New vaccine design based on defective genomes that combines features of attenuated and inactivated vaccines

Abstract

Background: New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260).

Principal finding: Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV.

Conclusions: The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms.

Figure 1. Immunogenicity of C-S8p260 in mice. C57BL/6 mice were inoculated in the FP with either 10³ or 10⁷ PFUs of C-S8p260, or PBS.

At 90 days post-immunization, mice were challenged with 10⁴ PFUs of FMDV C-S8c1 (procedures are described in Materials and Methods). A. Survival curves after immunization and after challenge (indicated in the abscissa, with arrow in the bottom panel). Continuous line: animals immunized with C-S8p260; discontinuous line: animals inoculated with PBS. B. IgG titers determined by ELISA at different days post-immunization and after challenge (abscissa and bottom panel). C. IgG titers in serum of mice immunized with BEI-inactivated C-S8p260. The day of the challenge with 10⁴ PFUs of C-S8c1 is indicated with an arrow; 16 animals were inoculated in each group. D. Survival of mice inoculated with C-S8p260p3d and C-S8c1; 21 mice were inoculated with 10⁷ PFUs C-S8p260p3d (squares), and 10 mice with 10³ PFUs of C-S8c1 (diamonds).

Figure 2. Serum IgG1, IgG2 and IgM-specific responses in pigs following immunization with C-S8p260.

Each column corresponds to the antibody titer for the indicated animal (numbers 1 to 14) at different times (D, day) post-immunization and post-challenge (white bars: D15 pi; light gray: D30 pi; dark grey: D32 pi (that corresponds to D2 post-challenge), and black: D37 pi (that corresponds to D7 post-challenge). Titers are expressed as the reciprocal of the highest dilution of serum (log₁₀) that gives an OD_A460 of twice the value obtained with the pre-immune serum of the corresponding animal.

Figure 3. Serum IgA-specific responses in pigs following immunization with C-S8p260.

Each column corresponds to the antibody titer for the indicated animal (numbers 1 to 14) at different times (D, days) post-immunization and post-challenge (white bars: D15 pi; light gray: D30 pi; dark grey: D32 pi (that corresponds to D2 post-challenge), and black: D37 pi (that corresponds to D7 post-challenge). Titers are expressed as the reciprocal of the highest dilution of serum (log₁₀) that gives an OD_A460 of twice the value obtained with the pre-immune serum of the corresponding animal.

Figure 4. FMDV-specific T cell responses in vaccinated pigs.

PBMCs were purified from blood of animals at day 30 (D30) post-immunization (pre-challenge) (white bars), and D11 post-challenge (grey bars). A. Lymphoproliferative responses of pigs 1 to 17 (groups 1, 2, 3, 4 and 5) to FMDV C-S8c1. Data are shown as stimulation index (see Material and Methods) and standard deviations are indicated. B. IFN-γ release by PBMC stimulated with FMDV C-S8c1. Values were determined at 72 h of in vitro stimulation. The detection level in control cultures (medium alone) were below the sensitivity of the assay (5 pg/ml).