Daniel J. Deer

Lipid nanoparticle siRNA treatment of Ebola virus Makona infected nonhuman primates

The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA.

Lipid nanoparticle delivery of anti-MARV nucleoprotein–targeting small interfering RNA can treat Marburg virus in nonhuman primates after symptom onset. Medicating Marburg Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever; however, there are currently no vaccines or drugs approved for human use to treat these devastating infections. Thi et al. now report that a lipid-encapsulated siRNA can treat MARV in nonhuman primates after symptom onset. They use lipid nanoparticles to deliver siRNA targeting the MARV nucleoprotein to treated animals at various time points after virus exposure. All animals that received the therapy survived MARV infection, including those that were treated 3 days after infection—a stage when animals are viremic and demonstrate the first clinical signs of disease. Therefore, this approach holds promise as a strategy to treat filovirus infection in humans. Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)–targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti–MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.

Monoclonal antibody therapy for Junin virus infection

Significance There are no Food and Drug Administration approved drugs available for preventing or treating Argentine hemorrhagic fever (AHF), and the current treatment option is limited to administration of immune plasma. With the expanding clinical use of monoclonal antibodies (mAbs) for acute and chronic conditions, it has become clear that mAbs offer a highly specific, potent, and generally safe drug platform for antivirals, and may be a useful alternative to immune plasma. Here, we show that mAbs are effective in the Junin virus guinea pig model and likely to be an economical therapy for AHF. Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic.

Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody

A single mAb is an effective therapy in nonhuman primates infected with Marburg or Ravn virus. Fighting filoviruses with antibody therapy Like other filoviruses such as Ebola virus, Ravn and Marburg viruses cause hemorrhagic fever in humans with high morbidity rates. Mire et al. tested the ability of previously identified human monoclonal antibodies to protect guinea pigs from lethal infection. One candidate antibody was then administered several days after lethal Marburg or Ravn infection in nonhuman primates and was able to reduce clinical symptoms and confer almost uniform protection. This antibody is a promising therapeutic that could be helpful in future filovirus outbreaks. As observed during the 2013–2016 Ebola virus disease epidemic, containment of filovirus outbreaks is challenging and made more difficult by the lack of approved vaccine or therapeutic options. Marburg and Ravn viruses are highly virulent and cause severe and frequently lethal disease in humans. Monoclonal antibodies (mAbs) are a platform technology in wide use for autoimmune and oncology indications. Previously, we described human mAbs that can protect mice from lethal challenge with Marburg virus. We demonstrate that one of these mAbs, MR191-N, can confer a survival benefit of up to 100% to Marburg or Ravn virus–infected rhesus macaques when treatment is initiated up to 5 days post-inoculation. These findings extend the small but growing body of evidence that mAbs can impart therapeutic benefit during advanced stages of disease with highly virulent viruses and could be useful in epidemic settings.

Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever

There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

Oral and Conjunctival Exposure of Nonhuman Primates to Low Doses of Ebola Makona Virus

Nonhuman primate (NHP) models of Ebola virus (EBOV) infection primarily use parenteral or aerosol routes of exposure. Uniform lethality can be achieved in these models at low doses of EBOV (≤100 plaque-forming units [PFU]). Here, we exposed NHPs to low doses of EBOV (Makona strain) by the oral or conjunctival routes. Surprisingly, animals exposed to 10 PFU by either route showed no signs of disease. Exposure to 100 PFU resulted in illness and/or lethal infection. These results suggest that these more natural routes require higher doses of EBOV to produce disease or that there may be differences between Makona and historical strains.

Rescue of non-human primates from advanced Sudan ebolavirus infection with lipid encapsulated siRNA

Although significant progress has been made in developing therapeutics against Zaire ebolavirus, these therapies do not protect against other Ebola species such as Sudan ebolavirus (SUDV). Here, we describe an RNA interference therapeutic comprising siRNA targeting the SUDV VP35 gene encapsulated in lipid nanoparticle (LNP) technology with increased potency beyond formulations used in TKM-Ebola clinical trials. Twenty-five rhesus monkeys were challenged with a lethal dose of SUDV. Twenty animals received siRNA-LNP beginning at 1, 2, 3, 4 or 5 days post-challenge. VP35-targeting siRNA-LNP treatment resulted in up to 100% survival, even when initiated when fever, viraemia and disease signs were evident. Treatment effectively controlled viral replication, mediating up to 4 log10 reductions after dosing. Mirroring clinical findings, a correlation between high viral loads and fatal outcome was observed, emphasizing the importance of stratifying efficacy according to viral load. In summary, strong survival benefit and rapid control of SUDV replication by VP35-targeting LNP confirm its therapeutic potential in combatting this lethal disease.

Nipah Virus C and W Proteins Contribute to Respiratory Disease in Ferrets

ABSTRACT Nipah virus (NiV) is a highly lethal paramyxovirus that recently emerged as a causative agent of febrile encephalitis and severe respiratory disease in humans. The ferret model has emerged as the preferred small-animal model with which to study NiV disease, but much is still unknown about the viral determinants of NiV pathogenesis, including the contribution of the C protein in ferrets. Additionally, studies have yet to examine the synergistic effects of the various P gene products on pathogenesis in animal models. Using recombinant NiVs (rNiVs), we examine the sole contribution of the NiV C protein and the combined contributions of the C and W proteins in the ferret model of NiV pathogenesis. We show that an rNiV void of C expression resulted in 100% mortality, though with limited respiratory disease, like our previously reported rNiV void of W expression; this finding is in stark contrast to the attenuated phenotype observed in previous hamster studies utilizing rNiVs void of C expression. We also observed that an rNiV void of both C and W expression resulted in limited respiratory disease; however, there was severe neurological disease leading to 60% mortality, and the surviving ferrets demonstrated sequelae similar to those for human survivors of NiV encephalitis. IMPORTANCE Nipah virus (NiV) is a human pathogen capable of causing lethal respiratory and neurological disease. Many human survivors have long-lasting neurological impairment. Using a ferret model, this study demonstrated the roles of the NiV C and W proteins in pathogenesis, where lack of either the C or the W protein independently decreased the severity of clinical respiratory disease but did not decrease lethality. Abolishing both C and W expression, however, dramatically decreased the severity of respiratory disease and the level of destruction of splenic germinal centers. These ferrets still suffered severe neurological disease: 60% succumbed to disease, and the survivors experienced long-term neurological impairment, such as that seen in human survivors. This new ferret NiV C and W knockout model may allow, for the first time, the examination of interventions to prevent or mitigate the neurological damage and sequelae experienced by human survivors.

siRNA rescues nonhuman primates from advanced Marburg and Ravn virus disease

Ebolaviruses and marburgviruses belong to the family Filoviridae and cause high lethality in infected patients. There are currently no licensed filovirus vaccines or antiviral therapies. The development of broad-spectrum therapies against members of the Marburgvirus genus, including Marburg virus (MARV) and Ravn virus (RAVV), is difficult because of substantial sequence variability. RNAi therapeutics offer a potential solution, as identification of conserved target nucleotide sequences may confer activity across marburgvirus variants. Here, we assessed the therapeutic efficacy of lipid nanoparticle (LNP) delivery of a single nucleoprotein–targeting (NP-targeting) siRNA in nonhuman primates at advanced stages of MARV or RAVV disease to mimic cases in which patients begin treatment for fulminant disease. Sixteen rhesus monkeys were lethally infected with MARV or RAVV and treated with NP siRNA-LNP, with MARV-infected animals beginning treatment four or five days after infection and RAVV-infected animals starting treatment three or six days after infection. While all untreated animals succumbed to disease, NP siRNA-LNP treatment conferred 100% survival of RAVV-infected macaques, even when treatment began just 1 day prior to the death of the control animals. In MARV-infected animals, day-4 treatment initiation resulted in 100% survival, and day-5 treatment resulted in 50% survival. These results identify a single siRNA therapeutic that provides broad-spectrum protection against both MARV and RAVV.

Postexposure Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against High and Low Doses of Marburg Virus Variant Angola in Nonhuman Primates

Abstract A recombinant vesicular stomatitis virus (rVSV) expressing the Marburg virus (MARV) Musoke variant glycoprotein fully protects macaques against 2 MARV variants and Ravn virus as a preventive vaccine and MARV variant Musoke as a postexposure treatment. To evaluate postexposure efficacy against the most pathogenic MARV variant, Angola, we engineered rVSVs expressing homologous Angola glycoprotein. Macaques were challenged with high or low doses of variant Angola and treated 20–30 minutes after exposure. A total of 25% and 60%–75% of treated macaques survived the high-dose and low-dose challenges, respectively. The more rapid disease progression of variant Angola versus variant Musoke may account for the incomplete protection observed.