Side-by-side comparison of gene-based smallpox vaccine with MVA in nonhuman primates

Abstract

Orthopoxviruses remain a threat as biological weapons and zoonoses. The licensed live-virus vaccine is associated with serious health risks, making its general usage unacceptable. Attenuated vaccines are being developed as alternatives, the most advanced of which is modified-vaccinia virus Ankara (MVA). We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus antigens, A33, B5, A27 and L1. This vaccine protects mice and non-human primates from lethal orthopoxvirus disease. Here, we investigated the capacity of the molecular adjuvants GM-CSF and Escherichia coli heat-labile enterotoxin (LT) to enhance the efficacy of the 4pox gene-based vaccine. Both adjuvants significantly increased protective antibody responses in mice. We directly compared the 4pox plus LT vaccine against MVA in a monkeypox virus (MPXV) nonhuman primate (NHP) challenge model. NHPs were vaccinated twice with MVA by intramuscular injection or the 4pox/LT vaccine delivered using a disposable gene gun device. As a positive control, one NHP was vaccinated with ACAM2000. NHPs vaccinated with each vaccine developed anti-orthopoxvirus antibody responses, including those against the 4pox antigens. After MPXV intravenous challenge, all control NHPs developed severe disease, while the ACAM2000 vaccinated animal was well protected. All NHPs vaccinated with MVA were protected from lethality, but three of five developed severe disease and all animals shed virus. All five NHPs vaccinated with 4pox/LT survived and only one developed severe disease. None of the 4pox/LT-vaccinated animals shed virus. Our findings show, for the first time, that a subunit orthopoxvirus vaccine delivered by the same schedule can provide a degree of protection at least as high as that of MVA.

Figure 1. Antibody responses against the 4pox vaccine targets in vaccinated mice.

A. Schematic showing the vaccination protocol. Mice were vaccinated by gene-gun twice at 3-week intervals with the 4pox vaccine and empty vector (4pox/WRG), 4pox plus GM-CSF (4pox/GM-CSF) or LT (4pox/LT) or a negative control vaccine containing irrelevant DNA. All mice were vaccinated with four cartridges per vaccination equaling ∼2 µg DNA/vaccination. Bleed dates are indicated by the down arrows. B. Purified 4pox antigens (L1, A33, B5, and A27) were plated in 96-well plates. Sera from mice vaccinated on week 3 (prime) or week 6 (boost) with the indicated vaccines were serially diluted tenfold (from 1∶100) and incubated with purified protein. Endpoint titers were calculated as described in the materials and methods. Data were plotted a scatter plot showing the distribution of the titer along with a horizontal line indicating the geometric mean titer. C. Pooled sera from vaccinated mice were serially diluted tenfold. Dilutions were incubated with duplicate plates containing the 4pox antigens. Secondary anti-mouse antibodies specific for either IgG1 or IgG2a were then incubated with the samples. Endpoint titers for each secondary antibody were calculated as in part B. The ratio of IgG1 to IgG2a was then determined and plotted. D. Sera from mice vaccinated on week 3 (prime) or week 6 (boost) with the indicated vaccines were serially diluted twofold and incubated with VACV strain IHD-J. 50% neutralization titers were calculated relative to the plaque count for virus that was not incubated with serum. Data were plotted as a mean titer for each group +/− standard deviation. All statistics were calculated as described in the material and methods and are denoted by an asterisks sign as well as a line showing which groups are being compared.

Figure 2. VACV challenge of vaccinated mice.

Groups of seven to eight mice vaccinated twice by gene-gun with the indicated DNA vaccine were challenged with 2×10⁶ pfu of VACV strain IHD-J by the i.n. route. Weights of individual mice were monitored for 21 DPI. The percent weights of surviving mice were calculated relative to starting weights (day 0). Mean % weight loss ± standard error of the mean (SEM) were plotted. On day 4 postinfection, there was a significant difference in weight loss between 4pox/WRG versus 4pox/GMCSF (p = 0.0336) or 4pox/LT (p = 0.0384), this point is denoted by an asterisk. Poison symbol indicates all animals in the group became moribund and were euthanized.

Figure 3. Vaccination of NHP with 4pox, MVA and ACAM2000.

A. Schematic showing the vaccination protocol. NHPs were vaccinated with the 4pox/LT vaccine or negative control DNA by the ND10 device twice at 4-week intervals. Another group of NHP was intramuscularly vaccinated with MVA (ACAM3000) 1×10⁸ pfu/dose twice at a 4-week interval. One NHP was vaccinated with a single dose of ACAM2000 and served as a positive control. The down arrows indicate bleed dates. B. Sera from individually vaccinated NHPs were collected at week 0, 4, 6 and 8 and level of antigen-specific antibodies was examined by protein-ELISA as in Figure 1, with the exception that a monkey secondary was used (1∶2000). Data were plotted as a line graph showing the GMT for each group. C. VACV strain WR infected-cell lysate or mock antigen was used as ELISA antigen. Serial 10-fold dilutions of individual NHP serum samples were evaluated by ELISA and the GMT for each group at each time-point were plotted. D. VACV- and MPXV-neutralizing antibody titers for weeks 6 and 8 were determined by PRNT as described in Figure 1. Mean PRNT50 titers from two experiments are shown for individual NHP at each time point. Error bars represent mean titers ±SEM. E. EEV neutralization. Sera from NHPs vaccinated with 4pox/LT (600, 1104, 0014, 854 and 112) or negative control (3868 and 0290) was diluted twofold starting at a 1∶40. Samples were incubated with fresh EV particles in the presence or absence of complement as indicated. Anti-MV antibodies (anti-L1 MAb-10F5) were included in all samples to remove residual MV. After incubation, EV particles were adsorbed to BSC-1 cell monolayers and incubated for 4 days when they were stained for plaque formation. Titers were determined as described in the material and methods. Each sample was run in duplicate. Mean values ±SD are shown. The limit of detection was a titer of 40 (dashed line).

Figure 4. MPXV challenge of vaccinated NHPs.

Groups of vaccinated NHPs were challenged with 2×10⁷ pfu of MXPV strain Zaire-79 by the intravenous route. A. Survival was monitored out to 28 DPI. Moribund animals were euthanized and the percent survival was plotted. The 4pox/LT and MVA vaccines exhibited a significant level of protection relative to the negative controls as measured by % survival (p-value 0.0145) and survival curve (p-value 0.0399) B. Pox lesions on infected NHPs were counted on days 0, 1, 2, 3, 6, 12, 15, 18, 21, and 28. Lesions were individually counted on the hands, feet, torsos, face, mouth arms and legs. The total lesion count per animal from each time-point is plotted. C. Shedding of viable virus from the throat was monitored on days 0, 1, 2, 3, 6, 12, 15, 18, 21, and 28 by plaque assay as described in the materials and methods. D. MPXV genomes in the whole blood of the animals were determined at the indicated time-points by quantitative PCR. The day 0 blood sample was collected immediately after i.v. injection of the challenge virus. The assay limit was 500 genomes per ml.