•We identify vinylsulfones as lead candidate inhibitors of Ebola virus and SARS-CoV.•K11777 inhibited Ebola virus and SARS-CoV entry in the sub-nanomolar range.•Potent inhibition correlated with the ...presence of a basic piperazine ring at P3.•Serine protease inhibitor and K11777 blocked coronavirus entry into caco-2 cells.•Camostat protected 6 out of ten mice from lethal infection with SARS-CoV.
In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl-2-{(E)-4-methylpiperazine-1-carbonylamino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
BACKGROUND
Transfusion of plasma from recovered patients after Ebolavirus (EBOV) infection, typically called “convalescent plasma,” is an effective treatment for active disease available in endemic ...areas, but carries the risk of introducing other pathogens, including other strains of EBOV. A pathogen reduction technology using ultraviolet light and riboflavin (UV+RB) is effective against multiple enveloped, negative‐sense, single‐stranded RNA viruses that are similar in structure to EBOV. We hypothesized that UV+RB is effective against EBOV in blood products without activating complement or reducing protective immunoglobulin titers that are important for the treatment of Ebola virus disease (EVD).
STUDY DESIGN AND METHODS
Four in vitro experiments were conducted to evaluate effects of UV+RB on green fluorescent protein EBOV (EBOV‐GFP), wild‐type EBOV in serum, and whole blood, respectively, and on immunoglobulins and complement in plasma. Initial titers for Experiments 1 to 3 were 4.21 log GFP units/mL, 4.96 log infectious units/mL, and 4.23 log plaque‐forming units/mL. Conditions tested in the first three experiments included the following: 1—EBOV‐GFP plus UV+RB; 2—EBOV‐GFP plus RB only; 3—EBOV‐GFP plus UV only; 4—EBOV‐GFP without RB or UV; 5—virus‐free control plus UV only; and 6—virus‐free control without RB or UV.
RESULTS
UV+RB reduced EBOV titers to nondetectable levels in both nonhuman primate serum (≥2.8‐ to 3.2‐log reduction) and human whole blood (≥3.0‐log reduction) without decreasing protective antibody titers in human plasma.
CONCLUSION
Our in vitro results demonstrate that the UV+RB treatment efficiently reduces EBOV titers to below limits of detection in both serum and whole blood. In vivo testing to determine whether UV+RB can improve convalescent blood product safety is indicated.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In addition to its surface glycoprotein (GP), Ebola virus directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. We ...recently reported that sGP actively diverts host antibody responses against the epitopes that it shares with GP and thereby allows itself to absorb anti-GP antibodies, a phenomenon we termed "antigenic subversion." To investigate the effect of antigenic subversion by sGP on protection against virus infection, we compared immune responses induced by different prime-boost immunization regimens with GP and sGP DNA vaccines in mice and their efficacy against lethal Ebola virus challenge. Similar levels of anti-GP antibodies were induced by 2 immunizations with sGP and GP DNA vaccines. However, 2 immunizations with GP but not sGP DNA vaccine fully protected mice from lethal challenge. Boosting with sGP or GP DNA vaccine in mice that had been primed by GP or sGP DNA vaccine augmented the levels of anti-GP antibody responses and further improved protective efficacy against Ebola virus infection. These results show that both the quality and the levels of anti-GP antibody responses affect the efficacy of protection against Ebola virus infection.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
ABSTRACT
Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection ...and preventing further transmission. Here, a one‐step qRT‐PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross‐reactivity being observed among them. The limits of detection of the assays ranged from 102 to 103 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture‐propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene‐ and strain‐specific. The RT‐PCR assays detected viral RNAs in blood samples from virus‐infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Sudan virus (SUDV), like the closely related Ebola virus (EBOV), is a filovirus that causes severe hemorrhagic disease. They both contain an RNA editing site in the glycoprotein gene that controls ...expression of soluble and full-length protein. We tested the consequences of cell culture passage on the genome sequence at the SUDV editing site locus and determined whether this affected virulence. Passage resulted in expansion of the SUDV editing site, similar to that observed with EBOV. We compared viruses possessing either the wild-type or expanded editing site, using a nonhuman primate model of disease. Despite differences in virus serum titer at one time point, there were no significant differences in time to death or any other measured parameter. These data imply that changes at this locus were not important for SUDV lethality.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are ...authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (polydi(carboxylatophenoxy)phosphazene, PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.
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