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

Results

Prechallenge EBOV neutralization titers were measured for the 26 nonhuman primates used in this study ( Table 1 ). Although all vaccinated animals seroconverted by immunoglobulin G enzyme-linked immunosorbent assay, neutralizing antibody (PRNT50) titers were very low. Only one macaque vaccinated with VRP-expressed EBOV GP had detectable neutralizing antibody. The marginal PRNT did not preclude challenge of the monkeys; however, in previous studies, similar results were obtained when cynomolgus macaques were vaccinated with the VRP expressing MBGV genes, yet the animals were protected from lethal disease[12].

All animals, including the four untreated macaques, were challenged with 1,000 PFU of EBOV. Timing of challenge varied because of differences in the optimal immunization regimens determined by preliminary testing in rodents. VRP-vaccinated animals were challenged 49 days after the third vaccine dose. At postchallenge day 3, all animals became ill; two animals from each vaccination group (i.e., GP, NP, GP + NP, influenza HA) died on day 6, and the remaining animals died on day 7 ( Table 2 ). VACV GP-inoculated macaques were challenged 45 days after the third vaccine dose, EBOV liposome-vaccinated animals 35 days after the third vaccine dose, and macaques vaccinated with the gamma-irradiated whole-virion preparation 43 days after the third vaccine dose. Again, all animals except one rhesus macaque, which received the gamma-irradiated virion preparation, became ill on the third day after challenge. Two cynomolgus macaques vaccinated with the gamma-irradiated virion preparation, one VACV-GP animal, and one untreated cynomolgus macaque died on postchallenge day 6 ( Table 2 ). The two remaining VACV-GP animals died at day 7 after challenge, as did two of the animals vaccinated with the EBOV liposome preparation and the remaining untreated cynomolgus macaque. The untreated rhesus macaques died on days 8 and 9 postchallenge; one rhesus vaccinated with the gamma-irradiated virion preparation died on day 9, and the other survived challenge. The remaining animal vaccinated with the EBOV liposome preparation died 11 days after challenge. The rhesus macaque that survived challenge did not become ill during the study and had a PRNT50 values >320 at day 26 postchallenge and 80 at days 26, 61, 99, and 902 postchallenge.

Conventional histopathologic and electron microscopic examination of lymphatic tissues, liver, and gastrointestinal tract showed no differences in lesions between the vaccinated animals and the unvaccinated EBOV-infected controls. Depletion and necrosis or apoptosis were noted in all lymphoid germinal centers in spleen, peripheral, and mesenteric lymph nodes, as described in other studies[17,18,19]. The spleen had copious deposits of fibrin throughout the red pulp, as well as abundant karyorrhectic cellular debris. By electron microscopy, widespread bystander lymphocyte apoptosis was a prominent feature in all the lymphatic tissues examined. Fibrin and fibrinocellular thrombi were also prominent in the submucosa of the gastrointestinal tract and in hepatic sinusoids, again consistent with well-documented findings[17,18].

We also evaluated retrospectively EBOV-infected rodent tissues in parallel. Although sites of infection and morphologic changes between guinea pigs, mice, and nonhuman primates had many similarities, the lack of fibrin thrombi in spleen and visceral vasculature was particularly striking in the EBOV-infected mice (Figure). Fibrin deposition was seen in guinea pigs as reported[20], but fibrin deposits and thrombi were considerably less prevalent compared with deposits in nonhuman primates (Figure). Lymphocyte apoptosis was also less frequently observed by electron microscopy in rodent lymphatic tissues than in nonhuman primates. EBOV was demonstrated in liver, spleen, kidney, lung, adrenal gland, and lymph nodes of all necropsied monkeys by immunohistochemistry, electron microscopy, or virus infectivity titration.

Sections of spleen from Ebola virus (EBOV)-infected animals. Top left, BALB/c mouse, note absence of polymerized fibrin (phosphotungstic acid [PTA] hematoxylin, original magnification X400). Field representative of five of five mice tested. Top right: guinea pig. Note discreet foci of polymerized fibrin (arrows) (PTA hematoxylin, original magnification X400). This field shows infrequent fibrin deposits; most fields in five of five animals examined showed no evidence of polymerized fibrin. Lower left: cynomolgus monkey. Note deposition of polymerized fibrin in red pulp (PTA hematoxylin, original magnification X400). Field representative of 25 of 25 monkeys. Lower right: cynomolgus monkey. Electron micrograph showing abundant fibrin deposits in red pulp (original magnification X5,300). Field representative of 11 of 11 monkeys examined.

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