Cysteine Cathepsin Inhibitors as Anti-Ebola Agents

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics

Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

<div><p>Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the <i>Ebolavirus</i> genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.</p></div

Cysteine Cathepsin Inhibitors as Anti-Ebola Agents

The recent Ebola virus outbreak in western Africa highlights the need for novel therapeutics that target Ebola virus and other filoviruses. Filoviruses require processing by host cell-derived cysteine cathepsins for productive infection. Here we report the generation of a focused library of cysteine cathepsin inhibitors and subsequent screening to identify compounds with potent activity against viral entry and replication. Our top compounds show highly potent and broad-spectrum activity against cysteine cathepsins and were able to effectively block entry of Ebola and Marburg viruses. These agents are promising leads for development as antifilovirus therapeutics

PolyICLC results in an increase of CD8α+ DC to the draining lymph node.

<p>(A) The percentage of CD8α DC in the popliteal lymph node after vaccination, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089735#pone-0089735-g006" target="_blank">Figure 6</a>. (B) CD8α DC percentages are the percentages of viable lymphocytes collected from the popliteal lymph node. Dot plots show CD11c on the y-axis and CD8α on the x-axis with samples from animals treated with saline, 0.1, 1, or 10 µg of polyICLC (dose level indicated by triangle) 24 hours or 72 hours before sample collection. 200,000–500,000 events were collected in duplicate for each animal. P values were determined using the Student’s T-test by comparing the percentage of responding cells in animals vaccinated with VLP alone versus vaccination with VLP and various doses of polyICLC. “*” indicates p<0.05, “**” indicates p<0.005, and “***” indicates p<0.0005. Four to five animals were utilized in each group for each iteration of this experiment.</p

Humoral responses of VLP-vaccinated macaques.

<p>The data are shown as individual animal responses (circles) or as the mean antibody units for each group (lines) at each time point the samples were drawn. (A-D) Serum titers of VLP-vaccinated macaques were measured using the purified (A) SUDV GPdTM, (B) SUDV VP40, (C) EBOV GPdTM or (D) EBOV VP40 proteins using an IgG detection ELISA. (E) Antibody response to live virus in VLP-vaccinated animals prior to challenge. An ELISA was performed using live virus (EBOV, SUDV, TAFV, and Lassa virus) as the coating antigen. The data in Panels A-D represent empirically defined EC₅₀ values as described in Materials and Methods and data in Panel E are expressed as the means of the endpoint dilution (endpoint defined as inverse of last dilution with an OD > 0.2).</p

Summary of the TAFV challenge study results through 15 days post challenge (study day 70–85).

<p><i>N</i>.<i>S</i>. = <i>no signs; N/A = not determined because the animal was deceased prior to the time point; fever was defined as a temperature more than 2</i>.<i>0°F over baseline; Temp drop was defined as a temperature more than 3</i>.<i>0°F below baseline; mild rash = focal areas of petechiae covering less than 10% of skin; moderate rash = areas of petechiae covering between 10% and 40% of the skin; severe rash = areas of petechiae or ecchymosis covering more than 40% of the skin</i></p><p><i>↑</i>, <i>2- to 3-fold increase</i></p><p><i>↑↑</i>, <i>4- to 5-fold increase</i></p><p><i>↑↑↑</i>, <i>> 5-fold increase</i></p><p><i>↓</i>, <i>2- to 3-fold decrease</i></p><p><i>WL</i>: <i>weight loss was the percentage compared to the weight at study initiation; BUN</i>: <i>blood urea nitrogen; ALT</i>: <i>alanine aminotransferase; AST</i>: <i>aspartate aminotransferase; ALP</i>: <i>alkaline phosphatase; ALB</i>: <i>albumin</i>, <i>Glu</i>: <i>glucose</i>, <i>Cre</i>: <i>creatinine</i>, <i>WBC</i>: <i>white blood cells; Plt</i>: <i>platelet</i>, <i>Lym</i>: <i>lymphocyte</i>.</p><p>Summary of the TAFV challenge study results through 15 days post challenge (study day 70–85).</p

PolyICLC augments VLP-mediated protection from ma-EBOV and gp-adapted-EBOV challenge.

<p>C57BL/6 mice were vaccinated IM twice, three weeks apart, with 1.25 µg of VLP with or without 10 or 1 µg of polyICLC (PIC), 10 µg of R848, or 10 or 1 µg of MPL (A) or with dose levels of polyICLC ranging from 100 µg to 100 ng; “Sal” indicates Saline vaccination (B). Challenge occurred four weeks after the vaccine boost. (C) Hartley guinea pigs were vaccinated two times, three weeks apart, with 5 µg of VLP, with or without 10 µg or 1 µg of polyICLC. Nine or ten animals received R848 or MPL as adjuvant, respectively (A); polyICLC groups were repeated and the n is noted in (B). All p-values were calculated using Fisher's exact test with multiple testing corrections performed by permutation based on the number of comparisons performed. Significant comparisons between vaccinations with 10 or 1 µg of adjuvant (A) or between the low dose group (red lines for both A and B) and the low dose group in combination with polyICLC (B and C) are shown. “***” indicates p<0.0005 and “**” indicates p<0.005.</p

SUDV VLP vaccine protects cynomolgus macaques from lethal infection with SUDV (Boniface isolate).

<p>Macaques were vaccinated with 3 mg of SUDV VLPs (total protein content) along with 100ug of QS-21 at 0 and 6 weeks. Antibody responses were determined using an ELISA to detect IgGs against purified SUDV GPdTM or VP40 in sera drawn immediately prior to challenge. All six macaques were challenged with ∼1000 pfu of SUDV 4 weeks after the final vaccination (week 10).</p><p>SUDV VLP vaccine protects cynomolgus macaques from lethal infection with SUDV (Boniface isolate).</p

Infectious virus detectable in the sera of NHPs vaccinated with QS-21 adjuvant and EBOV VLPs (A), SUDV VLPs (B), EBOV and SUDV VLPs (C), or adjuvant only (D) and challenged with TAFV on study day 70.

<p>Quantitation of virus infectivity was determined using a standard plaque assay and is represented as the values for individual monkeys on each study day after the challenge date (day 70).</p

PolyICLC induces transient increases in serum cytokine and chemokine levels.

<p>C57BL/6 mice were vaccinated with 1.25 µg of VLP, 10 µg of polyICLC, 0.1 µg of polyICLC, or 1.25 µg of VLP with 10, 1, or 0.1 µg of polyICLC. One hour, five hours, or twenty-four hours after vaccination, peripheral blood was collected and evaluated for cytokine and chemokine levels. Supernatant from splenocytes stimulated with PMA/ionomycin or LPS served as a positive control to evaluate consistency between assays and to confirm the efficacy of cytokine and chemokine detection. (A) Six cytokine and chemokines that consistently increased in mouse serum after polyICLC or VLP (1.25 µg) treatment. Red bars indicate levels five hours after vaccination; orange bars indicate levels twenty-four hours after vaccination. The gray bar indicates the levels detected in untreated mice. Mean and standard deviation are shown. Triangle indicates decreasing dose levels of polyICLC (10 µg, 1 µg, 0.1 µg) administered in combination with 1.25 µg of VLP. (B) IFNα and IFNβ levels in the serum were detected using an ELISA. Median is indicated with the black bar. Triangle indicates decreasing dose levels of polyICLC (10 µg, 1 µg, 0.1 µg) administered in combination with 1.25 µg of VLP. The unpaired, two-tailed Mann-Whitney test was used to determine p-values; comparisons are between the indicated sample and the untreated control sample. “*” indicates p<0.05, “**” indicates p<0.005, and “***” indicates p<0.0005. Four to five animals were utilized in each group for each of the two iterations of this experiment, and only cytokines and chemokines showing consistent changes in expression between iterations are shown. Other cytokines and chemokines that were analyzed and which did increase in the control sample but did not increase after experimental treatment include the following: IL12p40, IFNγ, IL5, IL1α, and IL1β.</p