Figure 1.
DF-1 cells support high-level growth of MVA.
(A) MVA yields following infection of DF-1 fibroblasts and 1° CEFs at MOI = 3. Data represent the means of duplicate samples. (The MVA stock was grown and titered on 1° CEFs). (B) MVA yields following infection of DF-1 fibroblasts, 1° CEFs, and BHK-21 cells at MOI = 3. Data represent the means of triplicate samples ±1 standard deviation. (The MVA stock was grown and titered on DF-1 cells).
Figure 2.
Construction and characterization of MVA-GZ.
(A) Genome map of MVA recombinant MVA-gz (top), which encodes gfpzeo (gz) under the control of an early modified H5 promoter (pH 5). Roman numerals (I-VI) represent the sites of the major genomic deletions in MVA as compared to its parental strain [3]. * = vaccinia virus early transcriptional stop signal (5′-TTTTTCT-3′) [70]. (B) Recombinant (MVA-gz) plaques visualized via fluorescence microscopy as GFP+ plaques on DF-1 cells at 4 days following infection; original magnification = 4X.
Figure 3.
Isolation of MVAΔudg recombinant viruses.
(A) Genome maps of wild type MVA and udg-deletion MVA recombinants with restriction fragment lengths in kilobases (kB). The StuI and KpnI restriction sites in wild type MVA denote genomic nucleotide positions 89,347 and 93921, respectively. (B) Diagnostic Southern blots that confirm genotypes of MVAΔudg isolates.
Figure 4.
MVAΔudg recombinants grow on the DF-1-derived udg-complementing cell line (CAN20), but do not grow on parental DF-1 cells.
Yields of MVAΔudg recombinants vDG013, vDG014, and udg+ recombinant MVA-gz were determined at the indicated times following infection of CAN20 cells (A) or DF-1 cells (B) at a ratio of 3 PFU per cell. Cell cultures were frozen at indicated times following infection and subsequently thawed, sonicated, and clarified by centrifugation (800 g). Virus titers were determined via plaque assay on CAN20 cells. Data represent the means of duplicate samples; error bars represent the ranges.
Figure 5.
MVAΔudg does not exhibit DNA replication during infection of non-complementing cells.
DF-1 cells (A, B, C, D) and CAN20 cells (E, F, G, H) were infected either with MVA in the absence (A, E) or presence (C, G) of the DNA synthesis inhibitor AraC (150 µM), MVAΔudg (vDG020) in the absence of AraC (B, F), or were mock infected (D, H) and labeled with BrdU between 2–6 hours following infection. Arrows denote cytoplasmic foci of viral DNA replication and arrowheads denote cell nuclei.
Figure 6.
MVAΔudg does not express viral late genes during infection of non-complementing cells in culture.
DF-1 and udg-complementing (CAN20) cells were infected with MVA (udg+) or MVAΔudg isolate vDG014 (Δ) at MOI = 10 in the absence or presence of the DNA synthesis inhibitor AraC (150 µM), as indicated. Infected cell proteins were metabolically labeled with 35S-methionine for 30 min immediately prior to harvesting at indicated times post infection. Proteins were separated via SDS-PAGE and visualized by autoradiography. Arrows denote viral late gene products as defined via AraC-mediated inhibition of expression.
Figure 7.
Immunization of mice with MVAΔudg elicits CD8+ T cell responses directed against early, but not late viral gene products.
(A) Representative intracellular cytokine staining (ICS) of peptide-stimulated splenocytes at 7 days following immunization with MVA or MVAΔudg, as indicated. Splenocytes were stimulated ex vivo with 0.5 uM of A3L270–277, B8R20–27, or A19L47–55 peptide for 5 hours in the presence of GolgiPlug secretion inhibitor and stained with fluorescently-labeled antibodies for flow cytometric analysis. Plots represent data from individual mice and denote the percentages of IFNγ-positive CD3+CD8+ splenocytes expressed as fractions of their corresponding overall CD3+CD8+ splenocyte populations. (B) Splenocytes from mice immunized with MVA (circles) or MVAΔudg (triangles) were analyzed by ICS assay (as above) following ex vivo stimulation with 0.5 µM A3L270–277, A42R88–96, B8R20–27, K3L6–15, or A19L47–55 peptide, or no stimulation, to determine the frequencies of antiviral CD8+ T cells present at 7 days following immunization. Data are organized by the kinetic class (Late, Early, or Unknown [Unk]) to which each viral gene belongs. Symbols represent data from individual mice; horizontal lines represent group means. Each dosage group (106, 108 PFU) presents data obtained from two independent immunization experiments. Statistical comparison of MVA vs MVAΔudg groups, for each CD8+ T cell epitope, was performed via nonparametric Mann-Whitney analysis; only P-values ≤0.05 are shown.
Figure 8.
Immunization of mice with MVAΔudg elicits MVA-specific neutralizing antibody responses that are of magnitudes similar to those elicited by MVA.
(A) Representative flow cytometric data from the GFP fluorescence-based MVA neutralization assay. Serial dilutions of serum from a mouse immunized 28 days earlier with 106 PFU MVA were mixed with a constant amount of GFP-expressing virus MVA-gz and incubated for 1 hour at 37°C. HeLa cells were then added to individual serum∶virus mixtures, incubated overnight at 37°C, and analyzed for GFP expression by flow cytometry. The gated percentages of GFP+ cells (shown) were also normalized to the average maximum response observed for cells infected with MVA-gz in the absence of test serum (normalized values expressed as percentages of the maximum response are shown parenthetically). Illustrative data representing serum dilutions 1∶16, 1∶256, 1∶512, and 1∶8,192, which constitute a subset of all serum dilutions analyzed, are shown. (B) Representative nonlinear regression analysis for the determination of EC50 neutralizing antibody (NAb) titers. Replicate normalized GFP+ response data from an individual mouse (described in (A)) was analyzed by non-linear regression. Goodness-of-fit value (R2) and the EC50 NAb titer, expressed as the dilution factor, are shown. (C) Mice were immunized once with the indicated doses of MVA (circles) or MVAΔudg (triangles). Titers of MVA-specific neutralizing antibodies were determined, as described above, for serum samples collected 28 days following immunization. Symbols represent NAb titers that were determined for individual mice; horizontal lines represent group means. Statistical comparison of MVA vs MVAΔudg groups, within each dosage group, was performed via nonparametric Mann-Whitney analysis and did not result in any significant (P≤0.05) differences.
Figure 9.
Immunization of rhesus macaques with MVAΔudg-gag elicits significantly higher frequencies of HIV Gag-specific CD8 and CD4 T cells.
Rhesus macaques (N = 6/group) were immunized at 0, 6, and 12 weeks with MVAΔudg-gag or MVA-gag (2×108 PFU per immunization). At the indicated times, PBMC samples were either stimulated ex vivo with a pool of matched overlapping HIV Gag peptides, or were not stimulated, and the frequencies of IFNγ- and IL2-producing CD8 and CD4 T cells were determined by intracellular cytokine staining/flow cytometric analysis as described. The frequencies of CD8 (A, C) and CD4 (B, D) T cells that co-expressed IFNγ (A, B) or IL2 (C, D) and the activation marker CD69 are shown. Symbols represent the means of replicate samples assayed for individual macaques; horizontal lines denote group medians. Statistical comparison of groups immunized with MVAΔudg-gag vs MVA-gag was performed at each timepoint via non-parametric Mann-Whitney analysis. P-values <0.07 are indicated as shown. Immunizations are denoted by vertical dashed lines.
Figure 10.
Immunization of rhesus macaques with MVAΔudg-gag elicits HIV Gag-specific antibody responses that are of magnitudes similar to those elicited by MVA-gag.
Rhesus macaques (N = 6/group) were immunized at 0, 6, and 12 weeks with MVAΔudg-gag or MVA-gag (2×108 PFU per immunization). At the indicated times, plasma samples were assayed to determine the titers of HIV Gag-specific binding antibodies via ELISA utilizing recombinant HIV Gag protein as the coating antigen, as described. Symbols represent the mean (of duplicate) absorbance (A450nm) values that were determined from 1∶25 dilutions of plasma samples from individual macaques; black lines denote group mean absorbance values. Immunizations are denoted by vertical dashed lines. Statistical comparison of groups immunized with MVAΔudg-gag vs MVA-gag was performed at each timepoint via non-parametric Mann-Whitney analysis and did not result in any significant (P≤0.05) differences.
Figure 11.
Immunization of rhesus macaques with MVAΔudg-gag elicits MVA-specific antibody responses that are of magnitudes similar to those elicited by MVA-gag.
Rhesus macaques (N = 6/group) were immunized at 0, 6, and 12 weeks with MVAΔudg-gag or MVA-gag (2×108 PFU per immunization). At the indicated times, heat-inactivated plasma samples were assayed to determine the titers of MVA-specific binding antibodies (A, B) via ELISA utilizing whole MVA virions as the coating antigen, or MVA-specific neutralizing antibodies (C, D) utilizing the MVA-lacZ neutralization assay, as described. Symbols represent data from individual macaques; black lines denote group geometric mean titers. Immunizations are denoted by vertical dashed lines. Statistical comparison of groups immunized with MVAΔudg-gag vs MVA-gag was performed at each timepoint via non-parametric Mann-Whitney analysis and did not result in any significant (P≤0.05) differences.