Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC

Abstract

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.

Bispecific antibodies directed to CD4 domain 2 and HIV envelope exhibit exceptional breadth and picomolar potency against HIV-1

“…This experiment was considered pertinent because TZM-bl cells express ∼2-to 10-fold more CD4 molecules than primary CD4 + T cells (37,38), which could influence their activity. Thus, we tested PG9-iMab against a subset (n = 23) of the 118 strains previously tested in the TZM-bl/pseudovirus assay, using replication-competent reporter (Env-IMC-LucR) viruses in a PBMC neutralization assay (39). This subset of viruses was shown to be representative of the full panel of HIV-1 strains (Fig.…”

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

“…3,27-31 However, we found that these EMCV IRES-containing reporter viruses vastly overexpress Nef 8 and exhibit both poor replication and stability of the reporter gene (personal observations and Brown et al 29 ). To overcome these challenges, we explored replication-competent HIV-1 reporter viruses that utilized the small ''ribosome skipping'' T2A peptide (18 amino acids) to mediate Nef expression in frame with, and release from (via ribosome skipping of the last peptide bond at the C-terminus of the T2A peptide 32,33 ), LucR.…”

“…of protection-encompassing proviral infectious molecular clones (IMC) of transmitter/founder (T/F) HIV-1 [4][5][6] and several forms of reporter virus derivatives expressing genes such as enhanced green fluorescent protein (EGFP) 7 and Renilla luciferase (LucR), 4,8 which underpin new immune monitoring assays and augment the performance of existing assays for various vaccine discovery approaches. 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).…”

“…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).…”

“…To overcome these challenges, we explored replication-competent HIV-1 reporter viruses that utilized the small ''ribosome skipping'' T2A peptide (18 amino acids) to mediate Nef expression in frame with, and release from (via ribosome skipping of the last peptide bond at the C-terminus of the T2A peptide 32,33 ), LucR. 8 Furthermore, the molecular strategy was designed to facilitate expression of heterologous env genes in an isogenic (NL4-3-derived) proviral backbone, collectively referred to as Env-IMC-LucR.T2A, or simply LucR.T2A reporter viruses. As previously reported, 3,8 this approach enables sensitive detection of infection, as measured by reporter gene expression, in assays that require replication-competent HIV-1, including those utilizing peripheral blood mononuclear cells (PBMCs) or other primary cells and nonreporter T cell lines.…”