Evaluation in Nonhuman Primates of Vaccines against Ebola Virus

Thomas W. Geisbert, Peter Pushko, Kevin Anderson, Jonathan Smith, Kelly J. Davis, and Peter B. Jahrling

Disclosures

Emerging Infectious Diseases. 2002;8(5) 

In This Article

Materials and Methods

Cynomolgus macaques (Macaca fascicularis) or rhesus macaques (M. mulatta) weighing 4 to 6 kg were used. For vaccine studies with VEE replicons, EBOV GP or NP genes were introduced into the VEEV RNA as described[10]. Groups of three cynomolgus macaques were vaccinated with VRP that expressed EBOV GP, EBOV NP, a mixture of EBOV GP and EBOV NP, or a control antigen (influenza hemagglutinin) that has no effect on EBOV immunity. Animals were vaccinated by subcutaneous injection of 107 focus-forming units of VRP in a total of 0.5 mL at one site. Vaccinations were repeated 28 days after the first injection and 28 days after the second.

In conducting research with animals, the investigators followed the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (1996). The animal facilities and animal care and use program of the U.S. Army Medical Research Institute of Infectious Diseases are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

For vaccine studies using primates, we adapted the optimal immunization regimens determined from the rodent studies. For the vaccine based on recombinant VACV, the EBOV GP gene was inserted into a VACV transfer vector plasmid, and recombinant VACV expressing EBOV GP were isolated as reported[13]. Three cynomolgus macaques were injected subcutaneously with the EBOV GP-expressing VACV vector. Injectionswere repeated at 28 and 53 days after the first injection.

For vaccine studies with inactivated EBOV whole-virion preparation, viral particles were concentrated from Vero cell culture fluids by ultracentrifugation in a sucrose density gradient. The infectivity titer of the preparation was approximately 8.0 log10 PFU/mL. The preparation was inactivated by exposure to 60Co gamma rays (6 x 106 rads). The absence of residual infectivity was proven by exhaustive testing for residual infectivity in assays in Vero cells[15,16]. Two cynomolgus monkeys and two rhesus monkeys were injected subcutaneously with a 50-µg dose of the gamma-irradiated virion preparation in RIBI adjuvant (Corixa, Hamilton, MT). As a further check on complete viral inactivation, blood samples taken from the monkeys 3 and 5 days after they received the vaccine were free of infectious viremia. Injections were repeated at days 7 and 35 after the initial injection.

For vaccine studies using a liposome formulation, three cynomolgus monkeys were vaccinated with gamma-irradiated virus encapsulated in liposomes containing lipid A, as described for previous studies in mice[14]. Animals received 1.0 mL of the liposome preparation by intravenous injections that were repeated at 28 and 55 days after the initial vaccination. Four macaques (two cynomolgus and two rhesus) served as unvaccinated controls for the VACV, gamma-inactivated virion, and liposome studies.

Anti-EBOV neutralizing antibody titers were monitored by measuring plaque reduction in a constant virus:serum dilution format[15]. All macaques received intramuscular injections in the leg with 1,000 PFU of the Zaire subtype of EBOV, which was isolated from a human patient in 1995[16]. Blood was obtained from all monkeys under Telazol anesthesia (Fort Dodge Laboratories, Fort Dodge, IA) at 2- or 3-day intervals postinfection to determine infectious viremia, neutralizing antibody titers, and standard hematologic and clinical pathology parameters. All terminally ill monkeys were killed and necropsied for pathologic examination. Virus infectivity assays on plasma and tissue homogenates were done by forming plaqueson Vero cell monolayers as described[15,16].

Tissues were immersion fixed in 10% neutral-buffered formalin and processed for histopathologic and immunohistochemical characteristics as described[17,18,19]. Replicate sections of spleen were stained with phosphotungstic acid hematoxylin to demonstrate polymerized fibrin. Sections of spleen from five EBOV-infected guinea pigs and five mice from previous studies[20,21] were similarly stained for polymerized fibrin. Portions of selected tissues from 11 monkeys were also immersion fixed in 4% formaldehyde and 1% glutaraldehyde and processed for transmission electron microscopy according to conventional procedures[17,18,19].

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