Impact of naturally occurring amino acid variations on the detection of HIV-1 p24 in diagnostic antigen tests.
BACKGROUND
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. Here we aimed to investigate the reason for this failure, hypothesising that it might be due to single amino acid variations in conserved epitopes.


METHODS
Using 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.


CONCLUSIONS
Changing 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.
Swiss National Center for Retroviruses (SNCR), Institute of Medical Virology, University of Zürich, Zürich, Switzerland. vetter.beatrice@virology.uzh.ch.
Beatrice N
BN
Vetter
Institute of Molecular Life Science, University of Zürich, Zürich, Switzerland. orlowski.vanessa@uzh.ch.
Vanessa
V
Orlowski
Blood Transfusion Service, Swiss Red Cross Berne (BSDSRK), Berne, Switzerland. christoph.niederhauser@bsd-be.ch.
Christoph
C
Niederhauser
Official Medicines Control Laboratory, Biologika, Swissmedic, Berne, Switzerland. louise.walter@swissmedic.ch.
Louise
L
Walter
Swiss National Center for Retroviruses (SNCR), Institute of Medical Virology, University of Zürich, Zürich, Switzerland. schupbach.jorg@virology.uzh.ch.
Jörg
J
Schüpbach
D015492:AIDS Serodiagnosis / Q000379:methods; D000596:Amino Acids / Q000235:genetics; D000956:Antigens, Viral / Q000276:immunology; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D016655:HIV Core Protein p24 / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011933:Reagent Kits, Diagnostic
D000596:Amino Acids; D000956:Antigens, Viral; D000939:Epitopes; D016655:HIV Core Protein p24; D011933:Reagent Kits, Diagnostic