Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine

Abstract

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

Figure 1. Immunization schedule and RABV/EBOV vaccine constructs.

(A) Schematic of the RABV vaccine constructs expressing EBOV GP used for prime and boost immunizations. (B) Experimental timeline: All NHPs were immunized on day 0 and challenged intramuscularly with 1,000 PFU of EBOV on day 56 or 70 as described in the Materials and Methods. The day of challenge (day 56 or 70) is referred to throughout the paper as challenge day 0. Group 4 was boosted with 250 µg of the inactivated construct on day 28.

Figure 2. Humoral immune response to RABV G and EBOV GP before Ebola Zaire challenge.

(A) Rhesus macaque total IgG immune response to RABV G. OD490 readings were compared to a World Health Organization (WHO) standard (human sera) for rabies. (B) Rhesus macaque total IgG immune response to EBOV GP compared to a control macaque which survived EBOV infection. All sera were diluted 1∶50 and analyzed in a 3 fold serial dilution via ELISA. (C) Neutralization assay for RABV G post immunization.

Figure 3. Survival curve and clinical findings in rhesus macaques after EBOV challenge.

Rhesus macaques were intramuscularly challenged with 1000 PFU of EBOV on day 56 or 70 (challenge day 0). The Kaplan-Meier survival curve (A) indicates that 100% of animals immunized with the replication-competent vaccine survived EBOV challenge. Clinical signs of infection such as body temperature (B), viremia (C), platelet count (D), serum alanine aminotransferase (ALT) levels (E), and serum aspartate aminotransferase (AST) levels (F) were monitored daily.

Figure 4. Humoral immune response to EBOV GP after challenge.

Rhesus macaque total IgG immune response to EBOV GP for post challenge day 0 (A), day 3 (B), day 6 (C), day 16 (D), and day 28 (E). NHP responses were compared to the response of a control macaque which survived EBOV infection. All sera were diluted 1∶50 and analyzed in a 3 fold serial dilution via ELISA. NHPs belonging to Group 1, those immunized with BNSP333, succumbed to EBOV infection by post challenge Day 9 whereas NHPs in (D) and (E) survived Ebola Zaire challenge.

Figure 5. Full length EBOV GP and EBOV GP-ΔMLD exhibit similar immunogenicity.

Total IgG response to full length EBOV GP (A) and EBOV GP-ΔMLD (B) on days 0, 3, and 6 post challenge.

Figure 6. IgG2/IgG1 isotype ratios in response to EBOV GP.

(A) Isotype ratios (IgG2/IgG1) at 1∶150 for post challenge days 0, 6, and 28. Ratios less than 1.0 indicate a bias towards a Th1 response. Group 1 animals did not show an IgG1 or IgG2 response to EBOV GP so the ratios are not shown. (B) Isotype ratios for protected animals versus unprotected animals after EBOV challenge. The isotype ratios of the protected animals were not statistically significant when compared to the ratios of the unprotected animals. When analyzing the isotype ratios of group 2 and group 4 protected animals to the unprotected animals in group 4, there is a significant difference (***, p<0.001). Statistical analysis was performed using unpaired t-test with Welch's correction to compare two groups. Results shown are presented as the mean. *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Avidity assay of total IgG immune response to EBOV GP.

Sera were analyzed from day 42 (A), day 0 (challenge) (B), day 3 post challenge (C), and day 28 post challenge (D) (study termination) with a NaSCN-displacement ELISA. Serum samples were diluted to an OD490 reading of 0.8 based on total IgG ELISA data.