linked immunosorbent assay (ELISA) based methods are limited by their single-analyte detection capacity and reduce sensitivity at low levels. Furthermore, the formation of sgp120 immune complex with circulating antibodies can obscure epitopes and hinder detection. To overcome these challenges, we have developed a novel multiplex fluorescence immunoassay (MFIA) capable of dissociating immune complexes and quantifying HIV antigen-specific antibodies and sgp120 levels in plasma and sera.
Methods
HIV antigens (gp140, gp41, gp120, p24, p51, Tat, ID2) or sgp120 specific antibodies (C11 and A32) were coupled to microsphere beads. HIV-specific IgG responses were detected using phycoerythrin (PE)-conjugated anti-human IgG secondary antibody. Sgp120 levels were quantified using PE-conjugated broadly neutralizing antibody N6. All measurements were performed on the Luminex MAGPIX system. Data were interpolated from standard curves using GraphPad Prism software and Spearman correlation statistical analysis was conducted using SPSS Statistics.
Results
The MFIA simultaneous detected HIV-specific IgG antibodies against all seven HIV antigens. Serum from PLWH (n=163) were analyzed, and HIV-specific antibodies were detected in 100% of the samples. Antibody responses to gp140 (p < 0.001), gp41 (p = 0.007), gp120 (p = 0.015), p24 (p = 0.038), and cluster A (p < 0.001) showed significant positive correlations with the percentage of CD8+ T cells. Picogram levels of sgp120 was detected in 76 of 163 samples (47%) and significantly associated with elevated levels of the proinflammatory cytokine IL-6 (p = 0.041).
Conclusions
Our MFIA can detect HIV-specific IgG antibodies and quantify low levels of sgp120 in serum samples compared to conventional ELISA. The presence of sgp120 was significantly associated with elevated IL-6 levels, suggesting a link between circulating sgp120 and immune activation. Additionally, the correlation between specific antibody responses and the percentage of CD8+ T cells support a potential role for antibody-dependent cellular cytotoxicity. This immunoassay provides a valuable tool for exploring mechanisms of HIV pathogenesis in ART treated individuals.