Development of Two Avidity-Based Assays to Detect Recent HIV Type 1 Seroconversion Using a Multisubtype gp41 Recombinant Protein

Abstract

Current laboratory methods to detect recent HIV-1 infection for the estimation of incidence have various limitations, including varying performance in different subtypes or populations. Therefore, new methods are needed to detect recent infections with increased specificity. We developed a recombinant protein, rIDR-M, that covered divergent sequences from the immunodominant region (IDR) of gp41 from all major subtypes and recombinants of HIV-1 group M and expressed in Escherichia coli. The rIDR-M protein was highly reactive with HIV antibodies in sera from different subtypes and equivalently detected antibodies to divergent subtypes B and AE from Thailand, in contrast to individual gp41 peptides derived from respective subtypes, suggesting that it can be used for incidence assays. The protein was used in two different assay formats to measure antibody avidity: (1) a two-well avidity index assay (AI-EIA) and (2) a new one-well limiting antigen avidity assay (LAg-avidity EIA), both with a pH 3.0 buffer to dissociate low-avidity antibodies present during early infection. Limiting the amount of antigen allowed detection of recent HIV-1 infection, with or without dissociation buffer, but the detection was most efficient when the pH 3.0 dissociation buffer was included. When a well-characterized 41-member seroincidence panel (20 recent and 21 long-term) was used, both the two-well AI-EIA and one-well LAg-avidity EIA efficiently distinguished recent and long-term infections. The new avidity-based assays using rIDR-M antigen may improve the accuracy of detecting recent HIV-1 infection and allow a better estimation of incidence in diverse HIV-1 subtypes.

A Comprehensive Evaluation of the Proficiency Testing Program for the HIV-1 BED Incidence Assay

“…Several groups have reported the development of different incidence assays (1,21,25,27,29,30). As these assays become implemented more widely, it will be important to ensure the quality of testing by multiple approaches, including the PT program.…”

What Do Chaotrope-Based Avidity Assays for Antibodies to HIV-1 Envelope Glycoproteins Measure?

“…We refer to it as the functional affinity index [FAI]. In some other studies, the level of Ab binding in the presence of a constant or varied chaotrope concentration relative to the level of binding without chaotrope is reported (42,44,47,48,73,(77)(78)(79)(80)(81)(82)(83)(84). Accordingly, we also calculated the reduction in Ab-binding plateaus, or B max values, and defined the B max index (BMI) as the B max with chaotrope divided by the B max without, expressed as a percentage.…”

Short Communication: The Impact of Viral Suppression and Viral Breakthrough on Limited-Antigen Avidity Assay Results in Individuals with Clade B HIV Infection

“…4 The newest commercially available form of this technology, also developed by the CDC, is a limiting antigen avidity enzyme immunoassay (LAg-Avidity) that measures the antigen-specific antibodies to very low concentration of recombinant gp41 peptide in the context of a chaotropic agent. 5 Cross-sectional incidence assays, specifically the LAgAvidity, have been shown to misclassify individuals infected >2 years who are virally suppressed as recently infected. 5,6 The antibody response to HIV (both titer and avidity) in individuals who start antiretroviral treatment (ART) early in HIV infection (<12 months) is often reduced.…”

“…5 Cross-sectional incidence assays, specifically the LAgAvidity, have been shown to misclassify individuals infected >2 years who are virally suppressed as recently infected. 5,6 The antibody response to HIV (both titer and avidity) in individuals who start antiretroviral treatment (ART) early in HIV infection (<12 months) is often reduced. 8 In contrast, individuals who start ART later in the course of infection have varied antibody responses, where titer decreases but avidity remains high.…”

“…10 This may reflect the fact that the LAg-Avidity assay and BED-CEIA use nearly identical target peptides. 3,5 Although the LAg-Avidity assay has a lower rate of misclassification than the BED-CEIA among elite suppressors and has a lower median decrease in OD-n values in virally suppressed individuals, the Lag-Avidity assay and BED-CEIA have similar patterns of misclassification in individuals with viral suppression and viral breakthrough. These results are supporting previously published data that suggested LAg Avidity fluctuated with viral load.…”