JOURNAL OF VIROLOGY, June 2008, p. 5664–5668 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.00456-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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Recombinant Vesicular Stomatitis Virus Vector Mediates Postexposure Protection against Sudan Ebola Hemorrhagic Fever in Nonhuman Primates䌤 Thomas W. Geisbert,1,2,3,4* Kathleen M. Daddario-DiCaprio,4,5 Kinola J. N. Williams,6 Joan B. Geisbert,1 Anders Leung,8 Friederike Feldmann,8 Lisa E. Hensley,5 Heinz Feldmann,7,8 and Steven M. Jones6,7,8 National Emerging Infectious Diseases Laboratories Institute,1 Department of Microbiology,2 and Department of Medicine3, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts; Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland4; Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland5; Department of Immunology6 and Medical Microbiology,7 University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, Canada; and Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada8 Received 2 March 2008/Accepted 21 March 2008
Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses. approaches based on small interfering RNA (12) and antisense oligomers (6, 22) have shown promising results in rodent models, but there have been no published reports of either treatment strategy being evaluated in the more stringent macaque models. Recently, we showed the first complete postexposure protection of nonhuman primates against a filovirus by administering a live-attenuated recombinant vesicular stomatitis virus (rVSV) vector expressing the MARV glycoprotein (GP) shortly after a high-dose MARV challenge (4). We subsequently demonstrated that an rVSV vector expressing the ZEBOV GP protected 50% of rhesus macaques when admin-
The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause severe and often fatal infections in humans and nonhuman primates. While there is a single species of MARV, there are four recognized species of EBOV: Ivory Coast ebolavirus (also known as Cote d’Ivoire ebolavirus), Reston ebolavirus, Sudan ebolavirus (SEBOV), and Zaire ebolavirus (ZEBOV) (7, 17). Until recently, nearly all EBOV outbreaks in humans have been caused by either SEBOV or ZEBOV. A possible fifth species of EBOV was associated with an outbreak in Uganda late in 2007, but little information is available regarding this new EBOV (1). Since 1976, there have been at least 10 outbreaks of ZEBOV, with case fatality rates approaching 90% (17). During the same period, there have been four outbreaks of SEBOV with mortality rates of approximately 50% (17). Remarkable progress has been made over the last few years in developing candidate preventive vaccines that can protect nonhuman primates against EBOV and MARV (2, 3, 14, 15, 18–21). Advances in development of postexposure treatments and therapies against the filoviruses have been much slower. Some degree of success has been achieved using strategies that mitigate the coagulation abnormalities that characterize filoviral infection (10, 11, 13). Several new postexposure treatment
* Corresponding author. Mailing address: Department of Microbiology, Boston University School of Medicine, 715 Albany Street, R514, Boston, MA 02118. Phone: (617) 638-4274. Fax: (617) 638-4286. E-mail:
[email protected]. 䌤 Published ahead of print on 2 April 2008.
FIG. 1. Kaplan-Meier survival curves for rhesus macaques given postexposure treatment for SEBOV infection. 5664
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TABLE 1. Clinical findings in rhesus monkeys infected with SEBOV and given postexposure treatment with an rVSV vector expressing the SEBOV GP or a VSV vector expressing a nonspecific GP Clinical finding(s) onb:
Subjecta Day 6
1
Fever, lymphopenia, thrombocytopenia, ALP1 Fever, lymphopenia Fever, lymphopenia, thrombocytopenia
2 3
Day 10
Day 14
Mild rash, lymphopenia, thrombocytopenia, ALP111, AST111, BUN1
Thrombocytopenia
Infection outcome
Survived
ALP1 Lymphopenia, thrombocytopenia, moderate rash, Lymphopenia, thrombocytopenia anorexia, ALP1, ALT111, AST111, BUN111, GGT1, CRE11 4 Fever, lymphopenia, ALP1 ALP1 Control 1 Fever, lymphopenia, Fever, mild rash, anorexia, depression, Fever, mild rash, anorexia, depression, thrombocytopenia lymphopenia, thrombocytopenia, ALP11, lymphopenia, thrombocytopenia, AST11, BUN1 ALP11, ALT11, AST111, GGT1, BUN111, UA1
Survived Survived Survived Died on day 17
a Subjects 1 to 4 underwent treatment with an rVSV vector expressing the SEBOV GP. Control 1 underwent treatment with a VSV vector expressing a nonspecific GP. b Fever was defined as a temperature more than 2.5°F over baseline or at least 1.5°F over baseline and ⱖ103.5°F. Mild rash was defined as focal areas of petechiae covering less than 10% of the skin, moderate rash as areas of petechiae covering between 10% and 40% of the skin, and severe rash as areas of petechiae and/or echymosis covering more than 40% of the skin. Lymphopenia and thrombocytopenia were defined by a ⱖ35% drop in the numbers of lymphocytes and platelets, respectively. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, ␥-glutamyltransferase; BUN, blood urea nitrogen; CRE, creatinine; UA, uric acid. 1, two- to threefold increase; 11, four- to fivefold increase; 111, more-than-fivefold increase.
istered shortly after a high-dose ZEBOV challenge (8). Here, we used a rhesus macaque model of SEBOV hemorrhagic fever (HF) to test the ability of rVSV vectors expressing the SEBOV GP to protect animals against a homologous SEBOV challenge. Nonhuman primate studies were performed with biosafety level 4 biocontainment at USAMRIID and were approved by the USAMRIID Laboratory Animal Care and Use Committee. Animal research was conducted in compliance with the Animal Welfare Act and other Federal statues and regulations relating to animals and experiments involving animals and adheres to the principles stated in the Guide for the Care and Use of Laboratory Animals (16). The facility used is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The rVSV expressing the GPs of SEBOV (strain Boniface) (VSV⌬G/SEBOVGP) or Lassa virus (LASV; strain Josiah) (VSV⌬G/LASVGPC) were generated as previously described (9). Five healthy, filovirus-seronegative, rhesus macaques (4 to 7 kg) were challenged by intramuscular (i.m.) injection with 1,000 PFU of homologous SEBOV. Approximately 20 to 30 min after SEBOV challenge, four of the animals received an i.m. injection at four different sites of the VSV⌬G/SEBOVGP
vector (⬃2 ⫻ 107 PFU). The remaining animal served as the experimental control and received an equivalent dose of the VSV⌬G/LASVGPC vector. Animals were monitored twice daily for clinical signs of SEBOV HF, including cutaneous rashes, hemorrhage, and reduced activity. Blood samples for viral infectivity titration, reverse transcription-PCR (RT-PCR), hematology, serum biochemistry, and immunoglobulin M (IgM) and IgG antibody were collected prior to SEBOV challenge and on days 3, 6, 10, 14, 23, and 35 after SEBOV challenge (4, 8). All four animals exposed to SEBOV and treated with the VSV⌬G/SEBOVGP vector developed a transient fever at day 6, but all survived SEBOV challenge, while the control animal succumbed on day 17 after SEBOV exposure (Fig. 1). Changes in hematology and blood chemistry were noticed in all treated and control animals during the course of the study (Table 1). Most values began to return to normal levels by day 14 in the VSV⌬G/SEBOVGP-treated animals, whereas values did not return to prechallenge levels in the control animal at any time after day 6. A transient recombinant VSV viremia was detected by RT-PCR in all five animals on day 3 (data not shown). Three of the four VSV⌬G/SEBOVGP-treated animals showed little change in appearance or behavior that indicated overt
TABLE 2. Viral load in rhesus monkeys infected with SEBOV and given postexposure treatment with an rVSV vector expressing the SEBOV GP or a VSV vector expressing a nonspecific GP Plasma viral load (log10 PFU/ml) onb:
Subjecta
1 2 3 4 Control 1
Day 3
Day 6
Day 10
Day 14
Day 17
Day 23
0 (⫺) 0 (⫺) 0 (⫺) 0 (⫺) 0 (⫺)
3.0 (⫹) 0 (⫹) 4.2 (⫹) 0 (⫺) 4.9 (⫹)
2.4 (⫹) 0 (⫺) 3.6 (⫹) 0 (⫹) 6.2 (⫹)
0 (⫹) 0 (⫺) 2.6 (⫹) 0 (⫺) 5.1 (⫹)
NT (NT) NT (NT) NT (NT) NT (NT) 5.0 (⫹)
0 (⫺) 0 (⫺) 0 (⫺) 0 (⫺) NA (NA)
a Subjects 1 to 4 underwent treatment with an rVSV vector expressing the SEBOV GP, and control 1 underwent treatment with a VSV vector expressing a nonspecific GP. b Viral load represents the log10 PFU of SEBOV/ml of plasma. Results in parentheses indicate whether the sample was positive (⫹) or negative (⫺) for SEBOV by RT-PCR. NT, not tested; NA, not applicable.
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FIG. 2. Serological response profile for rhesus macaques given postexposure treatment for SEBOV infection. (A) IgM. (B) IgG.
illness, and these animals remained healthy during the course of the study. One of these animals (subject 4) had a slight decrease in appetite on days 8 and 9, and another animal (subject 1) had a very small rash on one arm on days 9 and 10. Plaque assay was unable to detect any evidence of SEBOV in plasma of two of these three animals (Table 2); however, RT-PCR showed evidence of SEBOV in plasma of one of these animals at day 6 (subject 2) and the other animal at day 10 (subject 4). SEBOV was detected by plaque assay and RT-PCR in plasma of the third animal (subject 1) on days 6 and 10 (Table 2). The fourth animal treated with the VSV⌬G/ SEBOVGP vector (subject 3) developed symptoms consistent
with SEBOV HF, including anorexia and a macular rash. SEBOV was detected by plaque assay and RT-PCR in plasma of this animal on days 6, 10, and 14 after challenge (Table 2). However, viral load never exceeded 4.2 log10 PFU/ml, a value which is thought to be an important indicator for predicting survival based on results of previous studies with filovirusinfected monkeys (T. W. Geisbert, unpublished observation). This animal cleared the viremia and showed little evidence of illness by day 20. In contrast, the control animal became severely ill and developed classic symptoms of SEBOV HF, including dehydration, anorexia, depression, and the presence of a macular rash. The control animal was the only animal that
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was febrile on days 10 and 14. SEBOV was detected in plasma of this animal on days 6, 10, 14, and 17 by plaque assay and RT-PCR, and the viral load exceeded 6.1 log10 PFU/ml by day 10 (Table 2). The serological response profile of SEBOV infection after treatment was evaluated by IgM and IgG enzyme-linked immunosorbent assay. All four of the animals challenged with SEBOV and treated with the VSV⌬G/SEBOVGP vector demonstrated moderate to high levels of IgM by day 6 (1:100 to 1:1,000) (Fig. 2A) and IgG by day 14 (1:1,000), while the control animal did not mount a humoral response against SEBOV (Fig. 2B). Survival in this SEBOV study was better than the results of a similar study in rhesus macaques using the VSV⌬G/ ZEBOVGP vector as a treatment after a homologous ZEBOV challenge (8). In the ZEBOV study, we protected four of eight animals from lethal infection. The results of the current study were comparable to those of a study that we performed with rhesus macaques using the VSV⌬G/MARVGP vector against homologous MARV challenge in which all specifically treated animals survived (4), although in the MARV study, none of the animals treated with the VSV⌬G/MARVGP vector became viremic or clinically ill, whereas in the current study all of the animals treated with the VSV⌬G/SEBOVGP vector became viremic as a result of the SEBOV challenge and one animal became clinically ill. The difference in outcomes among these three studies (SEBOV, ZEBOV, and MARV) may be due to the length of the disease course in rhesus macaques. That is, the window of opportunity may be larger for SEBOV and MARV than for ZEBOV. In this study with SEBOV, the control animal died on day 17. While very few studies have examined the pathogenesis of SEBOV HF in rhesus macaques, the disease observed in the control animals in this study is consistent with sparse historical data in which controls have succumbed 12 to 17 days after exposure (5; Geisbert, unpublished). Experimental infection of rhesus monkeys with MARV (Musoke strain) produces a uniformly lethal disease 11 to 13 days (mean, 11.6 days) after i.m. exposure to 1,000 PFU of MARV (4; Geisbert, unpublished). In comparison, experimental infection of rhesus monkeys with ZEBOV produces a uniformly lethal infection in 7 to 10 days (mean, 8.3 days) after i.m. exposure to 1,000 PFU of ZEBOV (11). The exact mechanism of postexposure protection conferred by the VSV⌬G/SEBOVGP vector remains uncertain. There have been no cases of survival or delay in death among the cohort of positive control macaques receiving rVSV vectors expressing nonspecific GPs (3, 4, 8, 15). Interference between the rVSVs and wild-type filoviruses cannot be discounted. All of the rVSV vectors expressing filoviral GPs replicate much faster in vitro than their counterpart wild-type filovirus (9; H. Feldmann, unpublished observation). As these rVSV vectors contain a full-length filoviral GP, they presumably target and infect the same preferred host cells as wild-type filoviruses (9). However, it is clear that an adaptive immune response is important for protection as we have seen previously that MARVinfected macaques treated with VSV⌬G/ZEBOVGP died in parallel with untreated controls (4), showing that even though MARV and ZEBOV in general infect the same populations of host cells, an adaptive response is required to clear the virus and protect the host. Future studies to look at the biodistribu-
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tion of rVSV in macaques and possible studies looking at depleting different immune cell populations may shed further light on this most interesting finding. The current investigation was designed as a proof-of-concept study to compare the VSV⌬G/SEBOVGP vector in the rVSV postexposure format with previous studies that used VSV⌬G/MARVGP or VSV⌬G/ZEBOVGP vectors. Clearly, studies are needed to look at how far out treatment can be delayed. For example, if MARV-infected or SEBOV-infected macaques are first treated with a homologous rVSV vector 24 h after challenge instead of 20 to 30 min, is complete protection still achieved? Regardless of outcome of this type of study; however, this strategy may still prove useful in treating potential laboratory accidents or medical personnel exposed during case patient management where rapid treatment is certainly feasible. In addition, the overall strategy of postexposure treatment using rVSV vectors expressing filoviral GPs may be even more useful against filoviruses that are associated with reduced mortality rates and where disease progresses more slowly, as may be the case with Ivory Coast ebolavirus (17) and/or with the apparent new EBOV species that was recently identified in Uganda (1). From USAMRIID, we thank John Crampton and Carlton Rice for animal care and Anna Honko for assistance with biocontainment. From the National Microbiology Laboratory (NML) of the Public Health Agency of Canada (PHAC), we thank Allen Grolla for technical assistance with biocontainment. We are grateful to John Rose (Yale University) for kindly providing us with the vesicular stomatitis virus reverse genetics system and Peter Jahrling (NIAID/NIH) for helpful discussions. Work with filoviruses at USAMRIID was funded by the Defense Threat Reduction Agency (project number 04-4-7J-012). T.W.G. and J.B.G. were U.S. Government employees when portions of this work were performed at USAMRIID. Work with filoviruses at the NML was supported by PHAC, a grant awarded to H.F. from the Canadian Institutes of Health Research (MOP-39321), and a grant awarded to S.M.J. from Chemical, Biological, Radiological, and Nuclear (CBRN) Research and Technology Initiative (CRTI). Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by Boston University, the U.S. Army, or the Public Health Agency of Canada. REFERENCES 1. Alsop, Z. 2008. Ebola outbreak in Uganda “atypical”, say experts. Lancet 370:2085. 2. Bukreyev, A., P. E. Rollin, M. K. Tate, L. Yang, S. R. Zaki, W.-J. Shieh, B. R. Murphy, P. L. Collins, and A. Sanchez. 2007. Successful topical respiratory tract immunization of primates against Ebola virus. J. Virol. 81:6379–6388. 3. Daddario-DiCaprio, K. M., T. W. Geisbert, J. B. Geisbert, U. Stroher, L. E. Hensley, A. Grolla, E. A. Fritz, F. Feldmann, H. Feldmann, and S. M. Jones. 2006. Cross-protection against Marburg virus strains using a live, attenuated recombinant vaccine. J. Virol. 80:9659–9666. 4. Daddario-Dicaprio, K. M., T. W. Geisbert, U. Stroher, J. B. Geisbert, A. Grolla, E. A. Fritz, L. Fernando, E. Kagan, P. B. Jahrling, L. E. Hensley, S. M. Jones, and H. Feldmann. 2006. Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment. Lancet 367:1399–1404. 5. Ellis, D. S., E. T. Bowen, D. I. Simpson, and S. Stamford. 1978. Ebola virus: a comparison, at ultrastructural level, of the behaviour of the Sudan and Zaire strains in monkeys. Br. J. Exp. Pathol. 59:584–593. 6. Enterlein, S., K. L. Warfield, D. L. Swenson, D. A. Stein, J. L. Smith, C. S. Gamble, A. D. Kroeker, P. L. Iversen, S. Bavari, and E. Mu ¨hlberger. 2006. VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice. Antimicrob. Agents Chemother. 50:984–993. 7. Feldmann, H., T. W. Geisbert, P. B. Jahrling, H. D. Klenk, S. V. Netesov, C. J. Peters, A. Sanchez, R. Swanepoel, and V. E. Volchkov. 2004. Filoviridae, p. 645–653. In C. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.), Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, London, United Kingdom.
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