TY - JOUR
T1 - Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference
T2 - a proof-of-concept study
AU - Geisbert, Thomas W.
AU - Lee, Amy CH
AU - Robbins, Marjorie
AU - Geisbert, Joan B.
AU - Honko, Anna N.
AU - Sood, Vandana
AU - Johnson, Joshua C.
AU - de Jong, Susan
AU - Tavakoli, Iran
AU - Judge, Adam
AU - Hensley, Lisa E.
AU - MacLachlan, Ian
N1 - Funding Information:
Seven treatments with anti-ZEBOV siRNAs inhibited the replication of the virus and completely protected rhesus monkeys against death from haemorrhagic fever. In previous studies, ZEBOV-infected rhesus monkeys succumbed when viraemia on days 6–10 after exposure was greater than 4·5 log 10 pfu/mL, whereas animals treated therapeutically survived when the viraemia did not reach this level (Geisbert TW, unpublished). 19,21 In the current study, viraemia never exceeded 2·4 log 10 pfu/mL in any surviving animal. The development of treatments for EBOV-induced haemorrhagic fever has been slow and no previous candidate treatment has shown complete protection against ZEBOV haemorrhagic fever in non-human primates. Some success has been achieved with interventions that mitigate the coagulation disorders that characterise EBOV infection. 19,21 We protected 50% of non-human primates against ZEBOV by administering a live-attenuated recombinant vesicular stomatitis virus vaccine vector that expressed the ZEBOV glycoprotein shortly after a lethal ZEBOV challenge. 22 Several new postexposure treatments based on siRNA 11 and antisense oligomers 23,24 have shown promising results in rodent models, but there have been no reports of the assessment of either treatment strategy after EBOV challenge in non-human primates, which more faithfully reproduce human EBOV infections than do other animal models. 1,18 This model was rapid and uniformly lethal (death in all 21 naive rhesus macaques challenged with the same ZEBOV seed stock by the same dose and route as in the present study; Geisbert TW, unpublished) in which animals typically succumbed 6–10 days after challenge with the virus (mean time to death 8·3 days). The ZEBOV target in the siRNA postexposure treatment study in guineapigs was the L polymerase gene, and treatment initiated 1 h after ZEBOV challenge completely protected all animals. 11 Targeting only VP35 completely protected mice against ZEBOV challenge when the antisense oligomers were administered 24 h after ZEBOV exposure. 23 Targeting a combination of the L polymerase, VP24, and VP35 genes also completely protected mice and incompletely protected guineapigs when given 24 h after ZEBOV challenge. 24 Although RNA-interference-mediated treatment strategies show potential for combating EBOV infections, systemic administration of synthetically manufactured siRNA duplexes can activate the innate immune response, inducing high levels of inflammatory cytokines such as tumour necrosis factor α, interleukin 6, and interferons, particularly interferon α which might contribute to antiviral activity in vivo. 25–27 Off-target effects can be toxic to the host and also confound interpretation of preclinical data. siRNAs must therefore be modified to prevent detrimental immune activation in vivo, particularly because immunostimulation in human beings is much more toxic than in rodents. 14,25 We therefore tested immune stimulatory activity of SNALPs containing anti-ZEBOV siRNAs in mice and in human PBMC cultures. The ZEBOV cocktail was not immune stimulatory at the limit of sensitivity of the assays used. These data and the 5′RACE PCR data suggest that the antiviral effects in non-human primates are the result of specific RNA interference in reticuloendothelial cells and not due to immune stimulation or other off-target effects. Additionally, the death of the macaque given control Luc mod on day 10, which was within the normal range of expected time to death for historical untreated controls, also suggests that the ZEBOV-mediated protection was specific to the siRNAs and not related to off-target effects. When the immune toxicities associated with the siRNA component of SNALPs were abrogated through the use of selective chemical modification, the major remaining toxicity in mouse and non-human primate models was hepatotoxicity associated with the SNALP lipid components (MacLachlan I, unpublished). Preliminary tolerability studies in mice showed that seven daily doses of up to 4·3 mg/kg (30·0 mg/kg cumulative) of SNALPs containing ZEBOV siRNAs were well tolerated, corroborating the lack of hepatotoxicity (measured as increases in liver enzymes such as alanine aminotransferase and aspartate aminotransferase) in the treated macaques that survived infection. Our investigation was designed as a proof-of-concept study. The rhesus macaque model that we used represents a worse-case scenario such as an accidental needle stick exposure of a laboratory worker or first responder to a high infectious dose of ZEBOV, which has occurred several times during the past 5 years. 28–30 ZEBOV infection in human beings normally progresses slower than in macaques with case fatality rates in man ranging from 70–90%, 1 suggesting that the therapeutic window could be larger than in experimentally infected macaques. Nonetheless, the focus in future studies will need to be on investigation of whether anti-ZEBOV siRNAs will be beneficial if administered at the onset of symptoms. Here, we show a substantial progress in the treatment of ZEBOV infections in non-human primates compared with previously described postexposure methods that afforded only partial protection of non-human primates against ZEBOV. 19,21,22 To progress to license, studies will be done in guineapig and non-human primate models of ZEBOV infection in accordance with the US Food and Drug Administration's guidelines for the assessment of treatments for Centers for Disease Control and Prevention category A threats (number 21CFR314.610). Additionally, studies will need to be done to obtain data about pharmacology, dosing, and toxicology in uninfected non-human primates. Contributors TWG, ACHL, MR, AJ, LEH, and IM conceived and designed the experiments. TWG wrote the research programme plan and the animal protocol for the studies. LEH wrote modifications for the animal protocol. TWG and LEH selected the ZEBOV L polymerase as a target. TWG, ACHL, VS, MR, AJ, LEH, and IM selected ZEBOV VP24 and VP35 as targets. TWG co-designed the L polymerase siRNA, and ACHL and VS co-designed the VP24 and VP35 siRNAs. ACHL, VS, and AJ designed the modifications to the siRNAs. VS and ANH did the in vitro siRNA assays. JBG and JCJ did the Ebola challenge experiments in non-human primates and the clinical pathology assays. JBG did the Ebola virus infectivity assays. SDJ did the immunology and uptake assays. MR and IT did the 5′RACE assays. TWG, ACHL, MR, JBG, ANH, JCJ, AJ, LEH, and IM analysed the data. TWG and MR co-wrote the report. LEH and IM edited the report. All authors have seen and approved the final version of the report. Conflicts of interest TWG, ACHL, MR, VS, AJ, LEH, and IM claim intellectual property regarding RNA interference for the treatment of filoviral infections. ACHL, MR, VS, SDJ, IT, AJ, and IM are employees of Tekmira Pharmaceuticals. JCJ, ANH, and JBG declare that they have no conflicts of interest. Acknowledgments Work on filoviruses at US Army Medical Research Institute of Infectious Diseases was funded by the Defense Threat Reduction Agency (project number 04-4-7J-012 ). Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by Boston University or the US Army.
PY - 2010
Y1 - 2010
N2 - Background: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. Methods: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. Findings: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. Interpretation: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. Funding: Defense Threat Reduction Agency.
AB - Background: We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. Methods: A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV. Findings: Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. Interpretation: This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections. Funding: Defense Threat Reduction Agency.
UR - http://www.scopus.com/inward/record.url?scp=77952680862&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77952680862&partnerID=8YFLogxK
U2 - 10.1016/S0140-6736(10)60357-1
DO - 10.1016/S0140-6736(10)60357-1
M3 - Article
C2 - 20511019
AN - SCOPUS:77952680862
SN - 0140-6736
VL - 375
SP - 1896
EP - 1905
JO - The Lancet
JF - The Lancet
IS - 9729
ER -