Joshua C. Johnson

Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

Summary 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.

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail

Ebola virus–infected macaques were successfully treated with a cocktail of monoclonals manufactured in plants. Better Late than Never They say prevention is better than a cure, but sometimes, immediate action isn’t possible. This is especially the case for a deadly disease such as Ebola virus (EBOV) infection, where sporadic outbreaks make it hard to predict when and where treatment will be needed. In patients, neither preventative nor therapeutic options are currently available, but recent studies have shown that a cocktail of monoclonal antibodies may help if given within 1 to 2 days of challenge in macaques. Pettitt et al. now extend this window, showing that this antibody cocktail can be used as a therapeutic in nonhuman primates (NHPs) even after the onset of symptoms. The authors challenged NHPs with EBOV and didn’t begin treatment until after confirmation of infection and observation of fever. Although the controls here and all historical controls succumbed to infection, 43% of the treated animals survived the challenge. If these observations hold true in humans, these monoclonal antibodies could give hope to people exposed to EBOV. Ebola virus (EBOV) remains one of the most lethal transmissible infections and is responsible for high fatality rates and substantial morbidity during sporadic outbreaks. With increasing human incursions into endemic regions and the reported possibility of airborne transmission, EBOV is a high-priority public health threat for which no preventive or therapeutic options are currently available. Recent studies have demonstrated that cocktails of monoclonal antibodies are effective at preventing morbidity and mortality in nonhuman primates (NHPs) when administered as a post-exposure prophylactic within 1 or 2 days of challenge. To test whether one of these cocktails (MB-003) demonstrates efficacy as a therapeutic (after the onset of symptoms), we challenged NHPs with EBOV and initiated treatment upon confirmation of infection according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization and observation of a documented fever. Of the treated animals, 43% survived challenge, whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represent successful therapy of EBOV infection in NHPs.

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus–immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3+ T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8+ cells, as depletion of CD8+ cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4+ T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8+ cells have a major role in rAd5-GP–induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.

Recombinant Adenovirus Serotype 26 (Ad26) and Ad35 Vaccine Vectors Bypass Immunity to Ad5 and Protect Nonhuman Primates against Ebolavirus Challenge

ABSTRACT The use of adenoviruses (Ad) as vaccine vectors against a variety of pathogens has demonstrated their capacity to elicit strong antibody and cell-mediated immune responses. Adenovirus serotype C vectors, such as Ad serotype 5 (Ad5), expressing Ebolavirus (EBOV) glycoprotein (GP), protect completely after a single inoculation at a dose of 1010 viral particles. However, the clinical application of a vaccine based on Ad5 vectors may be hampered, since impairment of Ad5 vaccine efficacy has been demonstrated for humans and nonhuman primates with high levels of preexisting immunity to the vector. Ad26 and Ad35 segregate genetically from Ad5 and exhibit lower seroprevalence in humans, making them attractive vaccine vector alternatives. In the series of studies presented, we show that Ad26 and Ad35 vectors generate robust antigen-specific cell-mediated and humoral immune responses against EBOV GP and that Ad5 immune status does not affect the generation of GP-specific immune responses by these vaccines. We demonstrate partial protection against EBOV by a single-shot Ad26 vaccine and complete protection when this vaccine is boosted by Ad35 1 month later. Increases in efficacy are paralleled by substantial increases in T- and B-cell responses to EBOV GP. These results suggest that Ad26 and Ad35 vectors warrant further development as candidate vaccines for EBOV.

Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species

A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.

Evaluation of candidate vaccine approaches for MERS-CoV

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development. Supplementary information The online version of this article (doi:10.1038/ncomms8712) contains supplementary material, which is available to authorized users.

Interferon-β Therapy Prolongs Survival in Rhesus Macaque Models of Ebola and Marburg Hemorrhagic Fever

There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)-α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-β production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-β therapy in filovirus infection. Here we show that early postexposure treatment with IFN-β significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-β also significantly increased survival time after Marburg virus infection. IFN-β may have promise as an adjunctive postexposure therapy in filovirus infection.

The pathogenesis of Rift Valley fever virus in the mouse model.

Detailed studies describing the pathogenesis of Rift Valley fever (RVF) virus (RVFV) in the mouse model are lacking. A fully characterized small animal model of RVF is needed to evaluate potential vaccines and therapeutics. In this study, we characterized the pathogenesis of RVFV throughout the disease course in mice. Infection produced high-titer viremia and demonstrated RVFV tropism for a variety of tissue and individual cell types. Overwhelming infection of hepatocytes, accompanied by apoptosis, was a major consequence of infection. The majority of mice died or were euthanatized between days 3 and 6 postinfection with severe hepatitis. The remaining mice effectively cleared virus from the liver and blood, but exhibited neuroinvasion and developed panencephalitis. In addition, we characterized a number of other virological, clinicopathological, and histopathological features of RVFV infection in mice. The mouse model therefore mimics both the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF.

Postexposure treatment of Marburg virus infection.

Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus–based vaccine is administered 20–30 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection.