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. 2010 Dec 5;408(1):1-13.
doi: 10.1016/j.virol.2010.08.028. Epub 2010 Sep 21.

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

Tara G Edmonds  1 , Haitao Ding, Xing Yuan, Qing Wei, Kendra S Smith, Joan A Conway, Lindsay Wieczorek, Bruce Brown, Victoria Polonis, John T West, David C Montefiori, John C Kappes, Christina Ochsenbauer
Affiliations

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

Tara G Edmonds et al. Virology. .

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.

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Figures

Figure 1
Figure 1. Schematic representation of the insertion of the Renilla luciferase gene into the NL4-3 viral backbone and the “shuttling in” of heterologous env ectodomain sequences
The LucR gene and a T2A peptide (amino acid sequence underlined) were fused in-frame and inserted between the NL4-3 env and nef genes. * indicates the co-translational cleavage point between the penultimate and last amino acid of T2A and the arrow indicates the start codon of Nef. The nt sequence is depicted for the junction between the env TAA and LucR gene ATG. Furthermore, the “shuttling in” of heterologous env ectodomain sequences between the viral KpnI site (NL4-3 nt 6343) and the introduced silent BstBI site (NL4-3 nt 8301) in the membrane-spanning domain is depicted.
Figure 2
Figure 2. Viral gene expression in provirally transfected 293T
Western blot analysis of cell lysates prepared from 293T cells transfected with (1) NL4-3, (2) NL-LucR.T2A, (3) NL-BaL.ecto, (4) NL-LucR.T2A-BaL.ecto, (5) NL-SF162.ecto, (6) NL-LucR.T2A-SF162.ecto, (7) the nef frameshift negative control NL-LucR.T2A.FS, (8) mock-transfected cells and (9) NLENG1-IRES. (A) HIV-1 Nef antiserum, (B) anti-Renilla and (C) anti-Gag MAbs were used for detection.
Figure 3
Figure 3. Comparison of cell-encoded firefly luciferase and virally-encoded Renilla luciferase activity after infection of TZM-bl cells
TZM-bl cells were infected in triplicate with NL4-3, NL-LucR.T2A, NL-BaL.ecto, NL-LucR.T2A-BaL.ecto, NL-SF162.ecto and NL-LucR.T2A-SF162.ecto at a range of MOIs. Two days post infection the cell lysates were analyzed for (A) firefly luciferase expression and (B) LucR expression. All values are background corrected (160 RLU for firefly luciferase; 180 for LucR). Error bars represent the standard deviation.
Figure 4
Figure 4. Renilla luciferase encoding HIV-1 reporter viruses are replication-competent in PHA-P stimulated PBMC
Replication kinetics of the Env-IMC-LucR viruses (A) NL-LucR.T2A-SF162.ecto, (B) NL-LucR.T2A and (A, B) NL-LucR.T2A-BaL.ecto were compared to those of the respective non-reporter parental viruses NL4-3, NL-BaL.ecto and NL-SF162.ecto. PHA-P stimulated PBMC were infected overnight (MOI of 0.0025), washed twice and cultured for 14 days in 5 mL of medium. 2 mL supernatants were collected at the indicated time points to measure p24 antigen and the medium removed from the samples was replenished. (A) On day 6, the cell cultures were split 1:2 and fed with fresh medium and uninfected cells from the same donor. (B) On day 6, the cell cultures were split 1:2 and fed with fresh medium only. Data in A and B represent results from two independent experiments with PBMC from different donors.
Figure 5
Figure 5. The Renilla luciferase gene expression from the HIV-1 genome remains stable after multiple rounds of replication in PHA-P stimulated PBMC
293T transfection derived (TD) viruses, (A) NL-LucR.T2A-BaL.ecto and (B) NL-LucR.T2A-SF162.ecto, were allowed to replicate in PBMC for 14 days and culture supernatants collected on days 6, 9 and 14 after infection. The TD virus stock and the cell-free PBMC supernatants from each sample day were cryostored until used to infect TZM-bl cells in replicates. TZM-bl lysates were analyzed for expression of both luciferase reporters and the ratio of virally-encoded LucR to cell-encoded firefly luciferase (as a measure of overall infectivity) was determined for each time point. Error bars represent the standard deviation.
Figure 6
Figure 6. Neutralization assay in TZM-bl and NOMI cells utilizing Renilla luciferase expressing viruses
NL-LucR.T2A-BaL.ecto and NL-LucR.T2A-SF162.ecto were preincubated with serial dilutions of the T20 fusion inhibitor and the NAbs IgG1b12, 2G12 and 4E10 for 1 hour prior to infection. The virus/inhibitor mixture was then placed on either TZM-bl cells (MOI of 0.25) in the presence of DEAE-Dextran (40 μg/mL) or NOMI cells (MOI of 0.15) and incubated for 48 hours. After 48 hours, both cell types were lysed and the TZM-bl cells were analyzed for firefly (FF) and LucR expression and the NOMI cells were analyzed for LucR expression. (A and B) TZM-bl data were plotted as % neutralization as compared to virus-only infected cells (n=3). Scatter plots represent the FF (open symbols) and LucR (solid symbols) IC50 values obtained from 3 to 4 independent TZM-bl infections with (C) NL-LucR.T2A-BaL.ecto and (D) NL-LucR.T2A-SF162. The LucR and FF IC50 values for each experiment are depicted with corresponding shapes. The LucR IC50 values for 5 to 6 independent NOMI infections with (E) NL-LucR.T2A-BaL.ecto and (F) NL-LucR.T2A-SF162.ecto are also depicted. Error bars represent the standard deviation.
Figure 7
Figure 7. Neutralization assay in PHA-P stimulated PBMC utilizing Renilla luciferase expressing viruses
(A) NL-LucR.T2A-BaL.ecto and (B) NL-LucR.T2A-SF162.ecto were preincubated with dilutions of T20 and the NAbs IgG1b12, 2G12 and 4E10 for 1 hour. Each virus/inhibitor dilution was then placed (in triplicate) on PHA-P stimulated PBMC (MOI of 0.625) for 48 hours. After 48 hours, the cells were lysed and analyzed for LucR expression. Error bars represent the standard deviation of replica wells (n=3). (C) PBMC were infected with NL-LucR.T2A-SF162.ecto (MOI of 0.2) for 4 hrs, then the cells were washed to remove unbound virus and divided into two flasks, one of which was supplemented with 2 μM of Indinavir (IDV). Cells were collected and lysed at different time intervals and analyzed for LucR expression. All plotted values are background corrected (160 RLU) and the dotted line represents the RLU of the uninfected cell control.
Figure 8
Figure 8. ‘Four-day’ neutralization assay in PHA-P stimulated PBMC utilizing Renilla luciferase expressing viruses
NL-LucR.T2A-SF162.ecto was preincubated with dilutions of (A) sCD4, 4 different NAbs, HIVIG or (B) pooled patient plasma for 1 hour prior to infection. The inhibitor/virus mixture was then placed on PHA-P stimulated PBMC for four days and the cells were lysed and analyzed for LucR expression.
Figure 9
Figure 9. Variable responsiveness is observed when 75 donor PBMC are infected with HIV-1 in the presence of inhibitors
NL-LucR.T2A-BaL.ecto was preincubated with dilutions of IgG1b12, 4E10 and HIVIG for 1 hour prior to infection of PBMC obtained from 75 different healthy donors. Four days post infection, the PBMC were lysed and analyzed for LucR expression. Each bar represents an individual donor PBMC (same order A through D). (A) IgG1b12 inhibited in 75 of 75 (100%) of PBMC with a mean IC50 of 3.9 ± 4.2 μg/mL. (B) 4E10 inhibited in 57 of 75 (76%) of PBMC with a mean IC50 of 9.5 ± 6.5 μg/mL. (C) HIVIG inhibited in 75 of 75 (100%) of PBMC tested with a mean IC50 of 300.9 ± 369.4 μg/mL. (D) PBMC from the 75 donors were also infected with NL-LucR.T2A-BaL.ecto without the presence of inhibitors to analyze donor susceptibility to infection.
Figure 10
Figure 10. Analysis of inter-assay variation in two donor PBMC
NL-LucR.T2A-BaL.ecto was preincubated with dilutions of 4E10, 2F5, IgG1b12, 2G12 and sCD4 for 1 hour prior to infection. The inhibitor/virus mixture was then placed on PBMC from two different donors and four days post infection the cells were lysed and analyzed for LucR expression. For each donor, the IC50 values depicted are for 3 independent experiments.
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