Article Figures & Data
Figures
- FIG. 1.
Recombinant vectors and expression of HIV-1 envelope glycoproteins. (A) A schematic representation of rSFV vectors expressing the YU2gp120 and YU2gp140(-/GCN4) envelope glycoproteins is shown. The envelope genes are inserted downstream of the SFV nonstructural proteins (Replicase). The location of the SFV subgenomic promoter is indicated by an arrow. (B) Pulse-chase analysis of [35S]methionine-labeled BHK-21 cells infected with rSFV-YU2gp120 or rSFV-YU2gp140(-/GCN4) particles. Total labeled proteins at the different chase time points (0.5, 2, and 6 h) in cell lysates (left panel) and in TCA-precipitated supernatants (right panel) were separated by SDS-PAGE, and the proteins were visualized by autoradiography. (C) Supernatants from BHK-21 cells infected for 24 h with rSFV-YU2gp120 or rSFV-YU2gp140(-/GCN4) were immunoprecipitated with MAb IgGb12 and protein G Sepharose and eluted in SDS-PAGE sample buffer containing 2% BME, and the proteins were analyzed by SDS-PAGE and silver staining.
- FIG. 2.
Conformational integrity of rSFV-expressed envelope glycoproteins. Supernatants from [35S]methionine-labeled BHK-21 cells infected with rSFV-YU2gp120 or rSFV-YU2gp140(-/GCN4) were immunoprecipitated with pooled sera from HIV-1-infected persons (HIVIG) and MAbs IgGb12 and 2G12. The proteins were analyzed by SDS-PAGE.
- FIG. 3.
Oligomeric status of rSFV-expressed envelope glycoproteins. (A) Supernatants from [35S]methionine-labeled BHK-21 cells infected with rSFV-YU2gp120 or rSFV-YU2gp140(-/GCN4) were TCA precipitated and analyzed by SDS-PAGE (3 to 8%) under nonreducing conditions. (B) Unlabeled supernatants (10 μl) from rSFV-YU2gp120 or rSFV-YU2gp140(-/GCN4)-infected BHK-21 cells were analyzed on a blue native gel (4 to 12%), transferred to membranes, and subjected to Western blot analysis. (C) The supernatant (10 μl) from rSFV-YU2gp140(-/GCN4)-infected BHK-21 cells was also analyzed side-by-side with the purified YU2gp140(-/GCN4) protein (30 ng) by blue native/Western blot analysis.
- FIG. 4.
Immunization schedule. Schematic representation of the immunization regimens used in the mouse and rabbit experiments. (A) Mice (six per group) were immunized three times with 10 μg purified YU2gp140(-/GCN4) protein in Ribi (P) or two or three times with 107 IU rSFV-YU2gp140(-/GCN4) particles (SFV), followed by 10 μg purified YU2gp140(-/GCN4) protein in Ribi at the indicated intervals. Four control mice were immunized two times with 107 rSFV-lacZ (SFVlacZ) followed by 10 μg βgal in Ribi. (B) Rabbits (four per group) were immunized three times with 25 μg purified YU2gp140(-/GCN4) protein in Ribi (P) or two times with 5 × 107 IU rSFV-YU2gp140(-/GCN4) particles (SFV), followed by 25 μg or 5 μg purified YU2gp140(-/GCN4) protein in Ribi at the indicated intervals. Two control rabbits were immunized two times with 5 × 107 rSFV-lacZ (SFVlacZ) followed by 25 μg BSA in Ribi.
- FIG. 5.
Envelope-binding antibodies in sera from mice immunized with protein in Ribi or with rSFV particles followed by protein in Ribi. Mice were inoculated three times with 10 μg purified YU2gp140(-/GCN4) protein in Ribi (3×P), two times with 107 IU rSFV-YU2gp140(-/GCN4) followed by a single boost with 10 μg protein in Ribi (2×SFV+P), or three times with 107 IU rSFV-YU2gp140(-/GCN4) followed by a single boost with 10 μg protein in Ribi (3×SFV+P). (A) IgG ELISA end-point titers of envelope-binding antibodies in sera from individual mice 12 days after each immunization. The bar represents group geometric means; ND indicates not determined. (B) Titration of envelope-binding IgG1 antibodies (upper panels) and IgG2a antibodies (lower panels) before and after the final protein boost in three individual mice each in the Protein only group (2×P and 3×P) and in the 3×SFV+Protein group (3×SFV and 3×SFV+P). (C) IgG1:IgG2a ratios were calculated by dividing the IgG1 and IgG2a OD values for each individual mouse (black bars, 3×P group; white bars, 3×SFV+P group;) at the 1:6,250 serum dilutions.
- FIG. 6.
Analysis of CD4+ T-cell responses in mice immunized with rSFV prime-protein boost or protein only regimens. Recombinant YU2gp120 was used for in vitro stimulation of splenocytes from YU2gp140(-/GCN4)-immunized mice to measure envelope-specific CD4+ T cells responses. Cytokine-producing cells are shown as spot-forming units (SFU)/106 cells. (A) CD4+ T-cell depletion control. The contribution of CD4+ T cells in the IFN-γ ELISPOT assay was evaluated by comparing the response in total and CD4+ T-cell depleted splenocyte cultures. Data from representative mice (#1 and #2) stimulated with YU2gp120 or with ConA are shown. (B) Spleen cells from groups of mice immunized two or three times with 107 IU rSFV-YU2gp140(-/GCN4) (2×SFV and 3×SFV) were evaluated in an IFN-γ ELISPOT assay after stimulation with medium or with recombinant YU2gp120 protein. Data are presented as group means where the response from each individual mouse is determined as the mean ELISPOT value from triplicate wells. (C) IFN-γ, IL-4, and IL-2 ELISPOT analysis was performed on spleen cells harvested from groups of mice immunized three times with purified YU2gp140(-/GCN4) protein in Ribi (3×P) or three times with 107 IU rSFV-YU2gp140(-/GCN4) followed by a single boost with YU2gp140(-/GCN4) protein in Ribi (3×SFV+P). Control mice were immunized two times with rSFV-lacZ followed by a single boost with βgal protein in Ribi (Ctrl). Mean ELISPOT values from triplicate wells stimulated with medium or with YU2gp120 were calculated for each individual mouse. The data are presented as SFU/106 cells after subtracting the value obtained in medium-stimulated wells from the value obtained in YU2gp120 stimulated wells. ND (not detected) indicates that no positive responders were detected in a group.
- FIG. 7.
Envelope-binding antibodies in sera from rabbits immunized with protein in Ribi or with rSFV particles followed by protein in Ribi. Rabbits were inoculated three times with 25 μg purified YU2gp140(-/GCN4) protein in Ribi (Protein only; rabbits 1 to 4) or two times with 5 × 107 IU rSFV-YU2gp140(-/GCN4) followed by a single boost with 25 μg protein in Ribi (2×SFV+Protein; rabbits 5 to 8). The sera from the immunized rabbits, drawn 10 days after each inoculation, were analyzed by fivefold serial dilutions (starting at a 1/50 dilution) for envelope-binding antibodies. Data from individual rabbits at different time points are shown as follows: prebleeds, empty squares; after the first injection, filled squares; after the second injection, triangles; after the third injection, circles. The endpoint titers after three immunizations are marked with open arrowheads.
- FIG. 8.
In vitro neutralizing activity against a panel of HIV-1 isolates in sera from immunized rabbits. Neutralizing activity in 1:5 diluted sera from rabbits immunized three times with 25 μg purified YU2gp140(-/GCN4) protein in Ribi (Protein only; rabbits 1 to 4) or two times with 5 × 107 IU rSFV-YU2gp140(-/GCN4) followed by a single boost with 25 μg protein in Ribi (2 × SFV+Protein; rabbits 5 to 8). Control animals (rabbits 9 and 10) were immunized two times with 5 × 107 IU rSFV-lacZ followed by 25 μg BSA in Ribi. The numbers under each viral isolate represent percentages of neutralization, and color codes demonstrate different levels of neutralization (>80% neutralization, red; 60 to 80% neutralization, yellow; 40 to 59% neutralization, green). Preimmune sera were used to establish baseline-neutralizing activity in each individual rabbit, and these values were subtracted from the values shown. Neutralization equal to or less than zero is indicated as ≤0.
Additional Files
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Files in this Data Supplement:
- Supplemental file 1 - Legends MS Word document, 26K.
- Supplemental file 2
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Fig. S1. Repeated immunizations with rSFV in rabbits.
(A) Immunization schedule. (B) ELISA data.
Fig. S2. Effect of protein boost dose after priming with rSFV. (A) Immunization schedule. (B) ELISA data.
Zipped PowerPoint presentation, 483K.
