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

Beatrice N Vetter,Jörg Schüpbach,Vanessa Orlowski,Christoph Niederhauser,Louise Walter

doi:10.1186/s12879-015-1174-7

Beatrice N Vetter, Jörg Schüpbach + Show 3 more

Open Access

https://doi.org/10.1186/s12879-015-1174-7

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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.

Highlights

The 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
Amino acid variants To identify amino acid variations which might be responsible for the failure to detect p24 in a diagnostic test, the p24 sequences of all 43 virus-like particles (VLPs) panel members investigated in our previous study were aligned [4]
We identified a variation of Lysine (K) to Arginine (R) or Asparagine (N) at position 170, which might be accountable for the failure of the bioMérieux VIDAS DuoUltra and VIDAS HIV p24 II to detect VLPs pBV43-D, pBV60-D and pBV42-12BF

Summary

Introduction

The 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. The viral antigen is detected in a sandwich format, employing antibodies binding to distinct regions of the protein for capture and detection. These antibodies should bind to highly conserved regions of the target protein, minimizing the risk of reduced antigen sensitivity due to subtype-dependent sequence variability or evolutionary escape, both potentially leading to false-. We sought to identify amino acid variations that abrogated the detection of p24 in diagnostic HIV-1 p24 antigen-only or 4th-generation HIV screening assays, or by epitopespecific antibodies used in home-made tests

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