Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses

Abstract

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.

Fig. 1.

Virus constructs. Negative-sense RNA genomes are illustrated for the parental RABV vaccine constructs, BNSP (A) and BNSP333 (B) (28), and four RABV vaccine vectors expressing Zaire Ebola virus strain Mayinga GP with (C and D) or without RABV G (E and F). The 333 mutation results in an Arg→Glu change at amino acid 333 of RABV G, which has been shown to greatly attenuate neurovirulence of RABV vaccine vectors in adult mice (28, 29). ZEBOV GP is expressed authentically (C and E) or with the RABV GCD fused to its ectodomain and transmembrane domain (D and F). The GCD is depicted by a black box.

Fig. 2.

Results of the virus spread assay indicate ΔG viruses are growth restricted. BSR cells (A and C) or trans-complemented BSR cells expressing RABV G (B and D) were infected at an MOI of 0.001 with BNSPΔG-GP (A and B) or BNSPΔG-GP_GCD (C and D). Three days later, cells were fixed and immunostained for intracellular RABV nucleoprotein. Fluorescence microscope magnification, ×10.

Fig. 3.

Expression of GP by RABV vaccine constructs in vitro. (A) Western blot analysis of GP expressed by the indicated viruses at 24 to 72 h. Infected cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with polyclonal monkey anti-EBOV. (B) Coomassie blue-stained SDS-PAGE gel of purified virus particles with the indicated RABV proteins. The Western blots were probed with anti-EBOV sera or anti-RABV P sera. (C) BSR cells were infected with the indicated viruses and analyzed by dual-label immunogold electron microscopy. RABV G was detected by primary rabbit anti-RABV G followed by secondary anti-rabbit IgG labeled with 15-nm gold particles. ZEBOV GP was detected by primary human anti-GP antibody followed by secondary anti-human IgG labeled with 5-nm gold particles. Magnification, ×150,000 (BNSP333 and BNSP333-GP); ×210,000 (BNSP333-GP_GCD).

Fig. 4.

RABV vaccine viruses expressing GP are avirulent in mice after peripheral inoculation. (A) Groups of five BALB/c mice were inoculated i.m. with 5 × 10⁵ FFU live virus on day 0 or with 10 μg inactivated virus on days 0 and 14. Data are representative of two experiments. (B) Groups of 5 SW mice were inoculated with 1 × 10⁵ FFU of the indicated viruses. Data are representative of two experiments. (C) Groups of eight BALB/c mice were inoculated i.p. with 1 × 10⁶ FFU of the indicated viruses. Mice inoculated by each route were monitored daily for signs of morbidity and weighed periodically for the indicated time period.

Fig. 5.

RABV vaccines expressing GP induce RABV G- and ZEBOV GP-specific antibodies. Groups of 10 BALB/c mice were immunized i.m. with 5 × 10⁵ FFU of indicated live virus or 10 μg of inactivated virus on day 0 (1 dose) or on days 0 and 14 (2 dose) for two independent challenge experiments. Serum was drawn on day 30 postimmunization (blue bars) before RABV challenge (A and C) or MA-EBOV challenge (B and D), pooled, and analyzed by ELISA directed against ZEBOV GP at a 1:200 dilution (A and B) or RABV G at a 1:300 dilution (C and D). After RABV or MA-EBOV challenge, serum was collected, pooled, and tested in the same manner (red bars). Vehicle-Ch, mice immunized with vehicle but not challenged; Vehicle+Ch, mice immunized with vehicle and challenged with the indicated virus.

Fig. 6.

RABV vaccines expressing GP induce protective levels of RABV G-specific neutralizing antibodies. Groups of five BALB/c mice were immunized i.m. with 5 × 10⁵ FFU of the indicated live virus on day 0 or with 10 μg of inactivated virus on days 0 and 14 (2 dose). Serum was drawn on day 28 and analyzed in a RABV neutralization assay; results are expressed as IU/ml. The 0.5-IU level is considered protective by WHO standards.

Fig. 7.

RABV vaccines expressing GP confer protection from EBOV and RABV. Two groups of 10 BALB/c mice were immunized i.m. with 5 × 10⁵ FFU of the indicated live virus or 10 μg of inactivated virus on day 0 (1 dose) or on days 0 and 14 (2 dose). (A) On day 77 postimmunization, groups of 10 mice were challenged i.p. with 1,000 PFU of MA-EBOV. Mice were monitored for morbidity for 21 days. (B) On day 50 postimmunization, groups of 10 mice were challenged i.m. with virulent RABV virus strain CVS-N2c and monitored for morbidity for 21 days.

Fig. 8.

Weight loss after challenge with EBOV or RABV. Two groups of 10 BALB/c mice were immunized i.m. with 5 × 10⁵ FFU of the indicated live virus or 10 μg of inactivated virus on day 0 (1 dose) or on days 0 and 14 (2 dose). (A) On day 77 postimmunization, groups of 10 mice were challenged i.p. with 1,000 PFU of MA-EBOV. Mice were weighed daily for 9 days and every other day until day 21. (B) On day 50 postimmunization, groups of 10 mice were challenged i.m. with virulent RABV virus strain CVS-N2C and weighed daily for 21 days.