A Nucleic Acid-Based Orthopoxvirus Vaccine Targeting the Vaccinia Virus L1, A27, B5, and A33 Proteins Protects Rabbits against Lethal Rabbitpox Virus Aerosol Challenge

Abstract

In the age of COVID, nucleic acid vaccines have garnered much attention, at least in part, because of the simplicity of construction, production, and flexibility to adjust and adapt to an evolving outbreak. Orthopoxviruses remain a threat on multiple fronts, especially as emerging zoonoses. In response, we developed a DNA vaccine, termed 4pox, that protected nonhuman primates against monkeypox virus (MPXV)-induced severe disease. Here, we examined the protective efficacy of the 4pox DNA vaccine delivered by intramuscular (i.m.) electroporation (EP) in rabbits challenged with aerosolized rabbitpox virus (RPXV), a model that recapitulates the respiratory route of exposure and low dose associated with natural smallpox exposure in humans. We found that 4pox-vaccinated rabbits developed immunogen-specific antibodies, including neutralizing antibodies, and did not develop any clinical disease, indicating protection against aerosolized RPXV. In contrast, unvaccinated animals developed significant signs of disease, including lesions, and were euthanized. These findings demonstrate that an unformulated, nonadjuvanted DNA vaccine delivered i.m. can protect against an aerosol exposure. IMPORTANCE The eradication of smallpox and subsequent cessation of vaccination have left a majority of the population susceptible to variola virus or other emerging poxviruses. This is exemplified by human monkeypox, as evidenced by the increase in reported endemic and imported cases over the past decades. Therefore, a malleable vaccine technology that can be mass produced and does not require complex conditions for distribution and storage is sought. Herein, we show that a DNA vaccine, in the absence of a specialized formulation or adjuvant, can protect against a lethal aerosol insult of rabbitpox virus.

Keywords: DNA vaccines; aerosols; neutralizing antibodies; nucleic acid technology; poxvirus; rabbitpox; smallpox.

FIG 1

Evaluation of CpG ODNs as potential adjuvants for 4pox DNA vaccination in rabbits. (A) The number of IgM⁺/IgG⁺ splenocytes (“cell number × 10³”) was determined 5 days after stimulation with individual CpG oligodeoxynucleotides numbered 1 to 6 (x axis) by flow cytometry. (B) Supernatants were analyzed by ELISA to determine titers of secreted IgG. O.D. optical density. Results are representative of three independent experiments. Symbols represent the mean ± standard error from each group. *, P < 0.05 by t test.

FIG 2

The 4pox DNA vaccine is immunogenic in rabbits. (A) Experimental layout showing the vaccination protocol in weeks. Rabbits were vaccinated three times by i.m. EP at 3-week intervals with the 4pox vaccine (low and high doses), where five animals from each 4pox-vaccinated dose group received a combination of adjuvant CpGs 3, 4, and 6. Serum was collected (red arrows) on the indicated weeks. (B) Purified 4pox antigens (L1, A33, B5, and A27) were plated in 96-well plates. Sera from rabbits vaccinated with the indicated vaccines or vaccine-adjuvant combination at the indicated time points were serially diluted 10-fold (from 1:100) and incubated with purified protein. Endpoint titers were calculated as described in the Materials and Methods. Data were plotted using Prism software. Symbols represent the mean ± standard deviation from each group. (C) Sera from vaccinated rabbits were serially diluted 2-fold and incubated with MV of VACV strain IHD-J. PRNT₅₀s were calculated relative to the plaque count for virus that was not incubated with serum. Data were plotted as a geometric mean titer (GMT) from each group ± standard deviation. The dashed line indicates the limit of detection. (D) Comet inhibition was evaluated using serum from rabbit 6623, which received a low-dose 4pox vaccine without CpG. VACV strain IHD-J was adsorbed onto BSC-1 cells, and then medium containing week 0 and week 2 sera (1:20 dilution) was added to the wells. Plaques were visualized by staining with crystal violet 48 h after adsorption. As a positive control for comet inhibition, a semisolid methylcellulose overlay was added to control wells. Each sample was performed in duplicate and is representative of sera from other rabbits in the 4pox vaccine groups. EP, electroporation; “low,” 0.4-mg dose of 4pox vaccine; “low + CpG,” 0.4-mg dose of 4pox vaccine plus 0.5 mg CpGs (3, 4, and 6); “high,” 4-mg dose of 4pox vaccine; “high + CpG,” 4-mg dose of 4pox vaccine plus 0.5 mg CpGs (3, 4, and 6).

FIG 3

The 4pox vaccine administered i.m. by EP protects rabbits against lethal RPXV aerosol challenge. (A) Rabbits were aerosol challenged with a target dose of ∼1,000 PFU RPXV 28 weeks after the last DNA vaccination. Survival was plotted up to 20 days postinfection using Prism software. Symbols represent the mean ± standard error from each group. (B) Individual weights were calculated based on day 0 starting weight. Symbols represent the mean ± standard error from each group. (C) Mean temperatures on day −1 and day 3 were graphed for each group. Lines represent the geometric mean temperature for each group. The asterisk denotes statistical significance (P < 0.05 by t test). Blue data points represent animals that received CpG adjuvant with the 4pox vaccine.

FIG 4

The 4pox DNA vaccine delivered i.m. by EP prevents serum viremia and reduces viral load in peripheral tissues. (A) The presence of RPXV genomes was evaluated by qPCR in whole blood collected every other day following aerosol challenge. Each line represents a single rabbit. (B) Viral load in organs was determined by qPCR in organs collected from rabbits that succumbed to RPXV (unvaccinated controls) or from vaccinated rabbits that were euthanized at the conclusion of the study (days 27 to 29). Blood samples were collected from all rabbits on day 6 and are the same as those in panel A. Blue data points represent animals that received CpG adjuvant with the 4pox vaccine. ND, no genome was detected. (The superscript indicates the number of animals.) The limit of detection in the qPCR assay was 1,000 genomes.

FIG 5

Comparison of 4pox DNA vaccine immunogenicity when delivered by multiple platforms in rabbits. (A) Experimental design. Four groups of rabbits (n = 3) were vaccinated three times at 1-month intervals (black arrows) with 4pox DNA by i.m. EP, i.d. needle/syringe, and the DSJI i.d. or i.m. device. Serum was collected at weeks 0, 4, 6, 8, and 10 (red arrows). (B) Protein-specific ELISAs were performed as in Fig. 2B (C) PRNT₅₀s were determined with serum from the indicated group at the indicate time point as described in Fig. 2C. Geometric mean titers and standard deviations are shown.

FIG 6

Immunogenicity of 4pox DNA delivered by DSJI (PharmaJet ID) in rabbits. (A) Experimental design. Three groups of rabbits were vaccinated three times at 1-month intervals with the DSJI i.d. device (black arrows). Serum was collected at weeks 0, 3, 7, and 12 (red arrows). (B) Protein-specific ELISAs were determined as described in Fig. 2B. (C) PRNT₅₀s were determined with serum from the indicated group at the indicated time point as described in Fig. 2C. Geometric mean titers and standard deviations are shown. The y axis is log base 2.