BacMam Expressing Highly Glycosylated Porcine Interferon Alpha Induces Robust Antiviral and Adjuvant Effects against Foot-and-Mouth Disease Virus in Pigs

Abstract

Foot-and-mouth disease (FMD) is an acute contagious disease that affects cloven-hoofed animals and has severe global economic consequences. FMD is most commonly controlled by vaccination. Currently available commercial FMD vaccines contain chemically inactivated whole viruses, which are thought to be slow acting as they are effective only 4 to 7 days following vaccination. Hence, the development of a novel rapid vaccine or alternative measures, such as antiviral agents or the combination of vaccines and antiviral agents for prompt FMD virus (FMDV) outbreak containment, is desirable. Here, we constructed a recombinant baculovirus (BacMam) expressing consensus porcine interferon alpha (IFN-α) that has three additional N-glycosylation sites driven by a cytomegalovirus immediate early (CMV-IE) promoter (Bac-Con3N IFN-α) for protein expression in mammalian cells. Bac-Con3N IFN-α expressing highly glycosylated porcine IFN-α protein increased the duration of antiviral effects. We evaluated the antiviral effects of Bac-Con3N IFN-α in swine cells and mice and observed sustained antiviral effects in pig serum; additionally, Bac-Con3N IFN-α exhibited sustained antiviral effects in vivo as well as adjuvant effects in combination with an inactivated FMD vaccine. Pigs injected with a combination of Bac-Con3N IFN-α and the inactivated FMD vaccine were protected against FMDV at 1, 3, and 7 days postvaccination. Furthermore, we observed that in combination with the inactivated FMD vaccine, Bac-Con3N IFN-α increased neutralizing antibody levels in mice and pigs. Therefore, we suggest that Bac-Con3N IFN-α is a strong potential antiviral and adjuvant candidate for use in combination with inactivated FMD vaccines to protect pigs against FMDV. IMPORTANCE Early inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in pigs is highly desirable as FMDV transmission and shedding rates are higher in pigs than in cattle. However, commercial FMD vaccines require at least 4 to 7 days postvaccination (dpv) for protection, and animals are vulnerable to heterologous viruses before acquiring high antibody levels after the second vaccination. Therefore, the development of antiviral agents for use in combination with FMD vaccines is essential. We developed a novel antiviral and immunostimulant, Bac-Con3N IFN-α, which is a modified porcine IFN-α-expressing recombinant baculovirus, to improve IFN stability and allow its direct delivery to animals. We present a promising candidate for use in combination with inactivated FMD vaccines as pigs applied to the strategy had early protection against FMDV at 1 to 7 dpv, and their neutralizing antibody levels were higher than those in pigs administered the vaccine only.

Keywords: BacMam; foot-and-mouth disease virus; interferons; vaccines.

FIG 1

Plasmid construction and detection of protein expression in swine cells. (A) Genes of the cytomegalovirus immediate early 1 (CMV-IE) promoter, the signal sequence (ss), and consensus porcine IFN-α with N-glycosylation sites (4th, 23rd, and 78th amino acid positions) were cloned into the pFastBac dual vector by cleaving with the restriction sites SmaI and NheI to construct Bac-Con3N IFN-α. The baculovirus negative control (Bac-Neg con) was constructed by cloning the CMV-IE promoter by cleaving only with the restriction sites SmaI and XhoI. (B) LFBK cells were infected with Bac-Con3N IFN-α or Bac-Neg con at a multiplicity of infection (MOI) of 200. The supernatant was collected and concentrated at 48 h postinfection. Samples were treated with PNGase F or not to detect the presence of nonglycosylated or glycosylated protein and were used for Western blotting. To detect porcine IFN-α, swine IFN-α1 polyclonal antibody and rabbit anti-goat IgG(H+L) (HRP) were used as the primary and secondary antibodies, respectively.

FIG 2

Antiviral effects of Bac-Con3N IFN-α against FMDV in swine cells. LFBK cells were inoculated with recombinant baculoviruses (Bac-Neg con or Bac-Con3N IFN-α) at an MOI of 50, except for the FMDV control. After 18 h, the recombinant baculoviruses were removed, and cells were washed with DMEM. Thereafter, cells were infected with 100 TCID₅₀ of FMDV; after 1 h of FMDV infection, the inoculum was removed, and DMEM was added. Cells were incubated at 37°C for 24 or 48 h, and the supernatant was collected. To evaluate the effects on FMDV RNA replication, RNA extraction and quantitative real-time PCR were performed using the supernatant.

FIG 3

Enhanced duration of the antiviral effect of glycosylated porcine IFN-α protein in pig serum. The same units of protein produced from Bac-Con3N IFN-α in LFBK cells and the porcine IFN-α (mammalian) protein (R&D Systems, Minneapolis, MN, USA) expressed in mammalian cells were diluted to a 1:3 (vol/vol) ratio with swine serum. The porcine IFN-α proteins mixed with swine serum were incubated at 37°C for 6, 12, 24, or 48 h and immediately frozen at −80°C. After thawing the samples simultaneously, the biological activities of the IFN-α proteins were measured using the IFN biological assay. Error bars indicate standard deviations (SD) of the means. Unpaired t tests were performed for statistical analysis (*, P < 0.05; **, P < 0.01).

FIG 4

Induction of IFN-stimulated genes (ISGs) of Bac-Con3N IFN-α in swine cells. LFBK cells were inoculated with recombinant baculoviruses at an MOI of 50. Cells were collected at 6, 24, and 48 h postinfection, and RNA extraction, DNase I treatment, and quantitative real-time PCR were performed. To analyze ISG expression, quantitative real-time PCR was used to evaluate the levels of interferon beta (IFN-β) (A), 2′-5′-oligoadenylate synthetase (OAS) (B), and myxovirus resistance (Mx) (C) mRNAs. Error bars indicate standard deviations (SD) of the means. Unpaired t tests were performed for statistical analysis (*, P < 0.05).

FIG 5

Enhanced survival rates in mice injected with Bac-Con3N IFN-α. C57BL/6 mice were inoculated via intraperitoneal injection with 3 × 10⁸ PFU of Bac-Con3N IFN-α or Bac-Neg con and challenged with 200 LD₅₀ (50% lethal doses) of mouse-adapted FMDV O/VIT/2013 at 1 day (A) or 3 days (B) postinoculation. Animals were monitored for 7 days after FMDV infection.

FIG 6

Measurement of cytokine induction in mice after injection with Bac-Con3N IFN-α. BALB/c mice were inoculated via intraperitoneal injection with 3 × 10⁸ PFU of Bac-Con3N IFN-α or Bac-Neg con. Serum samples were collected at 0, 6, 16, and 24 h postinjection, and protein levels of mouse IFN-α, mouse IFN-γ, mouse IL-12, mouse IL-15, mouse IL-6, and mouse IL-18 were measured using an ELISA. Error bars indicate standard deviations (SD) of the means. An unpaired t test was performed to identify statistically significant differences between the 0-h posttreatment group and the 6-, 16-, or 24-h posttreatment group or between the Bac-Neg con group and the Bac-Con3N IFN-α group (*, P < 0.05; **, P < 0.01).

FIG 7

Measurement of cytokine induction in pigs after injection with Bac-Con3N IFN-α. Three pigs per group were inoculated via intramuscular injection with the O/SKR/Boeun/2017 vaccine or the O/SKR/Boeun/2017 vaccine in combination with 4 × 10¹⁰ TCID₅₀ of Bac-Con3N-poIFN-α, and blood collection was performed at 0, 3, or 7 days postvaccination. The vaccine was formulated with the O/SKR/Boeun/2017 vaccine antigen (15 μg/dose), 10% aluminum hydroxide gel, and Montanide ISA 206. Cytokine assays of serum samples were performed using the porcine IFN-γ ELISA kit, the porcine IFN-α ELISA kit, and the porcine IL-12/IL-23 P40 ELISA kit. Mean values and standard deviations (SD) are indicated with bars. Unpaired t tests were performed to identify statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.005).

FIG 8

Enhanced virus-neutralizing antibody (VN Ab) levels caused by the combination of Bac-Con3N IFN-α and an oil-adjuvant-based inactivated FMD vaccine in mice and pigs. (A) C57BL/6 mice were inoculated via intramuscular (i.m.) injection with a mixture of 3 × 10⁸ PFU of Bac-Con3N IFN-α (or Bac-Neg con) and the inactivated FMD vaccine or the FMD vaccine only. The FMD vaccine was formulated using ISA 206, 10% aluminum hydroxide gel, and 1 μg of the O/SKR/Boeun/2017 antigen. (B) Pigs were inoculated via i.m. injection with the mixture of 8 × 10⁹ PFU or 2 × 10¹⁰ PFU of Bac-Con3N IFN-α (or 2 × 10¹⁰ PFU of Bac-Neg con) and the inactivated FMD vaccine or the FMD vaccine only. The FMD vaccine was formulated using ISA 206 oil adjuvant, 10% aluminum hydroxide gel, and 10 μg of the O/SKR/Boeun/2017 antigen. Serum samples were collected at 1, 2, 3, and 4 weeks postvaccination in mice and at 1, 3, and 5 weeks postvaccination in pigs. The virus-neutralizing antibody test was performed using serum samples. The dotted line indicates the 1:45 (1.65 log₁₀ units) virus-neutralizing titer cutoff level. Error bars indicate standard deviations (SD) of the means. Unpaired t tests were performed to identify statistically significant differences between the vaccine group and others or among the combination groups (*, P < 0.05; **, P < 0.01; ***, P < 0.005).