Article Figures & Data
Figures
- FIG. 1.
Diagram of SFV genome and replicon constructs. (A) A diagram of the plus-stranded native RNA genome of SFV. (B) Representations of rSFV replicon vector constructs that encode prC epitope-tagged EV A33R, A34R, A56R, B5R, and GFP genes. The nonstructural genes encoding the replicase complex are indicated by nsP1, nsP2, nsP3, and nsP4. Helper vectors designated Helper C and Helper S encode the genes for the capsid (C) and the spike proteins (E1, E2, E3, and 6K), respectively. Large black arrow (SP6 pol) denotes the SP6 RNA polymerase promoter region; small black arrow denotes the 26S subgenomic promoter; black circle at 5′-end denotes CAP structure. An, polyA; PS, packaging signal; ΔnsP1-4, deleted nonstructural genes.
- FIG. 2.
Anti-Dryvax immune mouse serum recognizes rEV proteins. (A) Affinity-purified recombinant A33R, A34R, A56R, and B5R were analyzed under reducing conditions by using SDS-PAGE and SYPRO Ruby protein gel staining. Stained gel with lanes corresponding to (M) marker proteins and the indicated EV proteins is shown. Recombinant A33R, A34R, A56R, and B5R were treated with (+) and without (−) β-mercaptoethanol (β-ME) and were analyzed by SDS-PAGE and Western blotting using an anti-prC MAb (1/1,000 dilution) to the C-terminal prC epitope tag (B) or using a mouse anti-Dryvax antiserum (anti-Dryvax; 1/500 dilution) (C). Protein bands were detected by chemiluminescence after membranes were incubated with an anti-mouse IgG antibody conjugated to HRP (1/2,500 dilution). The positions and masses of the marker proteins are indicated on the left. Dots show the positions of EV protein bands that are consistent with the reported molecular mass for each of the indicated proteins (52).
- FIG. 3.
Analysis of immune serum from mice immunized with an HA deletion mutant vaccinia virus strain and noninfectious MV particles. (A) Hyperimmune antiserum anti-vA5Lint was obtained from BALB/c mice inoculated i.p. with a recombinant WR-strain vaccinia virus that lacks the A56R (HA) gene, vA5Lint, using a prime-boost schedule. (B) Hyperimmune antiserum anti-MV was obtained from mice inoculated s.c. with noninfectious WR strain vaccinia virus MV particles (4.0 × 107 PFU equivalents) using a prime-boost schedule. A33R, A34R, A56R, and B5R protein preparations were treated with (+) and without (−) β-mercaptoethanol (β-ME) prior to SDS-PAGE and protein transfer onto PVDF membranes. The PVDF membranes were incubated with mouse immune sera, followed by a second incubation with an anti-mouse IgG antibody conjugated to HRP. Protein bands were detected by chemiluminescence. Masses of protein markers are indicated on the left.
- FIG. 4.
EV-specific antibody response induced by vaccinia virus. (A) A set of pooled anti-vaccinia virus antisera referred to as anti-vaccinia, anti-vA5Lint, and anti-MV was produced in BALB/c mice (in groups of three) that were inoculated with the MVA-Dryvax combination (107 PFU), live vA5Lint (107 PFU), and noninfectious WR strain MV particles (4.0 × 107 PFU equivalents), respectively, using a prime-boost schedule. Antisera were collected 3 weeks after the last inoculation, and pooled antisera were analyzed by ELISA using plates coated with EV proteins A33R, A56R, or B5R or UV-inactivated Dryvax MV particles. As a control, ELISA plates were probed with normal (uninoculated) mouse serum (NMS). (B) A group of mice were immunized i.d. with a single dose of 106 PFU of Dryvax, and antisera were collected at weeks 3, 6, and 9 postvaccination and pooled. Levels of antibodies to indicated EV proteins were measured by ELISA. The results shown in panels A and B were derived from a single experiment. α, anti.
- FIG. 5.
EV-specific antibody titers in VIG. The EV-specific antibody titer in human VIG-IV was determined by ELISA for each indicated EV protein. Control human serum (NHS) is indicated.
- FIG. 6.
EV neutralization activity detected in anti-vaccinia virus hyperimmune sera. (A) Comet inhibition assays were performed using Vero cell monolayers infected with vaccinia virus (IHD-J) that were overlaid with medium alone, medium containing methyl cellulose (MC), or medium containing a 1/50 dilution of hyperimmune sera generated against Dryvax in mice (anti-Dryvax) or humans (Dryvax-VIG); (B) infected Vero cell monolayers were overlaid with medium containing a 1/50 dilution of mouse hyperimmune antisera anti-MV, anti-A5Lint, and anti-Dryvax (described in Fig. 3 legend). α, anti.
- FIG. 7.
Antibody response to rSFV infectious vectors expressing EV proteins. Mice in groups of five were inoculated s.c., using a prime-boost regimen, with 106 I.U. of indicated rSFV particles expressing GFP, A33R, A34R, A56R, or B5R or with 106 PFU of Dryvax. The inoculations were administered 3 weeks apart, and immune sera were collected 3 weeks after each injection and analyzed by single-EV-antigen ELISAs. (A) Antibody levels in antisera measured after the first inoculations. (B) Antibody levels measured after the second inoculations. Note: two out of five (2/5) mice inoculated once with rSFV-B5R seroconverted, whereas that none of the five (0/5) mice inoculated once with Dryvax had detectable antibody against B5R. After the second inoculation, all mice seroconverted.
- FIG. 8.
Western blot analysis of vaccinia virus-infected cells using rSFV vector-generated EV-specific antisera. Immune sera were collected from mice inoculated with two s.c. doses of 106 I.U. of rSFV particles encoding either A33R, A56R, or B5R and analyzed by Western blotting using uninfected and vaccinia virus-infected B-SC-1 cell lysates. B-SC-1 cells were infected at a multiplicity of infection of 5 with WR strain vaccinia virus (WR), Dryvax, and recombinant WR strain vaccinia virus vA5Lint (described in Fig. 3 legend). Indicated cell lysates were analyzed by Western blotting using antisera anti-A33R (A), anti-A56R (B), or anti-B5R (C) and an anti-mouse Ig-HRP conjugate. Indicated affinity-purified EV proteins A33R, A56R, and B5R were used as positive controls, and uninfected-cell lysate (uninf.) was used as a negative control. Molecular masses are indicated on the left. α, anti.
Tables
- TABLE 1.
Comparison of EV-specific antibody titers elicited by a single immunization with Dryvax or MVAa
Group Type of immunization Routea Dose Antibody titer (log10 ± SD) against EV antigen: A33R A56R B5R 1 PBS i.m. NA 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 2 Dryvax i.d. 106 3.40 ± 0.42 3.25 ± 0.21 2.50 ± 1.27 3 MVA i.m. 107 2.95 ± 0.21 3.11 ± 0.00 2.95 ± 0.21 4 MVA s.c. 107 2.05 ± 0.21 2.95 ± 0.21 3.11 ± 0.42 5 MVA i.d. 107 3.25 ± 0.64 3.11 ± 0.00 3.26 ± 0.21 6 MVA i.m. 108 3.40 ± 0.00 3.41 ± 0.00 3.26 ± 0.21 7 MVA s.c. 108 3.10 ± 0.00 3.11 ± 0.00 3.56 ± 0.21 8 MVA i.d. 108 3.86 ± 0.21 2.95 ± 0.21 3.71 ± 0.00 ↵ a BALB/c mice were immunized with a single dose of PBS, Dryvax, or MVA at week 0 by the indicated routes of vaccination. Antisera were collected at week 6 and pooled, and the antibody levels against the indicated EV antigens were measured by ELISA. The results are the average of two independent immunization experiments in which each group contained three mice.
- TABLE 2.
Comparison of EV-specific antibody titers elicited by single immunizations and prime-boost immunizationsa
Group Type of immunization Route Dose Wk(s) Antibody titer (log10 ± SD) against EV antigen: A33R A56R B5R 1 PBS i.m. NA 0 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 2 Dryvax i.d. 106 0 2.80 ± 0.00 2.80 ± 0.00 2.50 ± 0.00 3 Dryvax i.d. 107 0 3.26 ± 0.21 3.11 ± 0.42 2.95 ± 0.64 4 MVA i.d. 108 0, 3 3.11 ± 0.42 3.56 ± 0.21 4.01 ± 0.00 5 MVA i.d. 108 0, 6 3.86 ± 0.21 3.86 ± 0.63 4.31 ± 0.42 ↵ a BALB/c mice were immunized with a single dose of PBS, Dryvax, or MVA at week 0 using indicated routes of vaccination. Mice in groups 4 and 5 received two doses of MVA given 3 weeks apart and 6 weeks apart, respectively. Mice in group 6 were immunized once with Dryvax by scarification at week 0. In groups 1 through 5, the results represent the average of two independent immunization experiments in which each group contained three mice.
