Impact of naturally occurring amino acid variations on the detection of HIV-1 p24 in diagnostic antigen tests

Abstract

BackgroundThe detection of HIV-1 p24 antigen in diagnostic tests relies on antibodies binding to conserved areas of the protein to cover the full range of HIV-1 subtypes. Using a panel of 43 different virus-like particles (VLPs) expressing Gag from clinical HIV-1 isolates, we previously found that some highly sensitive tests completely failed to detect p24 of certain VLPs, seemingly unrelated to their subtype. Here we aimed to investigate the reason for this failure, hypothesising that it might be due to single amino acid variations in conserved epitopes.MethodsUsing amino acid alignment, we identified single amino acid variations at position 16 or 170 of p24, unique to those VLPs that failed to be detected in certain diagnostic tests. Through DNA-mutagenesis, these amino acids were changed to ones more commonly found at these positions. The impact of these changes on p24 detection was tested in commercial diagnostic tests as well as by Western Blot and ELISA, using epitope-specific antibodies.Results and ConclusionsChanging positions 16 or 170 to consensus amino acids restored the detection of p24 by the investigated diagnostic tests as well as by epitope-specific antibodies in Western Blot and ELISA. Hence, single amino acid changes in conserved epitopes can lead to the failure of p24 detection and thus to false-negative results. To optimise HIV diagnostic tests, they should also be evaluated using isolates which harbour less-frequent epitope variants.

Epidemiological trends of HIV‐1 infection in blood donors from Catalonia, Spain (2005‐2014)

“…The development of drug‐resistant isolates in patients receiving antiretroviral therapy and HIV‐1 strains with transmitted drug‐resistance mutations (TDRMs) in newly infected patients are major threats to the success of HIV‐1 prevention and treatment efforts . In addition, HIV‐1 sequence diversity can negatively affect the capacity of in vitro diagnostic and screening systems to detect and intercept HIV‐1–positive blood donations …”

“…13 In addition, HIV-1 sequence diversity can negatively affect the capacity of in vitro diagnostic and screening systems to detect and intercept HIV-1positive blood donations. 14 The aims of this study were to determine the prevalence and distribution of HIV-1 subtypes and TDRMs in blood donors diagnosed with HIV-1 infection in Catalonia and the associations with transmission risk factors and demographic parameters.…”

The Halo Library, a Tool for Rapid Identification of Ligand Binding Sites on Proteins Using Crystallographic Fragment Screening

“…A colorimetric assay (Roche), was used to determine the effect of fragment binders on RT polymerization activity. 39 The widely utilized PicoGreen assay 40 was not effective for assessing fragment inhibition, as unspecific quenching of PicoGreen fluorescence was caused by several Halo Library fragments (data not shown). The assay used follows a sandwich ELISA protocol that leads to the formation of a colorimetric product, causing an absorbance increase which is directly proportional to RT activity.…”

Computational Design of Epitope-Enriched HIV-1 Gag Antigens with Preserved Structure and Function for Induction of Broad CD8+ T Cell Responses

“…Thus, some epitopes may have been derived from defective viruses against which a CTL response could be primed anyway. However, it can neither be ruled out that the experimental assessment led to a false classification of an AAS by affecting binding of the antibodies used in the ELISA analysis 60 . Alternatively, some mutations causing a fitness defect could have been present in a Gag-variant also harboring a compensatory mutation, as has for instance been reported for the HLA-B27-restricted KK10 epitope 61 .…”