Successful topical respiratory tract immunization of primates against Ebola virus

Abstract

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

FIG. 1.

Detection of EBOV in peripheral blood samples from rhesus monkeys following EBOV challenge. Animal numbers (animals 1 to 8 or 9 to 16) are indicated at the top of the graphs. Blood was taken on the indicated days postchallenge in experiment 1 (A) and experiment 2 (B, C) and assayed for infectious EBOV titer determined by TCID₅₀ titration (A, B) or EBOV RNA detected by quantitative RT-PCR (C). For the samples in which EBOV was not detected, the value 1.5 log₁₀ TCID₅₀/ml were assigned. For the quantitative RT-PCR (QPCR) data, glyceraldehyde-3-phosphate dehydrogenase mRNA served as an endogenous reference molecule in calculating relative expression. X indicates that the monkey died after the last indicated blood sample was collected. Det. limit, detection limit.

FIG. 2.

Peripheral blood markers of EBOV disease in rhesus monkeys following EBOV challenge in experiments 1 (left) and 2 (right). Animal numbers (animals 1 to 16) are indicated at the top of the graph. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; BUN, blood urea nitrogen; CRE, creatinine. The values (in international units per liter or milligrams per decaliter as indicated) are shown for each animal on the indicated days postchallenge. Markers of liver function (*) and markers of kidney function (**) are indicated. X indicates that the monkey died after the last indicated blood sample was collected.

FIG. 3.

Detection of EBOV-specific cell-mediated responses to immunization in experiment 2. Peripheral blood mononuclear cells were isolated on the indicated days after primary or secondary immunization, stimulated in vitro with peptides to GP, and analyzed by flow cytometry for the indicated markers. Values for individual animals (animals 9 to 16) are shown, expressed as percentages of the CD8⁺ or CD4⁺ lymphocyte populations.