A Single-Dose Intranasal Combination Panebolavirus Vaccine

Abstract

Background: Ebolaviruses Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) cause severe human disease, which may be accompanied by hemorrhagic syndrome, with high case fatality rates. Monovalent vaccines do not offer cross-protection against these viruses whose endemic areas overlap. Therefore, development of a panebolavirus vaccine is a priority. As a vaccine vector, human parainfluenza virus type 3 (HPIV3) has the advantages of needle-free administration and induction of both systemic and local mucosal antibody responses in the respiratory tract.

Methods: To minimize the antivector immunity, genes encoding the HPIV3 envelope proteins F and HN were removed from the vaccine constructs, resulting in expression of only the ebolavirus envelope protein-glycoprotein. These second-generation vaccine constructs were used to develop a combination vaccine against EBOV, SUDV, and BDBV.

Results: A single intranasal vaccination of guinea pigs or ferrets with the trivalent combination vaccine elicited humoral responses to each of the targeted ebolaviruses, including binding and neutralizing antibodies, as well as Fc-mediated effector functions. This vaccine protected animals from death and disease caused by lethal challenges with EBOV, SUDV, or BDBV.

Conclusions: The combination vaccine elicited protection that was comparable to that induced by the monovalent vaccines, thus demonstrating the value of this combination trivalent vaccine.

Keywords: Ebola virus; intranasal vaccination; vaccine.

Figure 1.

Vaccine constructs and study design. A, Vaccine candidates were designed by inserting the GP gene of EBOV, SUDV, or BDBV between the P and M genes of HPIV3. B, The F and HN HPIV3 genes were removed so that the filovirus glycoprotein of interest is expressed as the sole transmembrane envelope protein in the vaccine constructs. C, Expression of filovirus GP proteins by cells infected with the monovalent or trivalent vaccine constructs evaluated by western blotting. Purified GP proteins were used as positive controls, and actin or GAPDH were used as loading controls. The experiment was performed independently twice with essentially similar results. D, Guinea pigs and ferrets were vaccinated intranasally on day 0 with the monovalent vaccines or with the trivalent combination, and the control group received the empty HPIV3 vector. The filovirus challenge occurred on day 33, via the intraperitoneal route for the guinea pig model and via the intramuscular route for the ferret model. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GP, glycoprotein; HPIV3, human parainfluenza virus type 3; SUDV, Sudan virus; WT, wild type.

Figure 2.

Antibody responses to HPIV3/ΔF-HN-EboGP, HPIV3/ΔF-HN/SUDV-GP, and HPIV3/ΔF-HN/Trivalent in guinea pigs. A, Binding IgG evaluated by ELISA. B, Binding IgA evaluated by ELISA. C, Virus-neutralizing antibody titers determined by plaque reduction assay. The GP antigens for ELISA or viruses for neutralization are indicated at the top of the figure, and the vaccines or vector control are indicated at the bottom. The dotted line indicates the level of nonspecific binding in pooled samples collected before vaccination. n = 5 except the HPIV3 group in EBOV and BDBV studies where n = 4. P values were determined by Kruskal-Wallis test followed by Dunn postcomparisons test. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; HPIV3, human parainfluenza virus type 3; Ig, immunoglobulin; SUDV, Sudan virus.

Figure 3.

Antibody responses to HPIV3/ΔF-HN/BDBV-GP and HPIV3/ΔF-HN/Trivalent in ferrets. A, Binding IgG evaluated by ELISA. B, Binding IgA evaluated by ELISA. C, Virus-neutralizing antibody titers determined by plaque reduction assay. The GP antigens for ELISA or viruses for neutralization are indicated at the top of the figure, and the vaccines or vector control are indicated at the bottom. Pooled serum samples collected prior to vaccination demonstrated no detectable binding. n = 5 animals per group except for IgA assays where n = 2 for HPIV3. P values were determined by Kruskal-Wallis test followed by Dunn postcomparisons test. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; HPIV3, human parainfluenza virus type 3; Ig, immunoglobulin; SUDV, Sudan virus.

Figure 4.

Fc-mediated responses to HPIV3/ΔF-HN-EboGP, HPIV3/ΔF-HN/SUDV-GP, and HPIV3/ΔF-HN/Trivalent in guinea pigs. A, Layout of the ADNP, ADCP, and ADCD assays. B, ADNP data. C, ADCP data. D, ADCD data. The GP proteins used in the assays are indicated at the top of panel B, and the vaccines or vector control are indicated at the bottom of the figure. n = 5 except for the EBOV challenge study where n = 4 for the HPIV3 group and n = 3 for HPIV3/ΔF-HN-EboGP group. P values were determined by Kruskal-Wallis test followed by Dunn postcomparisons test. The figures show representative data from at least 2 independent donors. The values indicate the mean from 2 technical replicates. Error bars are standard error of the mean (SEM). Abbreviations: Ab, antibody; BDBV, Bundibugyo virus; EBOV, Ebola virus; GP, glycoprotein; HPIV3, human parainfluenza virus type 3; SUDV, Sudan virus.

Figure 5.

Fc-mediated responses to HPIV3/ΔF-HN/BDBV-GP and HPIV3/ΔF-HN/Trivalent in ferrets. A, ADNP data. B, ADCP data. C, ADCD data. The GP proteins used in the assays are indicated at the top of the figure, and the vaccines or vector control are indicated at the bottom. n = 5 per group. P values were determined by Kruskal-Wallis test followed by Dunn postcomparisons test. The figures show representative data from at least 2 independent donors. The values indicate the mean from 2 technical replicates. Error bars are standard error of the mean (SEM). Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; GP, glycoprotein; HPIV3, human parainfluenza virus type 3; SUDV, Sudan virus.

Figure 6.

Protection against lethal EBOV, SUDV, or BDBV challenge induced by the homologous vaccines or HPIV3/ΔF-HN/Trivalent in guinea pigs or ferrets. A, Survival. B, Disease scores. C, Changes in body weight. D, Viremia; horizontal line indicates limit of detection. n = 5 animals per group except n = 4 guinea pigs for HPIV3 group in EBOV challenge study. For survival, P values are indicated for comparisons against the respective vector-control group based on log-rank Mantel-Cox statistical analyses. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; HPIV3, human parainfluenza virus type 3; PFU, plaque-forming unit; SUDV, Sudan virus.