News Article | November 23, 2016
KØBENHAVN, Danmark, 23. november 2016 - Bavarian Nordic A/S (OMX: BAVA) meddelte i dag, at selskabet har afsluttet rekrutteringen til et fase 3 klinisk forsøg, der er udformet til at påvise klinisk sammenlignelighed (non-inferioritet) mellem selskabets ikke-replikerende koppevaccine, IMVAMUNE og ACAM2000, som er den godkendte, replikerende koppevaccine i USA. Dette er det andet og sidste forsøg, som er aftalt med de amerikanske sundhedsmyndigheder, FDA, med henblik på at understøtte godkendelsen af flydende-frosset IMVAMUNE. Det første forsøg blev succesfuldt afsluttet i 2015 og var et lot consistency forsøg i 4.000 raske forsøgspersoner. Fase 3 forsøget rekrutterede 440 personer på et amerikansk militæranlæg i Sydkorea. Forsøget ledes af United States Army Medical Research Institute of Infectious Diseases (USAMRIID) i samarbejde med U.S. Defense Health Agency. Alle forsøgspersoner vil være afsluttet i forsøget i løbet af andet kvartal 2017 og toplinjeresultater forventes i andet halvår 2017. Paul Chaplin, administrerende direktør i Bavarian Nordic udtaler: "Afslutningen af rekrutteringen i dette forsøg udgør en vigtig milepæl i samarbejdet mellem Bavarian Nordic og den amerikanske regering. IMVAMUNE har været en hjørnesten for selskabet i det seneste årti, og vi fortsætter samarbejdet med amerikanerne med henblik på at imødegå deres målsætning om at beskytte 66 millioner borgere, som har brug for en mere sikker koppevaccine. Vi ser frem til at rapportere resultaterne fra dette forsøg samt det videre samarbejde med myndighederne i processen frem mod godkendelse i USA." Anerkendelse af støtte fra offentlige institutioner Fase 3 studiet, der sammenlignede sikkerheden og immunogeniciteten af IMVAMUNE med ACAM2000 er finansieret helt eller delvist af amerikanske offentlige midler fra Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under kontrakt nr. HHSO100200700034C Om Bavarian Nordic Bavarian Nordic er et fuldt integreret biotekselskab, der er fokuseret på udvikling, produktion og kommercialisering af cancer-immunterapier og vacciner mod infektionssygdomme baseret på selskabets virale vaccineplatform. Gennem mangeårige samarbejder, herunder med den amerikanske regering, har Bavarian Nordic udviklet en portefølje af vacciner mod infektionssygdomme, inklusive den ikke-replikerende koppevaccine IMVAMUNE®, der lagerføres til anvendelse i nødsituationer af den amerikanske stat samt andre regeringer. Vaccinen er godkendt i EU (under handelsnavnet IMVANEX®) samt i Canada. I partnerskab med Janssen udvikler Bavarian Nordic en ebola-vaccine, som er blevet fremskyndet af sundhedsmyndigheder verden over, og en vaccine til beskyttelse mod og behandling af HPV. Bavarian Nordic har desuden i samarbejde med National Cancer Institute udviklet en portefølje af aktive cancerimmunterapier, herunder PROSTVAC®, der er i fase 3 klinisk udvikling til behandling af fremskreden prostatacancer. Selskabet har indgået aftale med Bristol-Myers Squibb om potentiel kommercialisering af PROSTVAC. For yderligere information besøg www.bavarian-nordic.com eller følg os på Twitter @bavariannordic. Udsagn om fremtiden Denne meddelelse indeholder fremadrettede udsagn, som er forbundet med risici, usikkerheder og andre faktorer, hvoraf mange er uden for vores kontrol. Dette kan medføre, at faktiske resultater afviger væsentligt fra de resultater, som er omhandlet i ovennævnte fremadrettede udsagn. Fremadrettede udsagn omfatter udsagn vedrørende vores planer, mål, fremtidige begivenheder, præstation og/eller anden information, som ikke er historisk information. Alle fremadrettede udsagn skal udtrykkeligt vurderes i sammenhæng med de forbehold, der er taget eller henvist til i denne erklæring. Vi påtager os ingen forpligtelser til offentligt at opdatere eller revidere udsagn om fremtiden således, at disse afspejler efterfølgende begivenheder eller omstændigheder, undtagen i det omfang dette er foreskrevet ved lov.
News Article | November 23, 2016
COPENHAGEN, Denmark, November 23, 2016 - Bavarian Nordic A/S (OMX: BAVA, OTC: BVNRY) today announced the completion of enrollment of a Phase 3 clinical study designed to demonstrate non-inferiority between its investigational, non-replicating smallpox vaccine, IMVAMUNE and ACAM2000, the current U.S. licensed, and replicating smallpox vaccine. This is the second and final study agreed with the U.S. Food and Drug Administration (FDA) to support the registration of liquid-frozen IMVAMUNE. The first study, a lot consistency study in 4,000 healthy individuals, was successfully completed in 2015. The Phase 3 non-inferiority study enrolled 440 subjects at a U.S. military garrison in South Korea led by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) in collaboration with the U.S. Defense Health Agency. All subjects will have completed the study within second quarter of 2017, and top line data is anticipated in the second half of 2017. Paul Chaplin, President & Chief Executive Officer of Bavarian Nordic, said: "Completion of enrolment of this study represents a significant milestone in the collaboration between Bavarian Nordic, and multiple federal agencies. IMVAMUNE has served as the cornerstone for our Company over the past decade and we will continue to work with the U.S. Government to meet their stated goal of protecting 66 million Americans who are in need of a safer smallpox vaccine. We look forward reporting these data and working with the authorities in the process towards U.S. licensure." Bavarian Nordic has to-date delivered 28 million doses of liquid-frozen IMVAMUNE to the U.S. Strategic National Stockpile. Federal funding acknowledgments The Phase 3 study comparing the safety and immunogenicity of IMVAMUNE to ACAM2000 has been funded in whole or in part with Federal funds from the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract No. HHSO100200700034C About Bavarian Nordic Bavarian Nordic is a fully integrated biotechnology company focused on the development, manufacturing and commercialization of cancer immunotherapies and vaccines for infectious diseases, based on the Company's live virus vaccine platform. Through long-standing collaborations, including a collaboration with the U.S. government, Bavarian Nordic has developed a portfolio of vaccines for infectious diseases, including the non-replicating smallpox vaccine, IMVAMUNE®, which is stockpiled for emergency use by the United States and other governments. The vaccine is approved in the European Union (under the trade name IMVANEX®) and in Canada. Bavarian Nordic and its partner Janssen are developing an Ebola vaccine regimen, which has been fast-tracked, with the backing of worldwide health authorities, and a vaccine for the prevention and treatment of HPV. Additionally, in collaboration with the National Cancer Institute, Bavarian Nordic has developed a portfolio of active cancer immunotherapies, including PROSTVAC®, which is currently in Phase 3 clinical development for the treatment of advanced prostate cancer. The company has partnered with Bristol-Myers Squibb for the potential commercialization of PROSTVAC. For more information visit www.bavarian-nordic.com or follow us on Twitter @bavariannordic. Forward-looking statements This announcement includes forward-looking statements that involve risks, uncertainties and other factors, many of which are outside of our control, that could cause actual results to differ materially from the results discussed in the forward-looking statements. Forward-looking statements include statements concerning our plans, objectives, goals, future events, performance and/or other information that is not historical information. All such forward-looking statements are expressly qualified by these cautionary statements and any other cautionary statements which may accompany the forward-looking statements. We undertake no obligation to publicly update or revise forward-looking statements to reflect subsequent events or circumstances after the date made, except as required by law.
News Article | March 2, 2017
BOSTON, March 02, 2017 (GLOBE NEWSWIRE) -- Paratek Pharmaceuticals, Inc. (Nasdaq:PRTK), a biopharmaceutical company focused on the development and commercialization of innovative therapies based upon tetracycline chemistry, today reported financial results for the full year and quarter ended December 31, 2016. "We made excellent progress with the clinical development program of omadacycline in the fourth quarter and continued our work to prepare for a potential NDA submission in the first half of 2018,” said Michael Bigham, Chairman and Chief Executive Officer, Paratek. “With the enrollment of the pneumonia study now complete, we expect to release top-line data early in the second quarter. Our Phase 3 study of an oral-only dosing regimen in skin infections is progressing well. We continue to expect top-line data as early as the second quarter of this year from this study.” Fourth Quarter and Full Year 2016 Financial Results For the fourth quarter of 2016, Paratek reported a net loss of $26.5 million, or $1.16 per share, compared to a net loss of $21.1 million, or $1.20 per share, for the same period in 2015. For the year ended December 31, 2016, Paratek reported a net loss of $111.6 million, or $5.51 per share, compared to a net loss of $70.9 million, or $4.29 per share, for the same period in 2015. Research and development expenses were $19.7 million and $83.5 million for the quarter and year ended December 31, 2016, respectively, compared to $15.2 million and $50.8 million for the same periods in 2015. The increase in research and development expense for the year ended December 31, 2016 was primarily the result of ongoing development of omadacycline, including costs associated with clinical studies for the treatment of ABSSSI, CABP and UTI, production of omadacycline registration batches and manufacturing process validation work, other research and development activities, and employee compensation. General and administrative expenses were $6.5 million and $26.4 million for the quarter and year ended December 31, 2016, respectively, compared to $5.6 million and $20.0 million for the same periods in 2015. The increase in general and administrative costs was primarily the result of employee compensation. As of December 31, 2016, Paratek had cash, cash equivalents, and marketable securities of $128.0 million. Based on current assumptions, Paratek’s cash, cash equivalents and marketable securities will enable the Company to fund operating expenses and capital expenditure requirements through the first half of 2018. Paratek initiated sales of shares under a Controlled Equity Offering Sales Agreement with Cantor Fitzgerald & Co. in March 2016, and sold an aggregate of 860,014 shares of common stock through December 31, 2016, resulting in net proceeds of $11.6 million. As of February 24, 2017, an additional 870,078 shares were sold under the Sales Agreement subsequent to December 31, 2016, resulting in net proceeds of $13.1 million, which will be recognized during the first quarter of 2017. Conference Call and Webcast Paratek’s earnings conference call for the quarter ended December 31, 2016, will be broadcast at 8:30 a.m. EST on March 2, 2017. The live webcast can be accessed under "Events and Presentations" in the Investor Relations section of Paratek’s website at www.paratekpharma.com. Domestic investors wishing to participate in the call should dial 877-407-9039 and international investors should dial 201-689-8470. The conference ID is 13656270. Investors can also access the call at http://public.viavid.com/index.php?=123110. Replays of the call will be available through March 16, 2017. Domestic investors can access the replay by dialing 844-512-2921 and international investors can access the replay by dialing 412-317-6671. The PIN code to access the replay is 13656270. Website Information Paratek routinely posts important information for investors on the Investor Relations section of its website at www.paratekpharma.com. Paratek intends to use this website as a means of disclosing material, non-public information and for complying with its disclosure obligations under Regulation FD. Accordingly, investors should monitor the Investor Relations section of Paratek’s website, in addition to following its press releases, SEC filings, public conference calls, presentations and webcasts. The information contained on, or that may be accessed through, Paratek’s website is not incorporated by reference into, and is not a part of, this document. About Paratek Pharmaceuticals, Inc. Paratek Pharmaceuticals, Inc. is a biopharmaceutical company focused on the development and commercialization of innovative therapies based upon its expertise in novel tetracycline chemistry. Paratek's lead product candidate, omadacycline, is the first in a new class of tetracyclines known as aminomethylcyclines, with broad-spectrum activity against Gram-positive, Gram-negative and atypical bacteria. In June 2016, Paratek announced positive efficacy data in a Phase 3 registration study in acute bacterial skin and skin structure infections (ABSSSI) demonstrating the efficacy and safety of intravenous (IV) to once-daily oral omadacycline compared to linezolid. A Phase 3 registration study for community-acquired bacterial pneumonia (CABP) comparing IV-to-once-daily oral omadacycline to IV-to-oral moxifloxacin was initiated in November 2015 and completed enrollment in January 2017. Paratek will report top-line data from this study early in the second quarter of 2017. A Phase 3 registration study in ABSSSI comparing once-daily oral-only dosing of omadacycline to twice-daily oral-only dosing of linezolid was initiated in August 2016. Top-line data from this study are expected as early as the second quarter of 2017. A Phase 1B study in uncomplicated urinary tract infections (UTI) was initiated in May 2016 and positive top-line PK proof-of-principle data were reported in November 2016. The company plans to begin enrolling patients in a proof-of-concept Phase 2 study in complicated UTI as early as the fourth quarter of 2017. Omadacycline has been granted Qualified Infectious Disease Product designation and Fast Track status by the U.S. Food and Drug Administration for several indications. In October 2016, Paratek announced a new cooperative research effort with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) to study omadacycline against pathogenic agents causing infectious diseases of public health and biodefense importance. These studies are designed to confirm dosing regimens and assess efficacy of omadacycline against biodefense pathogens, including Yersinia pestis (plague) and Bacillus anthracis (anthrax). Omadacycline is a new once-daily oral and IV, well-tolerated broad spectrum antibiotic being developed for use as empiric monotherapy for patients suffering from serious community-acquired bacterial infections, such as acute bacterial skin and skin structure infections, community-acquired bacterial pneumonia, urinary tract infections and other community-acquired bacterial infections, particularly when antibiotic resistance is of concern to prescribing physicians. Paratek's second Phase 3 product candidate, sarecycline, is a well-tolerated, once-daily, oral, narrow spectrum tetracycline-derived antibiotic with potent anti-inflammatory properties for the potential treatment of acne and rosacea in the community setting. Allergan owns the U.S. rights for the development and commercialization of sarecycline. Paratek retains all ex-U.S. rights. Allergan initiated two identical Phase 3 registration studies in December 2014 for sarecycline for the treatment of moderate to severe acne vulgaris. Top-line Phase 3 data are expected in the first half of 2017. Forward Looking Statements This press release contains forward-looking statements including statements related to our overall strategy, product candidates, clinical studies, cash resources, prospects and expected results, including statements about the timing of advancing omadacycline and otherwise preparing for clinical studies, the potential for omadacycline to serve as an empiric monotherapy treatment option for patients suffering from ABSSSI, CABP, UTI, and other bacterial infections when resistance is of concern, the prospect of omadacycline providing broad-spectrum activity, and our having the resources to execute on our clinical studies. All statements, other than statements of historical facts, included in this press release are forward-looking statements, and are identified by words such as "advancing," "believe," "expect," "well positioned," "look forward," "anticipated," "continued," and other words and terms of similar meaning. These forward-looking statements are based upon our current expectations and involve substantial risks and uncertainties. We may not actually achieve the plans, carry out the intentions or meet the expectations or projections disclosed in our forward-looking statements and you should not place undue reliance on these forward-looking statements. Our actual results and the timing of events could differ materially from those included in such forward-looking statements as a result of these risks and uncertainties, which include, without limitation, risks related to (i) our need for substantial additional funding to complete the development and commercialization of our product candidates, (ii) our ability to raise the capital to do so, (iii) our ability to develop and manufacture our drug candidates for potential commercialization, (iv) the advancement of omadacycline Phase 3 studies for ABSSSI, (v) the potential for omadacycline to be successfully developed for use as an empiric monotherapy for patients suffering from serious community-acquired bacterial infections, (vi) the potential of omadacycline to become the primary antibiotic choice of physicians for the treatment of serious community-acquired bacterial infections, (vii) the potential use and effectiveness of sarecycline for the treatment of acne and rosacea in the community setting, and (viii) the timing of the Phase 3 program in moderate-severe acne for sarecycline, risks that data to date and trends may not be predictive of future results, risks related to the conduct of our clinical studies, and risks that our clinical studies and product candidates do not receive regulatory approval. These and other risk factors are discussed under "Risk Factors" and elsewhere in our Annual Report on Form 10-K for the year ended December 31, 2016, and our other filings with the Securities and Exchange Commission. We expressly disclaim any obligation or undertaking to update or revise any forward-looking statements contained herein.
News Article | March 16, 2016
No statistical methods were used to predetermine sample size for biochemical or cell-based assays, or for pharmacokinetic studies. Investigators were not blinded to outcome assessment during these investigations. For GS-5734 efficacy assessments in nonhuman primates, statistical power analysis was used to predetermine sample size, and subjects were randomly assigned to experimental group, stratified by sex and balanced by body weight. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals and outcome.
GS-5734, Nuc, and NTP were synthesized at Gilead Sciences, Inc., and chemical identity and sample purity were established using NMR, HRMS, and HPLC analysis (Supplementary Information). The radiolabelled analogue [14C]GS-5734 (specific activity, 58.0 mCi mmol−1) was obtained from Moravek Biochemicals (Brea, California) and was prepared in a similar manner described for GS-5734 using [14C]trimethylsilylcyanide (Supplementary Information). Small molecule X-ray crystallographic coordinates and structure factor files have been deposited in the Cambridge Structural Database (http://www.ccdc.cam.ac.uk/) and accession numbers are supplied in the Supplementary Information.
RSV A2 was purchased from Advanced Biotechnologies, Inc. EBOV (Kikwit and Makona variants), Sudan virus (SUDV, Gulu), Marburg virus (MARV, Ci67), Junín virus (JUNV, Romero), Lassa virus (LASV, Josiah), Middle East respiratory syndrome virus (MERS, Jordan N3), Chikungunya virus (CHIV, AF 15561), and Venezuelan equine encephalitis virus (VEEV, SH3) were all prepared and characterized at the United States Army Medical Research Institute for infectious diseases (USAMRIID). EBOV containing a GFP reporter gene (EBOV–GFP), EBOV Makona (Liberia, 2014), and MARV containing a GFP reporter gene (MARV–GFP) were prepared and characterized at the Centers for Disease Control and Prevention26, 27.
HEp-2 (CCL-23), PC-3 (CCL-1435), HeLa (CCL-2), U2OS (HTB-96), Vero (CCL-81), HFF-1 (SCRC-1041), and HepG2 (HB-8065) cell lines were purchased from the American Type Culture Collection. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in assays. HEp-2 cells were cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 100 U ml−1 penicillin and streptomycin. PC-3 cells were cultured in Kaighn’s F12 media supplemented with 10% FBS and 100 U ml−1 penicillin and streptomycin. HeLa, U2OS, and Vero cells were cultured in MEM supplemented with 10% FBS, 1% l-glutamine, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/streptomycin. HFF-1 cells were cultured in MEM supplemented with 10% FBS and 0.5 mM sodium pyruvate. HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 0.1 mM non-essential amino acids. The MT-4 cell line was obtained from the NIH AIDS Research and Reference Reagent Program and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 2 mM l-glutamine. The Huh-7 cell line was obtained from C. M. Rice (Rockefeller University) and cultured in DMEM supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and non-essential amino acids.
Primary human hepatocytes were purchased from Invitrogen and cultured in William’s Medium E medium containing cell maintenance supplement. Donor profiles were limited to 18- to 65-year-old nonsmokers with limited alcohol consumption. Upon delivery, the cells were allowed to recover for 24 h in complete medium with supplement provided by the vendor at 37 °C. Human PBMCs were isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Palo Alto, California) and maintained in RPMI-1640 with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin. Rhesus fresh whole blood was obtained from Valley Biosystems. PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation. Briefly, blood was overlaid on 15 ml Ficoll-Paque (GE Healthcare Bio-Sciences AB), and centrifuged at 500g for 20 min. The top layer containing platelets and plasma was removed, and the middle layer containing PBMCs was transferred to a fresh tube, diluted with Tris buffered saline up to 50 ml, and centrifuged at 500g for 5 min. The supernatant was removed and the cell pellet was resuspended in 5 ml red blood cell lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5). To generate stimulated PBMCs, freshly isolated quiescent PBMCs were seeded into a T-150 (150 cm2) tissue culture flask containing fresh medium supplemented with 10 U ml−1 of recombinant human interleukin-2 (IL-2) and 1 μg ml−1 phytohaemagglutinin-P at a density of 2 × 106 cells ml−1 and incubated for 72 h at 37 °C. Human macrophage cultures were isolated from PBMCs that were purified by Ficoll gradient centrifugation from 50 ml of blood from healthy human volunteers. PBMCs were cultured for 7 to 8 days in in RPMI cell culture media supplemented with 10% FBS, 5 to 50 ng ml−1 granulocyte-macrophage colony-stimulating factor and 50 μM β-mercaptoethanol to induce macrophage differentiation. The cryopreserved human primary renal proximal tubule epithelial cells were obtained from LifeLine Cell Technology and isolated from the tissue of human kidney. The cells were cultured at 90% confluency with RenaLife complete medium in a T-75 flask for 3 to 4 days before seeding into 96-well assay plates. Immortalized human microvascular endothelial cells (HMVEC-TERT) were obtained from R. Shao at the Pioneer Valley Life Sciences Institute28. HMVEC-TERT cells were cultured in endothelial basal media supplemented with 10% FBS, 5 μg of epithelial growth factor, 0.5 mg hydrocortisone, and gentamycin/amphotericin-B.
RNA POLII was purchased as part of the HeLaScribe Nuclear Extract in vitro Transcription System kit from Promega. The recombinant human POLRMT and transcription factors mitochondrial transcription factors A (mtTFA or TFAM) and B2 (mtTFB2 or TFB2M) were purchased from Enzymax. RSV ribonucleoprotein (RNP) complexes were prepared according to a method modified from ref. 29.
The intracellular metabolism of GS-5734 was assessed in different cell types (HMVEC and HeLa cell lines, and primary human and rhesus PBMCs, monocytes and monocyte-derived macrophages) following 2-h pulse or 72-h continuous incubations with 10 μM GS-5734. For comparison, intracellular metabolism during a 72-h incubation with 10 μM of Nuc was completed in human monocyte-derived macrophages. For pulse incubations, monocyte-derived macrophages isolated from rhesus monkeys or humans were incubated for 2 h in compound-containing media followed by removal, washing with 37 °C drug-free media, and incubated for an additional 22 h in media which did not contain GS-5734. Human monocyte-derived macrophages, HeLa and HMVEC were grown to confluence (approximately 0.5, 0.2, and 1.2 × 106 cells per well, respectively) in 500 μl of media in 12-well tissue culture plates. Monocyte and PBMCs were incubated in suspension (approximately 1 × 106 cells ml−1) in 1 ml of media in micro centrifuge tubes.
For adherent cells (HMVEC, HeLa, and monocyte-derived macrophages), media was removed at select time points from duplicate wells, cells washed twice with 2 ml of ice-cold 0.9% normal saline. For non-adherent cells (monocytes and PBMCs), duplicate incubations were centrifuged at 2,500g for 30 s to remove media. The cell pellets were re-suspended with 500 μl cell culture media (RPMI with 10% FBS) and layered on top of a 500 μl oil layer (Nyosil M25; Nye Lubricants) in a microcentrifuge tube. Samples were then centrifuged at room temperature at 13,000 r.p.m. for 45 s. The media layer was removed and the oil layer was washed twice with 500 μl water. The oil layer was then carefully removed using a Pasteur pipet attached to vacuum. A volume of 0.5 ml of 70% methanol containing 100 nM of the analytical internal standard 2-chloro-adenosine-5′-triphosphate (Sigma-Aldrich) was added to isolated cells. Samples were stored overnight at −20 °C to facilitate extraction, centrifuged at 15,000g for 15 min and then supernatant was transferred to clean tubes for drying in a MiVac Duo concentrator (Genevac). Dried samples were then reconstituted in mobile phase A containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine (DMH) in water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS).
LC-MS/MS was performed using low-flow ion-pairing chromatography, similar to methods described previously30. Briefly, analytes were separated using a 50 × 2 mm × 2.5 μm Luna C18(2) HST column (Phenomenex) connected to a LC-20ADXR (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies). A multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine over 8 min at a flow rate of 150 μl min−1 was used to separate analytes. Detection was performed on an API 4000 (Applied Biosystems) MS/MS operating in positive ion and multiple reaction monitoring modes. Intracellular metabolites alanine metabolite, Nuc, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate were quantified using 7-point standard curves ranging from 0.274 to 200 pmol (approximately 0.5 to 400 μM) prepared in cell extract from untreated cells. Levels of adenosine nucleotides were also quantified to assure dephosphorylation had not taken place during sample collection and preparation. In order to calculate intracellular concentration of metabolites, the total number of cells per sample were counted using a Countess automated cell counter (Invitrogen).
Antiviral assays were conducted in biosafety level 4 containment (BSL-4) at the Centers for Disease Control and Prevention. EBOV antiviral assays were conducted in primary HMVEC-TERT and in Huh-7 cells. Huh-7 cells were not authenticated and were not tested for mycoplasma. Ten concentrations of compound were diluted in fourfold serial dilution increments in media, and 100 μl per well of each dilution was transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers. The plates were transferred to BSL-4 containment, and the appropriate dilution of virus stock was added to test plates containing cells and serially diluted compounds. Each plate included four wells of infected untreated cells and four wells of uninfected cells that served as 0% and 100% virus inhibition controls, respectively. After the infection, assay plates were incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication was measured by direct fluorescence using a Biotek HTSynergy plate reader. For virus yield assays, Huh-7 cells were infected with wild-type EBOV for 1 h at 0.1 plaque-forming units (PFU) per cell. The virus inoculum was removed and replaced with 100 μl per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants were collected, and the amount of virus was quantified by endpoint dilution assay. The endpoint dilution assay was conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID ). To measure levels of viral RNA from infected cells, total RNA was extracted using the MagMAX-96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT–PCR) assay with primers and probes specific for the EBOV nucleoprotein gene.
Antiviral assays were conducted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard) in twofold dilution increments starting at 10 μM at 2 h before infection. The DMSO concentration in each well was normalized to 1% using an HP D300 digital dispenser. The assay plates were transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 cells. The assay plates were incubated in a tissue culture incubator for 48 h. Infection was terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described in Supplementary Table 1.
Antiviral assays were conducted in BSL-4 at USAMRIID. Primary human macrophage cells were seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate were added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO was normalized to 1% in all wells. The plates were transferred into the BSL-4 suite, and the cells were infected with 1 PFU per cell of EBOV in 100 μl of media and incubated for 1 h. The inoculum was removed, and the media was replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication was quantified by immuno-staining as described in Supplementary Table 1.
For antiviral tests, compounds were threefold serially diluted in source plates from which 100 nl of diluted compound was transferred to a 384-well cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells were added at a density of 5 × 105 cells per ml, then infected by adding RSV A2 at a titer of 1 × 104.5 tissue culture infectious doses (TCID ) per ml. Immediately following virus addition, 20 μl of the virus and cells mixture was added to the 384-well cell culture plates using a μFlow liquid dispenser and cultured for 4 days at 37 °C. After incubation, the cells were allowed to equilibrate to 25 °C for 30 min. The RSV-induced cytopathic effect was determined by adding 20 μl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25 °C, cell viability was determined by measuring luminescence using an Envision plate reader.
Antiviral assays were conducted in 384-or 96-well plates in BSL-4 at USAMRIID using a high-content imaging system to quantify virus antigen production as a measure of virus infection. A ‘no virus’ control and a ‘1% DMSO’ control were included to determine the 0% and 100% virus infection, respectively. The primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed in Supplementary Table 1. The primary antibody specific for a particular viral protein was diluted 1,000-fold in blocking buffer (1 × PBS with 3% BSA) and added to each well of the assay plate. The assay plates were incubated for 60 min at room temperature. The primary antibody was removed, and the cells were washed three times with 1 × PBS. The secondary detection antibody was an anti-mouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, catalogue number 405310). The secondary antibody was diluted 1,000-fold in blocking buffer and was added to each well in the assay plate. Assay plates were incubated for 60 min at room temperature. Nuclei were stained using Draq5 (Biostatus) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes were diluted in 1× PBS. The cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells were counter-stained with CellMask Deep Red (Thermo Fisher Scientific). Cell images were acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer) using a ×10 air objective to collect five images per well. Virus-specific antigen was quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei were quantified by measuring fluorescence emission at a 640 nm wavelength. Acquired images were analysed using Harmony and Acapella PE software. The Draq5 signal was used to generate a nuclei mask to define each nuclei in the image for quantification of cell number. The CellMask Deep Red dye was used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation. The viral-antigen signal was compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold were counted as positive for viral infection. The ratio of virus-positive cells to total number of analysed cells was used to determine the percentage of infection for each well on the assay plates. The effect of compounds on the viral infection was assessed as percentage of inhibition of infection in comparison to control wells. The resultant cell number and percentage of infection were normalized for each assay plate. Analysis of dose–response curve was performed using GeneData Screener software applying Levenberg–Marquardt algorithm for curve-fitting strategy. The curve-fitting process, including individual data point exclusion, was pre-specified by default software settings. R2 value quantified goodness of fit and fitting strategy was considered acceptable at R2 > 0.8.
All virus infections were quantified by immuno-staining using antibodies that recognized the relevant viral glycoproteins, as described in Supplementary Table 1.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 1 PFU per cell MARV, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.08 PFU SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.3 PFU per cell JUNV, which resulted in ~50% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period.
African green monkey (Chlorocebus sp.) kidney epithelial cells (Vero E6) were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of MERS virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period.
U2OS cells were seeded at 3,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20-h period.
HEp-2 (1.5 × 103 cells per well) and MT-4 (2 × 103 cells per well) cells were plated in 384-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. PC-3 cells (2.5 × 103 cells per well), HepG2 cells (4 × 103 cells per well), hepatocytes (1 × 106 cells per well), quiescent PBMCs (1 × 106 cells per well), stimulated PBMCs (2 × 105 cells per well), and RPTEC cells (1 × 103 cells per well) were plated in 96-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. Cells were cultured for 4–5 days at 37 °C. Following the incubation, the cells were allowed to equilibrate to 25 °C, and cell viability was determined by adding Cell-Titer Glo viability reagent. The mixture was incubated for 10 min, and the luminescence signal was quantified using an Envision plate reader. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in cytotoxicity assays.
RNA synthesis by the RSV polymerase was reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1–14 of the RSV leader promoter31, 32, 33 (3′-UGCGCUUUUUUACG-5′). RNA synthesis reactions were performed as described previously, except that the reaction mixture contained 250 μM guanosine triphosphate (GTP), 10 μM uridine triphosphate (UTP), 10 μM cytidine triphosphate (CTP), supplemented with 10 μCi [α-32P]CTP, and either included 10 μM adenosine triphosphate (ATP) or no ATP. Under these conditions, the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site. The NTP metabolite of GS-5734 was serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 μM as specified in Fig. 1f. RNA products were analysed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris–taurine–EDTA buffer, and radiolabelled RNA products were detected by autoradiography.
Transcription reactions contained 25 μg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl , 3 mM DTT, 2 mM EGTA, 50 μg ml−1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 μCi [α-32P]ATP (3,000 Ci mmol−1)). The radiolabelled nucleotide used in the transcription assay was selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
For a 25 μl reaction mixture, 7.5 μl 1 × transcription buffer (20 mM HEPES (pH 7.2–7.5), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol), 3 mM MgCl , 100 ng CMV positive or negative control DNA, and a mixture of ATP, GTP, CTP and UTP was pre-incubated with various concentrations (0–500 μM) of the inhibitor at 30 °C for 5 min. The mixture contained 5–25 μM (equal to K ) of the competing 33P-labelled ATP and 400 μM of GTP, UTP, and CTP. The reaction was started by addition of 3.5 μl of HeLa and extract. After 1 h of incubation at 30 °C, the polymerase reaction was stopped by addition of 10.6 μl proteinase K mixture that contained final concentrations of 2.5 μg μl−1 proteinase K, 5% SDS, and 25 mM EDTA. After incubation at 37 °C for 3–12 h, 10 μl of the reaction mixture was mixed with 10 μl of the loading dye (98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue), heated at 75 °C for 5 min, and loaded onto a 6% polyacrylamide gel (8 M urea). The gel was dried for 45 min at 70 °C and exposed to a phosphorimager screen. The full length product, 363 nucleotide runoff RNA, was quantified using a Typhoon Trio Imager and Image Quant TL Software.
Twenty nanomolar POLRMT was incubated with 20 nM template plasmid (pUC18-LSP) containing POLRMT light-strand promoter region and mitochondrial (mt) transcription factors TFA (100 nM) and mtTFB2 (20 nM) in buffer containing 10 mM HEPES (pH 7.5), 20 mM NaCl, 10 mM DTT, 0.1 mg ml−1 BSA, and 10 mM MgCl 34. The reaction mixture was pre-incubated to 32 °C, and the reactions were initiated by addition of 2.5 μM of each of the natural NTPs and 1.5 μCi of [32P]GTP. After incubation for 30 min at 32 °C, reactions were spotted on DE81 paper and quantified.
A homology model of RSV A2 and EBOV polymerases were built using the HIV reverse transcriptase X-ray crystal structure (PDB:1RTD). Schrödinger Release 2015-1: Prime, version 3.9 (Schrödinger, LLC), default settings with subsequent rigid body minimization and side-chain optimization. Loop insertions not in 1RTD of greater than 10 amino acids were not built.
For quantitative assessment of viral RNA nonhuman primate plasma samples, whole blood was collected using a K3 EDTA Greiner Vacuette tube (or equivalent) and sample centrifuged at 2500 (± 200) relative centrifugal force for 10 ± 2 min. To inactivate virus, plasma was treated with 3 parts (300 μl) TriReagent LS and samples were transferred to frozen storage (−60 °C to −90 °C), until removal for RNA extraction. Carrier RNA and QuantiFast High Concentration Internal Control (Qiagen) were spiked into the sample before extraction, conducted according to manufacturer’s instructions. The viral RNA was eluted in AVE buffer. Each extracted RNA sample was tested with the QuantiFast Internal Control RT–PCR RNA Assay (Qiagen) to evaluate the yield of the spiked-in QuantiFast High Concentration Internal Control. If the internal control amplified within manufacturer-designated ranges, further quantitative analysis of the viral target was performed. RT–PCR was conducted using an ABI 7500 Fast Dx using primers specific to EBOV glycoprotein. Samples were run in triplicate using a 5 μl template volume. For quantitative assessments, the average of the triplicate genomic equivalents (GE) per reaction were determined and multiplied by 800 to obtain GE ml−1 plasma. Standard curves were generated using synthetic RNA. The limits of quantification for this assay are 8.0 × 104 − 8.0 × 1010 GE ml−1 of plasma. Acceptance criteria for positive template control (PTC), negative template control (NTC), negative extraction control (NEC), and positive extraction control (PEC) are specified by standard operating procedure. For qualitative assessments, the limit of detection (LOD) was defined as C 38.07, based on method validation testing. An animal was considered to have tested positive for detection of EBOV RNA when a minimum of 2 of 3 replicates were designated as ‘positive’ and PTC, NTC, and NEC controls met specified method-acceptance criteria. A sample was designated as ‘positive’ when the C value was
News Article | March 2, 2017
PHILADELPHIA & ROCKVILLE, Md.--(BUSINESS WIRE)--Integral Molecular and Integrated BioTherapeutics have teamed up in the fight against the global health crises posed by Ebola and Zika viruses, signing a collaborative vaccine discovery agreement to help eradicate these threats. The two companies will leverage their complementary technologies to produce vaccine candidates that are specifically engineered to generate a maximally protective immune response in humans. The availability of such vaccines will prevent the recurrence of the deadly 2014-2016 Ebola epidemic that killed over 11,000 people in West Africa, and has the potential to curtail the spread of the ongoing Zika virus epidemic associated with severe fetal brain defects. Integral Molecular is an industry leader in the study of complex membrane proteins such as viral Envelope proteins. The company will use its proprietary Shotgun Mutagenesis protein engineering technology to generate and screen large panels of Envelope protein variants to identify an optimized protein that could serve as a highly immunogenic and protective vaccine, and will ultimately apply its high-resolution epitope mapping technologies to characterize the vaccine’s protective effects. Integrated BioTherapeutics, a leader in infectious disease research, will conduct preclinical studies to test the efficacy of vaccine candidates in disease models. “The vulnerability of human populations during the recent Ebola and Zika outbreaks highlighted the consequences of the lack of effective vaccines against these pathogens. The goal of our collaboration is to meet these concerns by creating efficacious vaccine candidates based on viral Envelope proteins,” said M. Javad Aman, President and CEO of Integrated BioTherapeutics. “We look forward to working with Integrated BioTherapeutics. Their experience in the development of a pipeline of antiviral products based on rationally designed and engineered viral proteins and antibodies will be a tremendous asset in our joint efforts towards producing Ebola and Zika vaccines,” continued Benjamin Doranz, President and CEO of Integral Molecular. Thus far, the two companies have engaged in highly successful collaborative research that has culminated in the pursuit of these vaccine candidates. This includes the development and characterization of the protective and cross-neutralizing pan-Ebola antibody FVM04, recently published in Cell Reports (Howell et al., 2016). Additional research resulting from this collaboration is expected to be published later this year. Integral Molecular is a research-driven biotechnology company creating innovative technologies and a pipeline of therapeutic antibodies against under-exploited membrane protein targets, including GPCRs, ion channels, transporters, and viral envelopes. This platform is built on the company’s Lipoparticle and Shotgun Mutagenesis technologies and over 15 years of experience optimizing membrane proteins. Integral Molecular discovers antibodies for partners in parallel with its own independent work developing antibodies for licensing. The company currently has therapeutic programs focused on pain, immunity, and infectious diseases. For more information, visit www.integralmolecular.com. IBT is a biotechnology company focused on the discovery of novel vaccines and therapeutics for emerging infectious diseases with a pipeline that includes promising product candidates for bacterial and viral infections including unique pan-filovirus monoclonal antibodies and vaccine candidates and a variety of other engineered product candidates for emerging viruses. IBT also operates a testing service business (www.ibtbioservices.com) focused on in vitro and in vivo models for viral agents such as Zika, dengue, yellow fever, influenza and RSV as wells as bacterial agents such as S. aureus, S. pneumoniae, E. Coli, and C. difficile. Located in Rockville, Maryland, IBT has a close working relationship with United States Government agencies including the National Institute of Allergy and Infectious Diseases (NIAID/NIH), National Cancer Institute (NCI), Department of Defense (DOD), United States Army Medical Research Institute of Infectious Diseases (USAMRIID) as well as many biotechnology and pharmaceutical companies and academic laboratories. For more information, visit www.integratedbiotherapeutics.com.
News Article | August 25, 2016
"Until now, multicomponent viruses were thought to infect only plants and fungi, as a result of relatively inefficient transmission," says first author Jason Ladner, a staff scientist from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). "Our finding that these viruses are present in mosquitoes is going to challenge us to re-evaluate some of our assumptions about them." Multicomponent viruses use a method of transmission that's different from other viruses known to infect animals. Instead of being contained in a single viral particle, their genomes are segmented and encapsulated among multiple particles. A yellow fever virus, for example, has all its genetic material packaged into a single particle. Therefore, one particle is enough to infect a cell. But in order for a multicomponent virus to establish an infection, the cell has to get infected with at least one particle of each type. The research is part of a global effort to monitor and prepare for outbreaks of unknown viral diseases. Mosquitoes and other insects can act as vectors for viral diseases, carrying them from place to place and transmitting them to human hosts via bites. Although the new virus does not appear to be a human pathogen, or even a mammalian one, the investigators say this work is a good exercise to help hone the tools and expertise needed to characterize novel infectious agents. In the study, the USAMRIID researchers worked with several other teams, including groups from the University of Texas Medical Branch and the New York State Department of Health, to isolate mosquitoes from different regions around the world. The newly discovered virus is named Guaico Culex after the Guaico region of Trinidad in which the mosquitoes that contained it were found. Guaico Culex was isolated by growing material obtained from the mosquitoes in cell culture. "This method has been useful particularly in finding new arboviruses, which are transmitted by mosquitoes and other arthropods to mammals," Ladner says. To identify arboviruses, cultures of mammalian cells are used. "We were also interested in viruses that may be found within mosquitoes but don't necessarily grow on mammalian cells, so we used cultures of insect cells, enabling us to find this new virus." Deep sequencing indicated that Guaico Culex belongs to a group of segmented viruses called Jingmenviruses, which were first discovered in 2014. In collaboration with a group at the University of Wisconsin-Madison, the USAMRIID researchers also showed for the first time evidence of a Jingmenvirus in the blood of a non-human primate, in this case a Ugandan red colobus monkey. This finding is also published in the current Cell Host & Microbe paper. Experts believe that the most likely infectious viruses to make the jump to humans are those that are already circulating in other mammals, especially non-human primates. Phylogenetic analysis indicated that this monkey virus shared a segmented common ancestor with Guaico Culex. However, researchers don't yet know if all Jingmenviruses are multicomponent like the Guaico Culex virus. It is also not known whether the Jingmenvirus isolated from the monkey had a pathogenic effect. "One of the things we're focused on at USAMRIID is rapid identification of pathogens from both clinical and environmental samples as well as characterization of novel viruses," says Gustavo Palacios, Director of the Center for Genome Sciences at USAMRIID and the study's senior author. "We're trying to make sure that we're not blindsided when the next virus comes around. With all of the diversity seen in these emerging viruses, we never know what the next one will be to have an impact on human health." Explore further: 'Good' mozzie virus might hold key to fighting human disease More information: Cell Host & Microbe, Ladner et al: "A Multicomponent Animal Virus Isolated from Mosquitoes" http://www.cell.com/cell-host-microbe/fulltext/S1931-3128(16)30310-9 , DOI: 10.1016/j.chom.2016.07.011
News Article | August 25, 2016
Scientists have identified a new "multicomponent" virus -- one containing different segments of genetic material in separate particles -- that can infect animals, according to research published in the journal Cell Host & Microbe. This new pathogen, called Guaico Culex virus (GCXV), was isolated from several species of mosquitoes in Central and South America. GCXV does not appear to infect mammals, according to first author Jason Ladner, Ph.D., of the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). However, the team also isolated a related virus -- called Jingmen tick virus, or JMTV -- from a nonhuman primate. Further analysis demonstrates that both GCXV and JMTV belong to a highly diverse and newly discovered group of viruses called the Jingmenvirus group. Taken together, the research suggests that the host range of this virus group is quite diverse--and highlights the potential relevance of these viruses to animal and human health. "Animal viruses typically have all genome segments packaged together into a single viral particle, so only one of those particles is needed to infect a host cell," Ladner explained. "But in a multicomponent virus, the genome is divided into multiple pieces, with each one packaged separately into a viral particle. At least one particle of each type is required for cell infection." Several plant pathogens have this type of organization, but the study published today is the first to describe a multicomponent virus that infects animals. Working with collaborators including the University of Texas Medical Branch and the New York State Department of Health, the USAMRIID team extracted and sequenced virus from mosquitoes collected around the world. The newly discovered virus is named for the Guaico region of Trinidad, where the mosquitoes that contained it were first found. In collaboration with a group at the University of Wisconsin-Madison, the USAMRIID investigators also found the first evidence of a Jingmenvirus in the blood of a nonhuman primate, in this case a red colobus monkey living in Kibale National Park, Uganda. The animal showed no signs of disease when the sample was taken, so it is not known whether the virus had a pathogenic effect. Jingmenviruses were first described in 2014 and are related to flaviviruses -- a large family of viruses that includes human pathogens such as yellow fever, West Nile and Japanese encephalitis viruses. "One area we are focused on is the identification and characterization of novel viruses," said the paper's senior author Gustavo Palacios, Ph.D., who directs USAMRIID's Center for Genome Sciences. "This study allowed us to utilize all our tools--and even though this virus does not appear to affect mammals, we are continuing to refine those tools so we can be better prepared for the next outbreak of disease that could have an impact on human health." While it is difficult to predict, experts believe that the infectious viruses most likely to emerge next in humans are those already affecting other mammals, particularly nonhuman primates.
News Article | October 26, 2016
In research published online today in Science, a team of scientists describe a new therapeutic strategy to target a hidden Achilles' heel shared by all known types of Ebola virus. Two antibodies developed with this strategy blocked the invasion of human cells by all five ebolaviruses, and one of them protected mice exposed to lethal doses of Ebola Zaire and Sudan, the two most dangerous. The team included scientists from Albert Einstein College of Medicine, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Integrated Biotherapeutics, Vanderbilt University Medical Center, and The Scripps Research Institute. Ebolaviruses cause a highly fatal disease for which no approved vaccines or treatments are available. About two dozen Ebola outbreaks have been documented since 1976, when infections first occurred in villages along the Ebola River in Africa. The largest outbreak in history--the 2014-2015 Western Africa epidemic--caused more than 11,000 deaths and infected approximately 29,000 people. Monoclonal antibodies, which bind to and neutralize specific pathogens and toxins, have emerged as the most promising treatments for Ebola patients. A critical problem, however, is that most antibody therapies target only one specific ebolavirus. For example, the most promising experimental therapy--ZMappTM, a cocktail of three monoclonal antibodies--is specific for Ebola virus Zaire, and doesn't work against the other two viruses (Sudan and Bundibugyo), which have both caused major outbreaks. The broad-spectrum antibodies developed by the research team represent an important advance against one of the world's most dangerous pathogens. In 2011, a team that included co-senior authors Kartik Chandran, Ph.D. professor of microbiology & immunology at Einstein, and John M. Dye, Ph.D., chief of viral immunology at USAMRIID, discovered that all filoviruses (the family to which ebolaviruses and the more distantly related Marburg virus belong) have an Achilles' heel: To infect and multiply in human cells, they must all bind to a host-cell protein called Niemann-Pick C1 (NPC1). But capitalizing on that knowledge required a completely new approach to targeting viruses: exploiting the fact that Ebola and many other viruses must enter host cell compartments called lysosomes. Once safely inside the lysosomes, the viruses transform and expose key portions of their exterior that the research team successfully targeted using monoclonal antibodies. To gain entry to cells, filoviruses bind to the host cell's outer membrane via glycoproteins (proteins to which carbohydrate chains are attached) that bristle from the virus's surface. (See illustration.) A portion of the cell membrane then surrounds the virus and pinches off, eventually developing into a lysosome--a membrane-bound, intracellular compartment filled with enzymes to digest foreign and cellular components. Filoviruses then use the host cells' resources to break out of their lysosomal "prisons" so they can enter the host cell's cytoplasm to multiply. Enzymes in the lysosome slice a "cap" from the virus's glycoproteins, unveiling a site that binds to the NPC1 embedded in the lysosome membrane. NPC1, which normally helps transport cholesterol within the cell, offers Ebola virus its only means of escaping the lysosome and multiplying. By fitting its protein "key" into the NPC1 "lock," the virus fuses itself to the lysosome membrane. (See illustration close-up.) Now the virus can propel its RNA from the lysosome and into the cell's cytoplasm, where it can finally replicate itself. The research team realized that monoclonal antibodies could potentially thwart all filovirus infections by neutralizing the viral protein that binds to NPC1, or by neutralizing NPC1 itself. There was just one problem: Reflecting Ebola's ingenuity, both targets reside only in lysosomes deep within cells--making them invisible to the immune system and shielded from attack by conventional antibodies. Dr. Chandran, Dr. Dye and co-senior author Jonathan R. Lai, Ph.D., associate professor of biochemistry at Einstein and an expert in engineering antibodies, devised a clever "Trojan Horse" strategy for overcoming the virus's invisibility cloak: Just as the citizens of Troy unwittingly pulled a wooden horse filled with Greek soldiers into their walled city, they tricked the viruses into carrying the means of their own destruction along with them into host cells. To do so, the research team synthesized two types of "bispecific" antibodies, each consisting of two monoclonal antibodies combined into one molecule. One bispecific antibody was devised to neutralize the viral protein that binds to NPC1, the other to target NPC1. Both had one monoclonal antibody in common: antibody FVM09, which binds to the surface glycoproteins of all ebolaviruses while the virus is outside cells, allowing the bispecific antibodies to hitch a ride with the virus into the lysosome. FVM09 was developed by co-senior author M. Javad Aman, Ph.D. at Integrated Biotherapeutics. Once in the lysosome, the bispecific antibodies are released from the viral surface when enzymes in the lysosome slice off the glycoprotein caps--allowing the business ends of the bispecific antibodies to swing into action. One bispecific antibody combined FVM09 with antibody MR72, which was isolated from a human survivor of Marburg virus infection by co-senior author James E. Crowe Jr., M.D., director of the Vanderbilt Vaccine Center. MR72 targets the NPC1-binding viral protein that is unveiled by all filoviruses in lysosomes. The second bispecific antibody links FVM09 to antibody mAb-548, developed at Einstein, which zeroes in on NPC1. With one bispecific antibody targeting the "lock" (NPC1) and the other targeting the "key" (the virus's NPC1-binding protein), both had the potential for preventing Ebola virus from interacting with NPC1 and escaping from the lysosome into the cytoplasm. The researchers then tested their bispecific antibodies against ebolaviruses in the lab. They initially used a harmless virus (vesicular stomatitis virus) that had been genetically engineered to display glycoproteins from all five ebolaviruses on its surface. The researchers incubated the bispecific antibodies with the Ebola-like viruses and then added the mixtures to human cells in tissue culture. Both bispecific antibodies successfully neutralized all five viruses. Work in the high-containment facilities at USAMRIID confirmed that these antibodies also blocked infection by the actual Zaire, Sudan, and Bundibugyo ebolaviruses. Next came studies at USAMRIID to test whether the two bispecific antibodies could protect mice infected with the two most dangerous ebolaviruses, Zaire and Sudan. Researchers, led by Dr. Dye, administered the bispecific antibodies two days after mice were exposed to a lethal dose of virus. The bispecific antibody that targeted the viral binding protein provided good protection to mice exposed to both viruses. As expected, the bispecific antibody that targeted NPC1 did not protect mice. It was designed to bind specifically to human NPC1, which differs slightly in structure from the NPC1 protein found in mice. As a next step, both bispecific antibodies will need to be tested in nonhuman primates, the current gold standard for anti-Ebola therapeutics.
News Article | November 1, 2016
MORRIS PLAINS, N.J., Nov. 1, 2016 /PRNewswire/ -- Xybion Corporation announced today that the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) Biosafety facility at Fort Detrick, Maryland has procured Xybion's Pristima® Suite for its scientific work to...