Generation of a Recombinant Gag Virus-Like-Particle Panel for the Evaluation of p24 Antigen Detection by Diagnostic HIV Tests

Abstract

BackgroundDetection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.MethodsWe generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.ResultsOur VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.ConclusionsThe HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Comparative multi-assay evaluation of Determine™ HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection

“…In only 18.8% (3/16) of the samples in this group, the old RDT identified the presence of HIV-1 p24CA. Even though the size of our cohort was limited, these data are in accordance with the results from other studies that reported poor HIV-1 p24CA sensitivity for the old RDT, ranging from 10.1 to 65.4% [22,24,26,28]. Two studies conducted in sub-Saharan Africa concluded that the old version of the RDT had 0% sensitivity in specimens from acutely infected patients [44,45].…”

“…The HIV-1 patient specimens evaluated in this study are mostly from subtype B, but also include the subtypes A1, C, CRF01_AE, CRF02_AG, F and URF. For future studies, it would be valuable to evaluate the performance of the RDTs with an even more diverse panel of HIV-1 specimens, especially since the old version of the RDT was shown to have impaired sensitivity to capsid antigen from non-B subtype HIV-1 [24].…”

“…One of the most frequently employed fourth-generation RDTs is the Determine™ HIV-1/2 Ag/Ab Combo (Alere) that works with either serum or whole blood samples. Several studies indicated a poor sensitivity for the detection of HIV-1 p24CA for the early version of this RDT [22][23][24][25][26][27][28], and therefore, Alere developed and released a modified version of this test. The sensitivity of this modified RDT to detect HIV-1/2 antibodies was shown to be high with low rates of false positive results [29][30][31].…”

The fourth generation Alere TM HIV Combo rapid test improves detection of acute infection in MTN-003 (VOICE) samples

“…Reported sensitivity for antigen detection was higher in samples from the United Kingdom (50%), the United States (45.5% and 75.8%), Italy (88.2%) and in mixed-subtype seroconversion panels (86.6%) suggesting clade differences [3,7–10]. Evaluation of Gag-expressing virus-like particles (VLP) showed that 6 of 7 subtype B VLPs were detected by Alere Determine™ HIV-1/2 Ag/Ab Combo, compared to only 1 of 5 subtype C, and 3 of 31 non B/non-C-subtypes [11]. …”

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

“…Manufacturers of diagnostic tests evaluate sequence-associated test sensitivity by testing HIV strains from different subtypes and frequent circulating recombinant forms (CRFs), thus hoping to cover a range of sequence variants. However, in a recent study conducted by our laboratory, using 43 virus-like-particles (VLPs) derived from clinical HIV-1 isolates expressing the Gag proteins of different subtypes and CRFs, we noticed that the failure of a diagnostic test to detect p24 of certain virus isolates was not necessarily related to their subtype, as even otherwise highly sensitive tests failed to detect a common subtype B VLP [ 4 ]. We hypothesised that detection failure could be due to subtype-independent amino acid variations and analysed our p24 sequences in relation to amino acid variation and detection failure.…”