A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

Abstract

Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.

Fig. 1.

Diagram illustrating the structure and the assembly of EIC. (A) EIC was designed and expressed by fusing Ebola GP1 to the C terminus of the H chain of 6D8 mAb (Left). The recombinant IgG can bind to Ebola GP1 and form an immune complex (Center). Female BALB/c mice were vaccinated with EIC s.c. with or without candidate adjuvant agents according to the designed vaccination schedule (B).

Fig. 2.

Codelivery of PIC with h-EIC in mice induced the highest anti-Ebola IgG antibody response relative to other TLR agonist combinations. Each group was vaccinated s.c. with h-EIC alone, h-EIC with CL097, h-EIC with PIC, or h-EIC with CL097 plus PIC on days 0, 21, 42, and 63. Anti-Ebola IgG titers were measured by ELISA on days 0, 14, 35, 56, and 84. GMTs were defined as the highest serum dilution giving a positive reaction. The GMTs for PBS solution negative control group and preimmune (i.e., day 0) serum in all groups were no greater than 1. Error bars indicate SD. Data presented are representative of three independent studies. Statistically significant differences (P < 0.05) in the level of IgG anti-Ebola were determined by Kruskal–Wallis test. Comparisons were made between vaccination groups relative to PBS solution control (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 3.

h-EIC codelivered with PIC significantly protected mice from Ebola lethal challenge. Vaccination with three or four doses of h-EIC with or without candidate adjuvant agents was performed in mice (n = 10 per group). After vaccination, mice were challenged i.p. with 1,000 pfu (30,000 LD₅₀) of mouse-adapted EBOV on day 84. (A) Survival was recorded for 30 d. (B) Percent body weight change was recorded for 14 d. Because body weight values are means from surviving mice, the loss of weight in the first week of the experiment for EIC-treated mice primarily corresponds to lack of eating of animals that would not survive challenge. Statistically significant differences (P < 0.05) in survival were determined by log-rank test and comparisons were made between vaccination groups relative to PBS solution control as indicated by asterisks.

Fig. 4.

Anti-Ebola IgG levels correlate with the level of protection afforded against Ebola challenge. Mice vaccinated as described for the challenge study were bled 2 wk after each dose and 3 wk after the last vaccination. Serum was analyzed for (A) total IgG and (B) neutralizing antibody specific to EBOV (n = 5 per group). (A) Individual serum samples for each mouse in each vaccination group were analyzed for total anti-Ebola IgG by ELISA on days 0, 14, 35, 56, and 84. The GMT for preimmune (i.e., day 0) serum in all groups were no greater than 1. Error bars indicate SD. Statistically significant differences (P < 0.05) in the level of IgG anti-Ebola were determined by Kruskal–Wallis test. Comparisons were made between vaccination groups relative to PBS solution control (*P < 0.05, **P < 0.01, and ***P < 0.001). (B) Virus neutralization assays were performed on serum collected on day 84. Pooled sera from groups of five mice were assayed. Serial dilutions of serum were mixed with EBOV and applied to Vero cell monolayers. Neutralization titers were determined to be the last dilution of serum that reduced the number of plaques by 80% compared with control wells.

Fig. 5.

PIC codelivered with h-EIC induced a mixed Th1/Th2 response that correlated with the level of protection against lethal EBOV challenge. Mice vaccinated as described for the challenge study were bled 2 wk after each vaccination (days 14, 35, and 56) and 3 wk after the last vaccination (day 84). Anti-Ebola antibody isotypes were measured by ELISA. IgG1 (blue), IgG2a (red), IgG2b (green), and IgG3 (orange) levels are presented for mice vaccinated with (A) h-EIC, (B) h-EIC codelivered with PIC, (C) h-EIC codelivered with alum and PIC, and (D) GP-VRP.