Article Figures & Data
Figures
- Fig. 1.
Representation of recombinant MVA virus genomes. Insertion of the expression cassette consisting of the sequences coding the SIVsmH-4 env precursor regulated by the synthetic early-late promoter (S.E/L) and the β-gal gene regulated by the vaccinia virus early-late P7.5 promoter is indicated by dashed lines to the site of deletion II within the MVA genome. Insertion of a cassette containing the sequences coding the SIVsmH-4gag-pol precursor regulated by the S.E/L promoter and the GUS gene regulated by the P7.5 promoter is indicated by dashed lines to the site of deletion III. The directions of promoters and open reading frames are indicated.
- Fig. 2.
Expression of SIV proteins in monkey cell line BS-C-1 infected with recombinant MVA viruses. Radioimmunoprecipitation of viral proteins from culture supernatants (panels A and B) and cell extracts (panel C) of BS-C-1 monkey cells infected with different MVA-SIV recombinant viruses. Extracts shown in panel A were immunoprecipitated with plasma from an SIV-infected macaque, and extracts shown in panels B and C were immunoprecipitated with a macaque SIVsm-gp120-specific monoclonal antibody, IgG-201 (23). Lanes 1, immunoprecipitation from mock-infected cells; lanes 2, cells infected with nonrecombinant MVA; lanes 3, recombinant MVA-env; lanes 4, original MVA-SIV recombinant virus; lanes 5, MVA-gag-pol-env; lanes 6, MVA-gag-pol. Molecular mass markers are listed at the right in kilodaltons.
- Fig. 3.
Production of SIV-like particles from BSC-1 cells 24 h after infection with the MVA-gag-pol-envrecombinant virus. Electron micrograph of infected cells with immature and mature SIV-like particles (panel A; magnification, ×54,000) and detailed pictures of mature VLPs (panel B; magnification, ×85,000) and stages of VLPs budding from the cell surface and maturing (panel C; magnification, ×68,000).
- Fig. 4.
Anti-SIVsmH-4 gp130 ELISA antibody titers in sera of immunized and control macaques. Six macaques per group were inoculated four times (open diamonds) with recombinant or nonrecombinant MVA and challenged 4 weeks later with SIVsmE660 (filled diamonds). Serial dilutions of plasma were incubated with recombinant SIVsmH-4 gp130 bound to microtiter plates treated by lectin from G. nivalis. End-point titers were defined as the reciprocal of the highest sera dilution that gave an optical absorbance at least two standard deviations greater in value than average values obtained with negative control sera.
- Fig. 5.
Plasma viral load in immunized and control macaques after challenge with uncloned SIVsmE660. Sequential levels of plasma viral RNA over the first 45 weeks after SIV challenge are shown for animals immunized prior to challenge with MVA-gag-pol, MVA-env, MVA-gag-pol-env, or MVA. Plasma viral load was determined by real-time RT-PCR as described in Materials and Methods. Results are expressed as number of copies of SIV genomic RNA equivalent per milliliter of plasma. Plasma samples having values under assay threshold sensitivity were given a value of 800 copy eq/ml. Animals sacrificed because of clinical manifestations of AIDS () and the animal that died of causes unrelated to AIDS (✞) are labeled.
- Fig. 6.
Geometric means of plasma viral load values for groups of immunized and control macaques after challenge with uncloned SIVsmE660. Significant reductions in geometric mean plasma viral load were observed in macaques immunized prior to SIV challenge with recombinant MVA expressing SIVsmH-4 proteins Gag-Pol, Env, or Gag-Pol-Env as compared to those immunized with nonrecombinant MVA (repeated measures ANOVA, P = 0.0011). The error bars represent the standard errors of the means of duplicated measurements for six macaques in each group.
- Fig. 7.
Peripheral blood CD4+ T-lymphocyte levels in immunized and control macaques infected with uncloned SIVsmE660. Sequential levels of peripheral CD4+ T cells over the first 45 weeks after SIV challenge are shown for animals immunized prior to challenge with MVA-gag-pol, MVA-env, MVA-gag-pol-env, or MVA. Animals sacrificed because of clinical manifestations of AIDS () and the animal that died of causes unrelated to AIDS (✞) are labeled. CD4+ T-lymphocyte levels were evaluated by fluorescence-activated cell sorting on whole heparinized blood samples using methods previously described (33).
- Fig. 8.
Survival rates for immunized and control macaques. Kaplan-Meier plot of cumulative survival rates in the first 590 days after challenge indicates significant differences between MVA-immunized- and MVA-SIV-immunized macaques.
Tables
- Table 1.
Production of SIV RT and CA protein in culture media of BS-C-1 cells infected with MVA-SIV recombinant virusesa
MVA recombinant RT activity (103cpm)b CA antigen (ng/ml)b MVA-SIV (Original) 54 ± 6 16 ± 1 MVA-gag-pol-env 1,683 ± 27 870 ± 12 MVA-gag-pol 1,706 ± 92 870 ± 12 a BS-C-1 cells were infected with 10 PFU of indicated MVA-SIV recombinant per cell, and samples were collected as described in Materials and Methods.
b Results are normalized to 106infected cells and are presented as average values of three independent experiments ± standard derivations.
- Table 2.
Neutralizing antibody titers to the vaccine (SIVsmH-4) and challenge (SIVsmE660) viruses on the day of challenge
Vaccine Macaque no. Reciprocal neutralizing dilution titer for: SIVsmH-4 SIVsmE660 MVA-env B1 <30 <30 B2 286 <30 B3 <30 <30 B4 <30 <30 B5 <30 <30 B6 86 <30 MVA-gag-pol-env C1 274 <30 C2 <30 <30 C3 <30 <30 C4 107 <30 C5 85 <30 C6 88 <30 - Table 3.
Summary of virus isolation results from PBMC of MVA-immunized macaques
Immunogen Macaque Week post-SIV Inoculation 1 2 3 4 6 8 12 16 20 28 36 44 MVA-gag-pol A1 + + + + − − − − − − − − A2 + + + + + + + + + + + + A3 + + + − − + + − − − − − A4 + + + + + + + + + + + + A5 + + + + + + + + + + + + A6 + + + + + + + + + + + + MVA-env B1 + + + + − + + − − − − − B2 + + + + + + + + + + + + B3 + + + + + + + + + + + + B4 + + + + + + + + + + + + B5 + + + + + + + + + + + + B6 + + + + + + + + + + + + MVA-gag-pol-env C1 + + + + + + + + + + + + C2 + + + + + + + + + + + + C3 + + + + + + + + + + + + C4 + + + + + + − − − − − − C5 + + + + + + + + + + + + C6 + + + + + + + + + + + + MVA D1 + + + + + + + + + + + + D2 + + + + + + + + + + Dead D3 + + + + + + + + + Dead D4 + + + + + + + + + + + Dead D5 + + + + + + + + Dead D6 + + + + + + + + + + + +
