High‐throughput quantitative analysis of HIV‐1 and SIV‐specific ADCC‐mediating antibody responses

Abstract

We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.

Anti-HIV Antibody–Dependent Activation of NK Cells Impairs NKp46 Expression

“…We now show that activation-induced MMP production decreases NKp46 expression on NK cells. We note that it will be useful to corroborate our findings of NKp46 downregulation on NK cells activated through CD16 with studies analyzing NKp46 expression on NK cells activated by Ab-coated HIV-infected cells (40)(41)(42). However, the currently presented observation raises several questions about the role of anti-HIV ADCC, and antiviral NK cell responses in general, in slowing the progression of chronic HIV infection toward AIDS.…”

Optimized ReplicatingRenillaLuciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

“…3,4,[8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Among formerly described replication-competent HIV-1 reporter vectors were those designed with a bicistronic EGFP-IRES-nef cassette in place of nef, in which the EGFP gene is downstream of env, and nef is under translational control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV).…”

“…8 In this regard, the LucR.T2A reporter virus technology offers several critical advantages and has found wide application, including enabling of a novel, highly sensitive T cell-based assay (referred to as the A3R5/Env-IMC-LucR neutralization assay) 9,10 for measuring NAb activity in vaccine sera from the RV144 and Vax003 HIV-1 vaccine trials in Thailand. 11,34,35 The reporter viruses have also underpinned the development of novel CD8 + T cell virus inhibition assays (VIA), 12,19 ADCC assays, [13][14][15] and a humanized mouse model of HIV-1 transmission. 16 The majority of current applications do not require functional Nef expression; however, the LucR.T2A strategy may be a limitation in certain applications such as correlates of protection discovery that require ''whole genome'' T/F reporter IMC.…”

Antibody functional assays as measures of Fc receptor-mediated immunity to HIV - new technologies and their impact on the HIV vaccine field.

“…Such assays include the rapid-fluorimetric ADCC assay (RFADCC) [150,151], NK viral inhibition assays [68,152], lactate dehydrogenase release assay [153], granzyme delivery assays [154,155], and NK cell activation assays measuring interferon-gamma and/or CD107a [13]. Such assays can be long, complex and difficult to standardize or reproduce precisely.…”

“…The GrB assay, marketed by OncoImmunin as GranToxiLux ® , measures delivery of the enzyme to target cells, which are preloaded with a GrB targeted substrate that becomes fluorescent upon cleavage. This method was evaluated by Pollara et al (2011) for measuring the ADCCmediating capacity of Abs against HIV and SIV in humans and non-human primate models [154].…”