Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus

Abstract

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Figures

Fig 1

VRP expression of Ebola virus and Sudan virus GP. BHK cells were infected with VRP expressing influenza virus hemagglutinin (A and B), EBOV GP (C), or SUDV GP (D). GP expression was detected using EBOV GP specific monoclonal antibody KZ52 (A and C) or SUDV GP specific monoclonal antibody 3C10 (B and D), and nuclei were stained with DAPI.

Fig 2

VRP-SUDV GP protects cynomolgus macaques from SUDV i.m. challenge. NHPs were vaccinated with VRP expressing irrelevant antigen (n = 2) or VRP expressing SUDV GP (n = 6). SUDV GP (A) or EBOV GP (B) specific serum antibody end titers were determined by ELISA at the indicated times postvaccination or challenge. Dotted line indicates assay limit of detection. Black and red arrows indicate time of SUDV challenge and EBOV back-challenge, respectively. (C) NHPs were challenged by i.m. injection of 1,000 PFU of SUDV 28 days postvaccination. (D) Surviving NHPs were then back-challenged by i.m. injection of 1,000 PFU of EBOV 30 or 35 days post-SUDV challenge. ***, P ≤ 0.001 (as determined by the Fisher exact test).

Fig 3

Dual vaccination with VRP-SUDV GP and VRP-EBOV GP protects cynomolgus macaques from SUDV i.m. challenge. NHPs were vaccinated with VRP expressing irrelevant antigen (n = 1) or VRPs expressing both SUDV GP and EBOV GP (n = 3). SUDV GP (A) or EBOV GP (B) specific serum antibody end titers were determined by ELISA at the indicated times postvaccination or challenge. Dotted line indicates assay limit of detection. Black and red arrows indicate time of SUDV challenge and EBOV back-challenge, respectively. (C) NHPs were challenged by i.m. injection of 1,000 PFU of SUDV 28 days postvaccination. (D) Surviving NHPs were then back challenged by i.m. injection of 1,000 PFU of EBOV 30 days post-SUDV challenge. **, P ≤ 0.01 (as determined by the Fisher exact test).

Fig 4

Dual vaccination with VRP-SUDV GP and VRP-EBOV GP protects cynomolgus macaques from EBOV i.m. challenge. NHPs were vaccinated with VRP expressing irrelevant antigen (n = 1) or VRPs expressing both SUDV GP and EBOV GP (n = 3). EBOV GP (A) or SUDV GP (B) specific serum antibody end titers were determined by ELISA at the indicated times postvaccination or challenge. Dotted line indicates assay limit of detection. Black and red arrows indicate time of EBOV challenge and SUDV back-challenge, respectively. (C) NHPs were challenged by i.m. injection of 1,000 PFU of EBOV 28 days postvaccination. (D) Surviving NHPs were then back challenged by i.m. injection of 1,000 PFU of SUDV 28 days post-EBOV challenge. **, P ≤ 0.01 (as determined by the Fisher exact test).

Fig 5

Histologic lesions typical of filovirus infection in experimental animal 10. (A) Liver has diffuse vacuolar degeneration of hepatocytes adjacent to a central vein (V), with foci of hepatocellular necrosis (arrowheads). (B) Spleen, with markedly decreased numbers of lymphocytes (i.e., lymphoid depletion) and hemorrhage in a lymphoid nodule (N). Deposits of large amounts of fibrin in the surrounding red pulp appear as amorphous to fibrillar pink material. (C and D) IHC of liver and spleen, respectively, fails to detect the presence of ebolavirus antigen. The greenish-brown granules present in some hepatocytes are bile pigment. (E and F) IHC of liver and spleen, respectively, reveals abundant marburgvirus antigen (brown staining) within hepatic Kupffer cells, endothelial cells, hepatocytes, and splenic histiocytes. Extracellular viral antigen is also present in areas of hepatic necrosis and in fibrin deposits in the spleen. Objective magnification: 40× for liver photos and 10× for spleen photos.

Fig 6

Real-time PCR detection of filovirus. RNA was purified from whole blood collected on day 7 postchallenge. One-step RT-PCRs were completed using filovirus species-specific primers. Reciprocal critical threshold (CT) values are reported. Dotted line indicates assay limit of detection (1 PFU/reaction).

Fig 7

Vaccination with VRP-SUDV GP protects cynomolgus macaques from SUDV aerosol challenge. NHPs were vaccinated with one (A and C) or two (B and D) doses of VRP expressing irrelevant antigen or VRP expressing SUDV GP. SUDV GP specific serum antibody end titers were determined by ELISA at the indicated times postvaccination with one (A) or two (B) doses of vaccine. Dotted line indicates assay limit of detection. (C) NHPs receiving one dose of irrelevant VRP (n = 1) or VRP-SUDV GP (n = 3) were challenged by aerosol exposure to 100 PFU of SUDV 28 days postvaccination. (D) NHPs receiving two doses of irrelevant VRP (n = 3) or VRP-SUDV GP (n = 3) were challenged by aerosol exposure to 100 PFU of SUDV 28 days following the second vaccination. **, P ≤ 0.01 (as determined by the Fisher exact test).

Fig 8

Pulmonary lesions associated with aerosolized SUDV. (A) Dorsal view of lungs and trachea (T) from experimental animal 13, after removal from the thoracic cavity. There is diffuse pulmonary edema and a large area of dark-red consolidation (arrow) in the caudal aspect of the left superior lung lobe (S). The pleural surface in this area and the left middle lung lobe (M) is covered with tan-white fibrinous exudate which causes adhesions between the left superior, middle, and inferior (I) lung lobes. (B) Histologic section of normal lung from a cynomolgus macaque that died after i.m. challenge with SUDV. Clear spaces are alveoli and are separated by thin-walled septa and small blood vessels (V). (C) Histologic section of lung from control animal 12, which died 7 days after challenge with aerosolized SUDV. The alveoli are filled by edema fluid, fibrin, and a mixture of macrophages, neutrophils, and red blood cells. Alveolar septa are congested and thickened with inflammatory cells. A small arteriole (A) is also present. (D) IHC of lung from control animal 12 reveals the presence of abundant intracellular and extracellular ebolavirus antigen (brown staining). (E) Histologic section of a pulmonary granuloma in experimental animal 18, which survived a challenge with aerosolized SUDV. The center of the granuloma (left 1/3 of the photo) contains suppurative inflammation, fibrin, and necrotic debris. The wall of the granuloma (right 2/3 of the photo) is composed of macrophages, lymphocytes, plasma cells, and fibrous connective tissue. (F) IHC of a replicate section of the image in panel E reveals ebolavirus antigen within macrophages and scattered extracellular antigen in the area of necrosis. Objective magnifications (B to F), ×20.