Differential Inhibition of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and TZM-bl Cells by Endotoxin-Mediated Chemokine and Gamma Interferon Production

Abstract

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1

“…Much of this discrepancy was demonstrated to be an artifact of endotoxin contamination affecting the PBMC assay (Geonnotti et al, 2010). Endotoxin, which is bacterial lipopolysaccharide stimulates susceptible PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, and can cause false-positive results in neutralizing antibody assays done in PBMCs, while assays performed in TZM-bl cells are unaffected (Geonnotti et al, 2010). …”

Purification and functional characterization of mucosal IgA from vaccinated and SIV-infected rhesus macaques

“…3). This assay is unaffected by endotoxin present in the samples [50]. In the vaccinated cohort, 4 of 5 fecal IgA samples exhibited neutralizing activity, with the exception being animal P889 (Fig.…”

Towards an AIDS vaccine: The transmembrane envelope protein as target for broadly neutralizing antibodies

“…The influence of endotoxin on the results of neutralization assays was well studied. 61 Endotoxin in the form of lipopolysaccharides may be released into sera by contaminating bacteria and can induce beta-chemokines in blood cell-based neutralization assays inhibiting HIV-1 infection as shown by. 61 In addition, interferon gamma released from blood cells after interaction with endotoxin may give false positive results in TZM-bl cell-based neutralizing assays.…”

“…61 Endotoxin in the form of lipopolysaccharides may be released into sera by contaminating bacteria and can induce beta-chemokines in blood cell-based neutralization assays inhibiting HIV-1 infection as shown by. 61 In addition, interferon gamma released from blood cells after interaction with endotoxin may give false positive results in TZM-bl cell-based neutralizing assays. Therefore, preparation, handling, transport, freeze-thawing and storage of preimmune and immune sera are of great importance when their neutralizing activity is investigated.…”