New virologic reagents for neutralizing antibody assays

Abstract

Comprehensive assessment of anti-HIV-1 antibody potency and breadth is essential for evaluating vaccine immunogens, the advancement of vaccine candidates into clinical trials, and ultimately the development of effective vaccine strategies. Env-pseudovirion and recombinant reporter cell line neutralization assays are important tools for rapid and standardized measurement of neutralizing antibody activity. However, recent studies indicate that reporter cell lines fail to detect neutralization activity of certain antibodies observed when analyzed in peripheral blood mononuclear cells and may yield results on neutralizing antibody breadth that are discordant with peripheral blood mononuclear cell assays. Importantly, it remains unknown whether current in-vitro assays may be predictive of a protective neutralizing antibody response elicited by vaccine immunogens. This situation underscores the significance of standardizing existing, complementary methods as well as developing new assay concepts that assess neutralization in primary cells. Thus, this chapter focuses on new virologic reagents that promise to facilitate reaching this goal.

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

“…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.…”

“…5 and 6), we investigated alternative reporter virus strategies that utilized an IRES bicistronic reporter system. We and others have found the performance of previous EMCV IRES HIV-1 reporter viruses (e.g., NLENG1i) 3,[27][28][29] to be suboptimal with respect to replication, stability of the EGFP reporter gene (personal observations), and supraphysiological overexpression of Nef.…”

“…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).…”

T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone

“…KK10-specific TCR expression by the transduced CD8 ϩ T cells was determined by sequential staining with PE-labeled HLAB*2705/KK10 dextramer for 10 min at room temperature and with Pacific Blue-labeled anti-CD8a antibody (BD Biosciences) for 20 min at 4°C followed by washing with PBS and analysis by flow cytometry. In parallel, HLA-B*2705-expressing GXR cells (1 ϫ 10 5 cells/well) were infected in 96-well plates with HIV-LucR (MOI, ϳ0.5), an infectious HIV-1 molecular clone that expresses the HIV-1 JR-CSF Env and a Renilla reniformis luciferase (LucR) reporter gene (67,68). One day later, the HIV-1-infected GXR cells and either mock-transduced or lentivirus-transduced CD8 ϩ T cells were added at an effector-to-target ratio of 1:1 in quadruplicate and cultured for an additional 3 days.…”

Characterization of Simian Immunodeficiency Virus Variants Anatomically Compartmentalized in Plasma and Milk in Chronically Infected African Green Monkeys

“…For certain experiments, replication competent reporter viruses encoding SIV Env and Renilla luciferase (LucR) were utilized. pNL-LucR.T2A-SIVagm.sab92018.ps was constructed by modifying the previously described pNL-LucR.T2A-Env.ecto HIV-1 reporter virus approach (29,30) such that SIV env genes of interest could be expressed in an established robust, isogenic reporter virus backbone. It has been previously reported that SIV Env cytoplasmic tail truncations to 22 or 23 amino acids (aa) are selected for by replication of SIVmac in human cells, including PBMCs (31).…”