text:
Post-exposure treatment of Ebola virus disease in guinea pigs using EBOTAb, an ovine antibody-based therapeutic
Ebola AntigensJawahar Raina Jul 24, 2020
Products Related to West Nile, Dengue, Malaria, T.B, Chikungunya, Sars, Zika
Product# 12003 Ebola Recombinant Gycoprotein, GP1 Full Length, amino acids, 60 to 495 (E.Coli)
Product# 12002 Ebola Recombinant Fusion Glycoprotein GP2 Antigen (E.Coli)
Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. An ovine polyclonal antibody therapy has been developed against EBOV, named EBOTAb. When tested in the stringent guinea pig model of EBOV disease, EBOTAb has been shown to confer protection at levels of 83.3%, 50% and 33.3% when treatment was first started on days 3, 4 and 5 post-challenge, respectively. These timepoints of when EBOTAb treatment was initiated correspond to when levels of EBOV are detectable in the circulation and thus mimic when treatment would likely be initiated in human infection. The effects of EBOTAb were compared with those of a monoclonal antibody cocktail, ZMapp, when delivered on day 3 post-challenge. Results showed ZMapp to confer complete protection against lethal EBOV challenge in the guinea pig model at this timepoint. The data reported demonstrate that EBOTAb is an effective treatment against EBOV disease, even when delivered late after infection.
Ebola virus (EBOV) has recently been responsible for its largest outbreak in West Africa, first recognised in March 20141, causing more deaths than all previously known outbreaks combined. Whilst the first outbreak of EBOV was identified in 19762, there are still no approved therapeutics; however, during the 2014 EBOV outbreak the World Health Organisation approved immunotherapy in the form of homologous polyclonal antibodies (pAb)3. Nevertheless, due to difficulties with the human-derived antibody treatments4, alternative immunotherapeutic strategies are required.
In order to be of value in EBOV infection, treatments should be tested after exposure to the virus in order to demonstrate therapeutic effects. Post-exposure therapies against filoviruses described in human clinical trials and/or animal model systems have previously been reviewed5 and consist of: recombinant proteins involved with anticoagulation6 and human activated protein C7; RNA interference by phosphorodiamidate morpholino oligomers8 and stable nucleic acid-lipid particles targeting the EBOV L protein9,10; mannose-binding lectin11; and small molecule inhibitors12,13. These treatments range from treating clinical symptoms, inhibiting viral processes, boosting host immune responses and limiting viremia14. Vaccination approaches have also been demonstrated to confer post-exposure protection against EBOV, such as a recombinant vesicular stomatitis virus vector expressing the EBOV glycoprotein which protected 50% of guinea pigs following treatment up to 24 hours after lethal challenge15.
Cocktails of monoclonal antibodies (mAb) are currently the most studied post-exposure EBOV treatments reported and are the only therapy that has demonstrated substantial benefits in non-human primates when administered greater than 24 hours post-EBOV exposure16. Verified protection as late as 3 days post-infection has been reported in the guinea pig model17. Based on success in non-human primates, ZMapp and ZMAb have been used under emergency compassionate protocols in humans to treat EBOV infections originating from outbreak (25 were treated on compassionate ground, 22 survived and only 1 died after receiving at least 2 doses), six patients have been treated with ZMAb, with all surviving and all administrations were reported as well tolerated18. While still not clear if the survival can be directly attributed to treatment with the mAb cocktails, this production and clinical testing of anti-EBOV cocktails is being accelerated. However, mAb therapies suffer from several disadvantages including high production costs and risk of escape mutants, particularly for RNA viruses such as EBOV which have high mutation frequencies19; polyclonal antibody (pAb) approaches are therefore an alternative option. An ovine pAb-based product, EBOTAb, has previously been described based on purified IgG from sheep immunised with mammalian-expressed recombinant EBOV glycoprotein20. This approach offers a cost-effective method of treating EBOV infection and is economically viable for developing regions facing epidemic EBOV disease. Similar intact ovine pAb have been used in West Africa for several years to treat >40,000 patients envenomated by carpet vipers, with the resultant product EchiTAb being one of the most cost-effective therapies currently available21.
EBOTAb has previously been demonstrate to bind to both the GP1 and GP2 subunits of the EBOV glycoprotein20; and since this is a pAb preparation it contains antibodies against multiple epitopes. This reduces the likelihood that escape mutations of EBOV can arise as has been reported for individual mAb contained within ZMapp22. Since different epitopes are recognised at different stages of viral infection, the pAb approach is likely to confer multiple effects including inhibition of host cell attachment, obstructing enzymatic cleavage and blocking the cleaved forms of glycoprotein, thus obstructing the activation of endosomal virus-cell fusion while limiting the emergence of escape mutants23,24.
Whilst an initial study documented the use of EBOTAb delivered 6, 48 and 72 hours post-challenge, this report covers our assessment of the protective efficacy of EBOTAb up to 5 days post-challenge and compares EBOTAb delivery with ZMapp, a mAb-based therapy.
Efficacy of antibody treatment delivered 3 days post-EBOV challenge
The EBOTAb or ZMapp preparation was first delivered to guinea pigs on the third day after infection with a lethal dose of EBOV. Survival analysis showed that all untreated animals met humane endpoints by day 11 (Fig. 1a). Treatment with EBOTAb starting on day 3 and with repeat administrations on days 4, 5, 7, 9 and 11 demonstrated 83.3% survival (Fig. 1a), a significance different as compared to untreated animals (P = 0.003, Log-Rank survival analysis). When EBOTAb was given on day 3 post-challenge alone, survival was reduced to 33% but this was still significant compared to the untreated controls (P = 0.032, Log-Rank survival analysis). ZMapp administered on day 3 resulted in complete protection (P < 0.001 compared to untreated animals, Log-Rank survival analysis). There was no statistical difference between the ZMapp-treated animals and those given EBOTAb at day 3 followed by repeat doses (P = 0.317, Log-Rank survival analysis). All animals treated with single or multiple doses of EBOTAb showed signs of disease, including weight loss and temperature increases (Fig. 1b,c, respectively). In the EBOTAb-treated groups there was an apparent delay in temperature spiking and a reduction in peak temperature in the group which received multiple doses of EBOTAb. Clinical signs were assessed in surviving animals from each of the groups and signs of illness were detected in the animals treated with EBOTAb, albeit at lower levels to untreated animals (Fig. 1d). In our analysis, ZMapp treated animals remained clinically healthy throughout the course of the study.
Open in a separate window
Survival and clinical observations of EBOV-challenged guinea pigs after treatment with EBOTAb or ZMapp starting after 3 days.
Guinea pigs were challenged with EBOV and treated with EBOTAb or ZMapp after 3 days. One group received 0.5 ml (51 mg/ml, equating to 75–86 mg/kg) EBOTAb twice per day on days 3, 4, 5, 7, 9 and 10 post-challenge. Two groups received 0.5 ml of EBOTAb (48 mg/ml, equating to 68–77 mg/kg) and ZMapp (3.4 mg/ml, equating to 5–6 mg/kg), respectively, in each of three doses delivered within 10 hours with the concentrations arranged so the same amount of EBOV-specific antibody was present and both groups received 10 mg of specific-antibody on day 3 post-challenge. (a) Survival analysis between EBOTAb and ZMapp treated groups compared to untreated animals (n = 6 per group). (b) Weight changes, showing percentage differences from values on the day of challenge. (c) Temperature differences in animals compared to values on the day of challenge. (d) Clinical scores of animals after challenge. In panels b–d, mean results are shown for animals still surviving in all groups, with error bars denoting standard error.
Efficacy of EBOTAb treatment delivered 4 and 5 days post-EBOV challenge
EBOTAb was first delivered to guinea pigs 4 and 5 days after infection with a lethal dose of EBOV, with repeat (two per day) doses being given on three consecutive days followed by another six doses (two per day) every other day. Unfortunately, ZMapp was not available for this second experiment. Survival analysis showed that treatment with EBOTAb starting on day 4 and 5 resulted in 50% and 33% protection, respectively (Fig. 2a) (P = 0.002 and P = 0.134 compared to untreated animals, respectively; Log-Rank survival analysis). The survival in the EBOTAb treatment group starting on day 5 post-challenge was not significant due to a single guinea pig meeting humane clinical endpoint before any of the untreated control animals. All guinea pigs treated with EBOTAb showed signs of disease, including weight loss and temperature increases (Fig. 2b,c, respectively). Clinical signs were detected in the animals treated with EBOTAb at these later timepoints (Fig. 2d).
Survival and clinical observations of EBOV-challenged guinea pigs after treatment with EBOTAb starting after 4 and 5 days.
Guinea pigs were challenged with EBOV and treated with EBOTAb starting after 4 or 5 days. 0.5 ml (51 mg/ml, equating to 71–84 mg/kg) EBOTAb was administered twice per day on three consecutive days followed a further 3 doses every other day. (a) Survival analysis between EBOTAb treated groups compared to untreated animals (n = 6 per group). (b) Weight changes, showing percentage differences from values on the day of challenge. (c) Temperature differences in animals compared to values on the day of challenge. (d) Clinical scores of animals after challenge. In panels B–D, mean results are shown for animals still surviving in all groups, with error bars denoting standard error.
Presence of EBOV RNA in the peripheral circulation
Prior to the administration of antibody treatment on days 3–5 post-challenge, blood was withdrawn and the presence of EBOV RNA detected using a quantitative RT-PCR assay. Results showed viremia at day 3 post-challenge, which increased on day 4 and 5 post-challenge (Fig. 3a). On day 8 post-challenge, blood was sampled from all surviving animals with patent catheters and viral RNA levels assessed (Fig. 3b). The levels of viral RNA in EBOTAb treated animals were similar to untreated animals across all groups (P > 0.05, Mann-Whitney statistical test). No viral RNA was detectable within the limitations of the assay in the circulation of ZMapp treated animals 8 days post-challenge. Blood samples were collected at day 21 from the animals surviving until the scheduled end of the study and with their intravenous catheters still patent to allow blood withdrawal (EBOTAb given at days 3, 4 and 5 post-challenge, n = 3, n = 2 and n = 2, respectively; EBOTAb given on day 3 only, n = 1; and ZMapp given on day 3 only, n = 2). No viral RNA was detected in any of the blood of samples collected at 21 days post-challenge.
Viral genome copies in the blood of EBOV-challenged guinea pigs prior to treatment and on day 8 post-challenge.
(a) RNA levels in the blood of EBOV challenged groups prior to administration of any antibody treatments on day 3, 4 and 5 post-challenge. (b) RNA levels in the blood at day 8 post-EBOV challenge of animals treated with EBOTAb and ZMapp. Bars show mean results with error bars denoting standard error.
Histological changes and presence of viral antigen in local sites of infection
Sections of liver and spleen were taken to assess the architecture of cells within the primary sites of EBOV infection in guinea pigs and stained for the presence of viral antigen (Table 1). In the untreated group, microscopic lesions and the presence of viral antigen were noted in the spleen and liver in all animals (Fig. 4a,b). In animals which received EBOTAb at day 3 post-challenge with repeat dosing, the treatment was generally effective (Fig. 4c,d); although one animal was severely affected (89314). Giving only a single dose of EBOTAb on day 3 or delaying dosing to days 4 and 5 resulted in microscopic lesions and EBOV antigen being detectable in most animals. Treatment with ZMapp showed fewer histological changes, although microscopic changes were noted, and no viral antigen was observed with immunohistochemical staining (Fig. 4e,f).
Histology and immunohistochemical staining of liver and spleen samples from EBOV infected guinea pigs treated with EBOTAb and ZMapp.
(a) Liver, untreated (ID03904). Focal necrotic lesions (arrows) with mineralisation (arrowheads) and scattered, macrovesicular vacuolation of hepatocytes, consisent with fat. Inset, numerous cells staining strongly for Ebola viral antigen. (b) Spleen, untreated (ID03917). Marked congestion and single cell death within the red pulp (arrows), and patchy, PMN cell infiltrates (circle). Inset,numerous cells staining strongly for Ebola viral antigen. (c) Liver, EBOTAb day 3 treated (ID89303). Focal, mineralised areas (arrows). Inset, rare cells staining positive for Ebola viral antigen (arrows). (d) Spleen, EBOTAb day 3 treated (ID89258). Red pulp appears normal, with a minimal reduction in lymphocytes within the white pulp (not shown in image). Inset, small numbers of cells staining positive for Ebola viral antigen. (e) Liver, ZMapp treated on day 3 (ID03905). Normal. Inset, negative staining for viral antigen. (f) Spleen, ZMapp treated on day 3 (ID03905). Normal. Inset, negative staining for viral antigen. Images show H&E with insets of IHC stained samples.
Histological changes and immunohistochemistry staining in the liver and spleen of EBOV challenged guinea pigs treated with EBOTAb and ZMapp.
Animal ID (day*)
Lipid vacuoles
Focal necrosis
Focal mineral
Red pulp single cell death
Red pulp foci PMN
White pulp single cell death
White pulp lymphocyte depletion
Untreated 03893 (7) − + − +++ +++ +++ ++ +++ +++ +++
03915 (7) ++++ ++ − ++ ++ ++ +++ + + +++
03917 (7) +++ + + ++ ++++ ++++ +++ ++ +++ ++
89256 (7) +++ ++ + +++ +++ ++++ +++ +++ +++ +++
89299 (8) ++ + − ++ +++ ++ − +++ ++++ ++
03904 (11) ++++ ++ +++ ++ +++ ++ +++ + + +
EBOTAb
3 days post−
challenge**
89314 (8) + ++++ +++ ++++ +++ ++++ − ++++ ++++ ++++
89273 (21) − − ++ − + − − − − +
89209 (21) + − − − − − − − − +
89303 (21) − − ++ + − + + +++ ++ +
89258 (21) − ++ − +*** − − − − ++ +
89265 (21) − − − − − − − − − −
89271 (9) +++ ++++ ++++ ++++ +++ ++++ ++ ++++ ++ ++++
89318 (12) +++ ++++ +++ ++ − ++++ − ++++ +++ +++
89311 (13) + +++ ++++ +++ ++ +++ ++++ + +++ +
89278 (21) + + ++++ + − − − − − +
89349 (21) − − ++++ + ++ ++ − + ++ +
89336 (21) − ++ − − − − − − − +
EBOTAb starting
post-challenge**
89253 (6) +++ ++++ − +++ − ++ +++ + ++ ++
03892 (7) +++ ++++ + ++ ++ ++++ + − − ++
89344 (9) + ++++ ++++ +++ ++++ +++ + +++ +++ +++
89352 (12) +++ ++++ +++ +++ +++ ++++ +++ ++ ++ +
89245 (21) − − ++++ + − − − ++ − +
89257 (21) − − − + − − − − − −
EBOTAb, day
03911 (8) + + + − +++ ++ − − + −
89279 (9) +++ +++ ++ ++ +++ ++++ +++ ++++ ++ ++
89282 (9) − ++++ − +++ ++++ ++ − +++ ++ +++
03898 (12) ++++ + ++++ + +++ ++++ +++ +++ ++++ +
03883 (21) − − + − − − − − − −
89312 (21) − − ++++ + − − − − − +
ZMApp,
03905 (21) +++ − + − − − − − − −
03886 (21) + − − − + − − − − −
03907 (21) − − − − − − − ++ − −
03971 (21) +++ − − − − − − − − −
03913 (21) + − − − − − − − ++ −
03887 (21) + − − − − − − − − −
Abbreviations:-within normal limits; +minimal; ++mild; +++moderate; ++++marked.
*day post-challenge samples were collected; **repeat dosing of EBOTAb was conducted on 5 subsequent days; ***unusual intra-nuclear immunostaining.
Assessment of EBOV RNA levels in the liver and spleen of animals surviving at day 21 post-challenge
At the scheduled end of the study, 21 days post-EBOV challenge, samples of liver and spleen were assessed for the presence of EBOV RNA in animals which survived. Several animals in the groups treated with EBOTAb had no evidence of viral RNA in the liver or spleen; however, in other animals viral RNA was detectable with higher levels being observed in the liver (Fig. 5). In the ZMapp treated group, EBOV RNA was detected in two animals, but only in the spleen.
Viral genome copies in the liver and spleen of EBOV-challenged guinea pigs treated with EBOTAb and ZMapp and surviving to day 21.
At the end of the scheduled study, sections of spleen and liver were taken from surviving animals and homogenised prior to assessment of the viral RNA levels by RT-PCR assay. Bars denote the mean value from triplicate wells quantified by RT-PCR. Abbreviations: ND, not done. *repeat dosing of EBOTAb was undertaken on 5 subsequent days.
Determination of anti-EBOTAb specific antibodies
In animals which survived until day 21, blood was withdrawn from catheters that remained patent; 2 animals treated with ZMapp on one day, 2 animals treated with EBOTAb on one day and 8 animals which received EBOTAb on six separate days. EBOTAb-specific IgG was detected in the sera of animals treated with EBOTAb compared to those animals which received ZMapp as a control (Fig. 6). No difference in the generation of anti-EBOTAb antibody levels were observed in the groups treated on either a single day or those which received sequential treatments on 6 separate days.
Measurement of anti-EBOTAb specific antibodies in the sera of animals surviving to day 21 post-challenge.
At the end of the scheduled study, sera was isolated from animals where catheters retained patency from animals which had received ZMapp on a single day (n = 2), EBOTAb on a single (n = 2) or EBOTAb on six separate days (n = 8). Results show the mean values with error bars denoting standard error.
In this study, we investigated in further detail the delayed use of EBOTAb, a cost-effective antibody treatment for EBOV. Whilst responses when EBOTAb has been delivered 6, 48 and 72 hours post-challenge have previously been reported20, in this study dosing was started at either 3, 4 or 5 days post-challenge. Our results show that prior to initiating treatment, viral RNA was present in the bloodstream 3 days post-challenge, before rising subsequently after 4 and 5 days. This is consistent with other guinea pig studies, where viremia was first detected within 2 days post-challenge and increased to a peak on day 725. In this study we assessed viral load with the standard diagnostic PCR assay for EBOV26; thus, our study followed the same methodology used in human EBOV infection to ensure that the guinea pigs were first given EBOTAb in the same treatment window used in the clinical setting.
Delayed treatment with a cocktail of 3 pooled mAbs has demonstrated protection of 67% (4/6 guinea pigs surviving) when given 3 days post-challenge27; similar to our results with EBOTAb which demonstrated 83% efficacy (5/6 guinea pigs surviving) when treatment was also first administered day 3 post-challenge in the current study and 75% efficact (3/4 guinea pigs surviving) in a previous study20. In the pooled mAb study, treatment was initiated at -1, 1, 2 and 3 days post-challenge and demonstrated that survival peaked with delivery starting at day 2 post-challenge with 100% protection, but when delivered 1 day prior or 1 day after challenge the efficacy dropped to 50%27. Our results are more in line with expectations of efficacy waning with increasing time to initiating treatment, with efficacies of 50% and 33.3% observed when EBOTAb was first delivered at days 4 and 5 post-challenge, respectively.
In our study, we compared the effects of EBOTAb with ZMapp, one of the leading mAb therapies against EBOV which contains a mixture of 3 mAb produced from the plant, Nicotiana benthamiana28. Our results demonstrate that when EBOTAb and ZMapp are first administered 3 days post-challenge, there is no statistical difference in survival outcomes if the EBOTAb is then administered throughout the course of infection. When EBOTAb was given only on day 3 post-challenge, it still provided a statistically significant level of protection although this was not as effective as repeat dosing of EBOTAb or with ZMapp delivered on a single day. Interestingly, despite their being no significant difference in survival at day 3 of EBOV infected guinea pigs between those treated with 12 doses of EBOTAb or with a single dose of ZMapp, the clinical condition of animals which received ZMapp was superior. Thus, animals treated with ZMapp showed no loss in weight, no temperature fluctuations nor clinical signs throughout the period of study, unlike those that received EBOTAb. Whilst survival is the most important aspect of an EBOV therapy, these results also indicate that treatment with EBOTAb might require supportive care in addition to direct symptom management associated with disease.
The use of a pAb therapy negates limitations associated with mAb-based therapies, particularly the risk that mutations can arise in epitopes that could render escape from therapeutic regime and necessitates the development of multiple antibody cocktails29. From several studies of mAb treatment using a combined total of 36 NHPs, the occurrence of escape mutations was suggested to be 3%. While this was only detected in a single animal30, their development is significant. Additional evidence was produced in a study using cynomolgus macaques treated with a combination of three mAbs (ZMAb) where mutations in the epitope binding sites of these antibodies were detected31, suggesting the level of escape mutations to be higher. Similar mutations have also been identified in rhesus macaque studies using MB-003, a similar mAb combination therapy to ZMAb and ZMapp22. Given that NHP studies are controlled and conducted within a relatively short timescale, it could be speculated that escape mutations following treatment with mAb-based therapeutics in human EBOV infection could potentially compromise the ability to contain outbreaks in affected populations. This might be exacerbated by the ability of EBOV to lie dormant within immune privileged sites after clinical disease32,33.
As filoviruses are endemic in developing and third-world countries, it is important that therapeutic costs are kept to a minimum so that stockpiling of treatments for emergency purposes is not an economic burden14. It has been suggested that existing contract manufacturing organisations under ideal conditions could produce mAbs at an unit cost as low as US$60/g34. However, the costs incurred to develop mAbs into approved treatments are high. For example the US Biomedical Advanced Research and Development Authority (BARDA) provided financial support of US$18.9 m over 18 months to Mapp Biopharmaceutical to generate a relatively small stock of clinical grade (cGMP) ZMapp (<1000 doses) to advance the product through clinical trial evaluation35. Additionally the production of mAb from tobacco plants is not a straightforward process36, a cheaper approach for the production of mAb is to use mammalian expression systems. However, it has also been shown that when one of the ZMapp precursor mAb combinations (MB-003) was expressed in a mammalian expression system there was a threefold difference in effectiveness when tested in rhesus macaques compared to the same mAb cocktail being expressed with tobacco virus28. This observation has also been reported in mice37. The only change between ZMapp produced in tobacco plants and in Chinese hamster ovary (CHO) cells is the glycosylation of the antibodies. The absence of core fucose in the ZMapp antibodies is known to increase binding to the FcγRIII, resulting in a dramatic improvement in antibody-dependent cellular cytotoxicity (ADCC)28. As mammalian cells in general produce only a small percentage of antibodies lacking core fucose, it might be for this reason that ZMapp performs better than EBOTAb in reducing viremia and clinical signs of EBOV in the guinea pig studies we report within.
EBOTAb and the ZMapp-precursor mAb combinations have both previously been tested at 3 days post-infection in EBOV-infected guinea pigs, demonstrating 75%20 and 33–67%18,38 efficacies, respectively. To the best of our knowledge there is currently no data where antibodies against EBOV have been administered after day 3 post-challenge in the guinea pig model. The finding that EBOTAb exerts a level of protection as late as day 5 post-challenge is therefore a significant finding. A major difference between the effects of EBOTAb and ZMapp was on viral load at day 8 post-challenge; a timepoint chosen since circulating viral loads are high8 and, historically, this is when guinea pigs begin to meet humane clinical endpoints. Whilst there was no difference in circulating viral RNA levels in the EBOTAb treated animals, no EBOV RNA was detected in those which received ZMapp. Therefore, EBOTAb might have different mechanisms of action. Both EBOTAb and ZMapp are known to have high neutralisation activity against live EBOV11,16; however, in order to directly compare, further work would be required to test both compounds in identical assay systems. Since EBOTAb is a pAb preparation it binds to multiple epitopes on the EBOV glycoprotein which play different roles in the course of viral infection24.
The finding that EBOV RNA was present in liver and spleen (the only organs sampled and tested) indicates residual virus present at the end of the study, including in the animals treated with ZMapp. The range of responses observed is typical due to the use of outbred guinea pigs. However, given that no EBOV antigen was detected by immunostaining in several of the samples showing positive PCR, it is likely that the PCR assay detected viral nucleic acid fragments remaining from the infection. A similar finding has been reported by others who showed detection of viral RNA at 28 days post-challenge but live virus was not recovered39. Interestingly, no viremia was detectable in the blood of animals tested at 21 days post-challenge, the scheduled end of the study. This is important, as negative results of PCR assays in the blood are currently one of the main parameters for the discharge of EBOV convalescent patients40.
In our study, we have evaluated the antibody preparations as a sole treatment option. It may be possible that the protective efficacy of EBOTAb could be increased by supplementing with other compounds. For example: A human adenovirus serotype 5 which expresses recombinant interferon-α (IFN-α), termed DEF201, has been shown to enhance protection of an adenovirus-based vaccine against EBOV when delivered 30 minutes post-exposure in guinea pigs41. Rapid administration of DEF201 has also been shown to result in complete survival of EBOV infected guinea pigs when mAbs were delivered within 7 days post-infection17. A similar result was observed with DEF201 and mAbs in NHPs42. The DEF201 could constitutively produce high levels of IFN-α for days in a cost efficient manner, as recombinant IFN-α is expensive and has a short half-life of 8 hours in vivo43.
Whilst an uninfected EBOTAb treated group was not conducted, it is unlikely that any adverse effects would be detected. Our previous studies have demonstrated that when treatment is first given either 6 or 48 hours post-challenge with EBOV, no clinical signs were detected when EBOTAb was delivered on 10 or 8 occasions, respectively20. A similar based therapy against Clostridium difficle has also been developed using the same platform44 and has completed GLP toxicity studies in rats with no adverse findings reported. Whilst a case of severe anaphylactic/anaphylactoid reaction has been reported to an ovine polyvalent immune preparation after a snakebite injury45, this was later challenged by other authors with the clinical findings to be more consistent with angioedema caused by angiotensin converting enzyme (ACE) inhibitors46. Therefore, this example of a serious adverse reaction to an ovine antibody-based preparation was compromised by the patient an anti-hypertensive medicaction with a design based on venom, was envenomated, and was then treated with another venom-derived medication45. Additionally, our results demonstrated an antibody response being raised against EBOTAb. However, despite the small numbers of animals studied, there was no evidence of titres increasing with repeated deliveries of EBOTAb.
In this work, we have used the guinea pig model as a suitable small animal model for EBOV disease. While murine models exist, these are less predictive to study efficacy of treatments and vaccines compared to guinea pigs and non-human primates (NHPs)38,47,48. The guinea pig model displays the same tissue tropism as in NHPs and recapitulates the lesions observed in tissues of EBOV infected NHPs and humans25. The histological findings of diffuse lymphocyte depletion, necrosis and congestion in the spleen and liver samples of EBOV infected guinea pigs in this report are also features observed in EBOV infected NHPs49,50. Nevertheless, future studies are underway to analyse the efficacy of EBOTAb in the NHP model.
Whilst a post-exposure treatment would confer significant public health benefits to communities affected by EBOV, a medical countermeasure would also be of use in the event of laboratory exposure to the virus as has previously occurred, including institutions in the US51, Côte d'Ivoire52, England53 and Germany54. The recent outbreak in West Africa has also highlighted the nosocomial transmission of this virus to healthcare workers55. In addition, EBOV has historically, and in the present day, been listed as a possible agent for bioterrorism56.
Our results demonstrate the potential importance of EBOTAb as a cost-effective therapy against EBOV and further strengthen the evidence base showing protective effects as late as 5 days post-challenge. The advantages of the pAb technique in reducing the risk of escape virus mutants further strengthens the continued development of EBOTAb as a potential therapy for EBOV. In addition, EBOTAb was developed rapidly in response to the EBOV outbreak in a fraction of the time that has been spent developing alternatives, which demonstrates the feasibility of the ovine pAb approach to other emerging pathogens that may arise in the future.
Animal studies were performed under Containment Level 4 conditions with all procedures being undertaken according the United Kingdom Animals (Scientific Procedures) Act 1986. Studies were conducted under Establishment Licence reference PEL PCD 70/1707 with Project Licence PPL 30/3247. Studies were approved by the Public Health England ethics committee and the Project Licence approved by a UK Home Office inspector.
Virus and antibody preparations
EBOV strain Yambuku-Ecran (previously named ME71857) was passaged five times in guinea pigs to achieve lethality, as previously described58. Virus was titrated by 50% tissue culture infective dose (TCID50) assay in VeroE6 cells (European Collection of Cell Cultures, UK). EBOTAb was generated as previously reported20. ZMapp was supplied by Mapp Biopharmaceutical, Inc. Where required, EBOV and antibodies were diluted with sterile PBS.
Female adult Dunkin-Hartley guinea pigs (Harlan Laboratories, United Kingdom) were used for in vivo studies, with an average mean starting weight of 325 g (range 288–359 g). Animals were supplied with catheters inserted into the jugular vein to allow intravenous access59. For procedures, guinea pigs were anaesthetised with 1.5%–2% isoflurane in oxygen until full sedation was achieved. Food and sterile water were available ad libitum. Animals were weighed and temperatures recorded daily via an indwelling temperature chip. Clinical signs were monitored at least twice daily, and the following numerical score was assigned for analysis: 0 (normal); 2 (ruffled fur); 3 (lethargy, pinched, hunched and wasp waisted); 5 (labored breathing). EBOV stock was diluted in sterile phosphate buffered saline (PBS) to prepare 102 TCID50 in a 0.2 mL volume and subcutaneously inoculated in the lower right quadrant of the back of each animal. Virus was back titrated on VeroE6 cells to confirm the dose. EBOTAb was delivered intravenously starting at either 3, 4 or 5 days following EBOV challenge. Repeat administrations (2 doses) were delivered on each of the following two days and then on alternate days for another 3 occasions. On each day of administration, twice-daily dosing of 0.5 mL EBOTAb (51 mg/mL) was delivered with at least 5 hours between doses. One group of animals received EBOTAb on day 3 post-challenge only. EBOTAb was diluted to 48 mg/mL and 0.5 mL was delivered over three occasions separated by at least 3 hours. ZMapp was diluted to 3.4 mg/mL, and administered identically to the animals that received EBOTAb on day 3 only. For the animals which received compounds only on day 3 post-challenge, the amounts received were 72 mg/animal EBOTAb (5 mg/mL EBOV-specific antibody) and 5 mg/animal ZMapp.
EBOV titration by RT-PCR
Blood samples were taken in RNAprotect animal blood tubes (Qiagen, UK), mixed and stored at −80 °C until processing. For processing, 200 μL of tissue homogenate or blood solution was transferred to 600 μL RLT buffer (Qiagen) for RNA extraction and PCR. Extraction of RNA was performed using the MagnaPure 96 small volume RNA kit (Roche). Plates were loaded onto the MagnaPure 96 automated extraction robot and RNA was eluted in 60 μL nuclease free water. Target amplification was performed using primers to EBOV glycoprotein60 using the Fast Virus qRT-PCR Kit (Qiagen). Analysis was performed using the ABi 7500 (Applied Biosystems) at the following cycling conditions; 50 °C for 10 minutes, 95 °C for 30 seconds followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 3 seconds. Temperature cycling was set to maximum ramp speed and data was acquired and analysed using the ABi 7500 on-board software with a threshold set to 0.05. A standard curve of RNA transcript at known quantities was run in parallel on each PCR plate for assessing genome copies per reaction.
Samples of liver and spleen were placed in 10% neutral buffered formalin for at least 21 days and processed routinely to paraffin wax. Sections were cut at 3–5 μm, stained with haematoxylin and eosin (HE) and examined microscopically. For immunohistochemistry, sections were stained for EBOV antigen using the Leica BondMax (Leica Biosystems) and the Leica Bond Polymer Refine Detection kit (Leica Biosystems). An antigen retrieval step was included for 10 minutes using the Bond Enzyme Pretreatment kit, enzyme 3 (3 drops). A rabbit polyclonal, anti-EBOV VP40 antibody (IBT Bioservices) (dilution 1:2000) was incubated with the slides for 60 minutes. DAB chromogen and haematoxylin counterstains were used to visualise the slides.
Anti-EBOTAb antibody ELISA
High binding ELISA plates (Maxisorb, Nunc) were coated with 10 μg/ml EBOTAb diluted in PBS overnight at 4 °C. Unbound antibody was removed by washing three times with PBS and a three-fold dilution of sera was conducted starting at 1:10 and added to the plate. All conditions were tested in triplicate wells. After 1 hour at 37 °C plates were washed three times with PBS and a peroxidase-labelled donkey anti-guinea pig IgG antibody solution added (Jackson ImmunoResearch). After 1 hour at 37 °C plates were washed 5 times with PBS and 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) substrate (Insight Biotechnology) added and left for 15 minutes at room temperature before the reaction was stopped by the addition of ABTS stop solution (Insight Biotechnology). Absorbances were read on a plate spectrophotometer at a wavelength of 405 nm.
To determine group sizes for the in vivo studies, a power calculation with the Fisher exact test was performed using software G*Power, version 3.0.10. A group size of 6 met a power of 0.8 and an α of 0.05. The Log-Rank test for nonparametric survival was used to compare differences between groups of animals using Minitab, version 16 and applying right-censoring with a time censor of 21 days.
How to cite this article: Dowall, S. D. et al. Post-exposure treatment of Ebola virus disease in guinea pigs using EBOTAb, an ovine antibody-based therapeutic. Sci. Rep. 6, 30497; doi: 10.1038/srep30497 (2016).
The views expressed are those of the authors and are not necessarily those of the employing organisations. We would like to thank Larry Zeitlin from Mapp Biopharmaceuticals for the generous supply of ZMapp used in the studies.
Work was funded by the Public Health England pipeline fund and from MicroPharm Ltd. J.L., I.C., R.C. and I.A. are employees of MicroPharm Ltd.
Author Contributions The following authors designed the studies: S.D.D., I.T., J.L., I.C., R.C., G.K., R.H. and M.W.C. The work was conducted and analysed by S.D.D., A.B., E.R., G.H., I.A., V.A.G. and I.T. The manuscript was written and reviewed by S.D.D., J.L., I.C., G.K., R.H. and M.W.C.
Baize S. et al.. Emergence of Zaire Ebola virus disease in Guinea. The New England journal of medicine 371, 1418–1425, doi: 10.1056/NEJMoa1404505 (2014). [PubMed] [CrossRef] [Google Scholar]
WHO. Ebola haemorrhagic fever in Zaire, 1976. Bulletin of the World Health Organization 56, 271–293 (1978). [PMC free article] [PubMed] [Google Scholar]
Gulland A. First Ebola treatment is approved by WHO. Bmj 349, g5539, doi: 10.1136/bmj.g5539 (2014). [PubMed] [CrossRef] [Google Scholar]
Kreil T. R. Treatment of Ebola virus infection with antibodies from reconvalescent donors. Emerging infectious diseases 21, 521–523, doi: 10.3201/eid2103.141838 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Friedrich B. M. et al.. Potential vaccines and post-exposure treatments for filovirus infections. Viruses 4, 1619–1650, doi: 10.3390/v4091619 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Geisbert T. W. et al.. Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet 362, 1953–1958, doi: 10.1016/S0140-6736(03)15012-X (2003). [PubMed] [CrossRef] [Google Scholar]
Hensley L. E. et al.. Recombinant human activated protein C for the postexposure treatment of Ebola hemorrhagic fever. The Journal of infectious diseases 196 Suppl 2, S390–S399, doi: 10.1086/520598 (2007). [PubMed] [CrossRef] [Google Scholar]
Warren T. K. et al.. Advanced antisense therapies for postexposure protection against lethal filovirus infections. Nature medicine 16, 991–994, doi: 10.1038/nm.2202 (2010). [PubMed] [CrossRef] [Google Scholar]
Geisbert T. W. et al.. Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference. The Journal of infectious diseases 193, 1650–1657, doi: 10.1086/504267 (2006). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Geisbert T. W. et al.. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet 375, 1896–1905, doi: 10.1016/S0140-6736(10)60357-1 (2010). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Michelow I. C. et al.. High-dose mannose-binding lectin therapy for Ebola virus infection. The Journal of infectious diseases 203, 175–179, doi: 10.1093/infdis/jiq025 (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Warren T. K. et al.. Antiviral activity of a small-molecule inhibitor of filovirus infection. Antimicrobial agents and chemotherapy 54, 2152–2159, doi: 10.1128/AAC.01315-09 (2010). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Aman M. J. et al.. Development of a broad-spectrum antiviral with activity against Ebola virus. Antiviral research 83, 245–251, doi: 10.1016/j.antiviral.2009.06.001 (2009). [PubMed] [CrossRef] [Google Scholar]
Wong G., Qiu X., Olinger G. G. & Kobinger G. P. Post-exposure therapy of filovirus infections. Trends in microbiology 22, 456–463, doi: 10.1016/j.tim.2014.04.002 (2014). [PubMed] [CrossRef] [Google Scholar]
Feldmann H. et al.. Effective post-exposure treatment of Ebola infection. PLoS pathogens 3, e2, doi: 10.1371/journal.ppat.0030002 (2007). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Qiu X. et al.. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514, 47–53, doi: 10.1038/nature13777 (2014). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Qiu X. et al.. Monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in Ebola virus-infected guinea pigs. Journal of virology 87, 7754–7757, doi: 10.1128/JVI.00173-13 (2013). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Davidson E. et al.. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies. Journal of virology 89, 10982–10992, doi: 10.1128/JVI.01490-15 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Alfson K. J., Worwa G., Carrion R. Jr. & Griffiths A. Determination of the Spontaneous Mutation Frequency of Ebola virus and Exploitation of this Therapeutically. Journal of virology, doi: 10.1128/JVI.02701-15 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Dowall S. D. et al.. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection. The Journal of infectious diseases, doi: 10.1093/infdis/jiv565 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Habib A. G. Public health aspects of snakebite care in West Africa: perspectives from Nigeria. The journal of venomous animals and toxins including tropical diseases 19, 27, doi: 10.1186/1678-9199-19-27 (2013). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Kugelman J. R. et al.. Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail. Cell reports 12, 2111–2120, doi: 10.1016/j.celrep.2015.08.038 (2015). [PubMed] [CrossRef] [Google Scholar]
White J. M. & Schornberg K. L. A new player in the puzzle of filovirus entry. Nature reviews. Microbiology 10, 317–322, doi: 10.1038/nrmicro2764 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Gonzalez-Gonzalez E. et al.. Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead. Critical reviews in biotechnology, 1–16, doi: 10.3109/07388551.2015.1114465 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Connolly B. M. et al.. Pathogenesis of experimental Ebola virus infection in guinea pigs. The Journal of infectious diseases 179 Suppl 1, S203–S217, doi: 10.1086/514305 (1999). [PubMed] [CrossRef] [Google Scholar]
Vogt F. et al.. Assessment of the MSF triage system, separating patients into different wards pending Ebola virus laboratory confirmation, Kailahun, Sierra Leone, July to September 2014. Euro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 20, doi: 10.2807/1560-7917.ES.2015.20.50.30097 (2015). [PubMed] [CrossRef] [Google Scholar]
Qiu X. et al.. Ebola GP-specific monoclonal antibodies protect mice and guinea pigs from lethal Ebola virus infection. PLoS neglected tropical diseases 6, e1575, doi: 10.1371/journal.pntd.0001575 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Olinger G. G. Jr. et al.. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America 109, 18030–18035, doi: 10.1073/pnas.1213709109 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Qiu X. & Kobinger G. P. Antibody therapy for Ebola: is the tide turning around? Human vaccines & immunotherapeutics 10, 964–967 (2014). [PMC free article] [PubMed] [Google Scholar]
Audet J. et al.. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus. Scientific reports 4, 6881, doi: 10.1038/srep06881 (2014). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Qiu X. et al.. Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies. Science translational medicine 4, 138ra181, doi: 10.1126/scitranslmed.3003876 (2012). [PubMed] [CrossRef] [Google Scholar]
Varkey J. B. et al.. Persistence of Ebola Virus in Ocular Fluid during Convalescence. The New England journal of medicine 372, 2423–2427, doi: 10.1056/NEJMoa1500306 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
MacIntyre C. R. & Chughtai A. A. Recurrence and reinfection-a new paradigm for the management of Ebola virus disease. International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases 43, 58–61, doi: 10.1016/j.ijid.2015.12.011 (2015). [PubMed] [CrossRef] [Google Scholar]
Kelley B. Industrialization of mAb production technology: the bioprocessing industry at a crossroads. mAbs 1, 443–452 (2009). [PMC free article] [PubMed] [Google Scholar]
McCarthy M. U. S. signs contract with ZMapp maker to accelerate development of the Ebola drug. Bmj 349, g5488, doi: 10.1136/bmj.g5488 (2014). [PubMed] [CrossRef] [Google Scholar]
Fulton A., Lai H., Chen Q. & Zhang C. Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana. Journal of chromatography. A 1389, 128–132, doi: 10.1016/j.chroma.2015.02.013 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Zeitlin L. et al.. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proceedings of the National Academy of Sciences of the United States of America 108, 20690–20694, doi: 10.1073/pnas.1108360108 (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Feldmann H., Jones S., Klenk H. D. & Schnittler H. J. Ebola virus: from discovery to vaccine. Nature reviews. Immunology 3, 677–685, doi: 10.1038/nri1154 (2003). [PubMed] [CrossRef] [Google Scholar]
Wong G. et al.. Ebola virus transmission in guinea pigs. Journal of virology 89, 1314–1323, doi: 10.1128/JVI.02836-14 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
O'Dempsey T., Khan S. H. & Bausch D. G. Rethinking the discharge policy for Ebola convalescents in an accelerating epidemic. The American journal of tropical medicine and hygiene 92, 238–239, doi: 10.4269/ajtmh.14-0719 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Richardson J. S. et al.. Evaluation of Different Strategies for Post-Exposure Treatment of Ebola Virus Infection in Rodents. Journal of bioterrorism & biodefense S1, 007, doi: 10.4172/2157-2526 (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Qiu X. et al.. mAbs and Ad-vectored IFN-alpha therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Science translational medicine 5, 207ra143, doi: 10.1126/scitranslmed.3006605 (2013). [PubMed] [CrossRef] [Google Scholar]
Reddy K. R. et al.. Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C. Hepatology 33, 433–438, doi: 10.1053/jhep.2001.21747 (2001). [PubMed] [CrossRef] [Google Scholar]
Roberts A. et al.. Development and evaluation of an ovine antibody-based platform for treatment of Clostridium difficile infection. Infection and immunity 80, 875–882, doi: 10.1128/IAI.05684-11 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Lepak M. R., Bochenek S. H. & Bush S. P. Severe adverse drug reaction following Crotalidae Polyvalent Immune Fab (Ovine) administration for copperhead snakebite. The Annals of pharmacotherapy 49, 145–149, doi: 10.1177/1060028014555711 (2015). [PubMed] [CrossRef] [Google Scholar]
Mullins M. E., Liss D. B. & Schwarz E. S. Comment: severe anaphylactic reaction following crotalidae polyvalent immune fab (ovine) administration for copperhead snakebite. The Annals of pharmacotherapy 49, 496, doi: 10.1177/1060028015571964 (2015). [PubMed] [CrossRef] [Google Scholar]
Geisbert T. W. & Jahrling P. B. Exotic emerging viral diseases: progress and challenges. Nature medicine 10, S110–S121, doi: 10.1038/nm1142 (2004). [PubMed] [CrossRef] [Google Scholar]
Bray M. & Paragas J. Experimental therapy of filovirus infections. Antiviral research 54, 1–17 (2002). [PubMed] [Google Scholar]
Baskerville A., Bowen E. T., Platt G. S., McArdell L. B. & Simpson D. I. The pathology of experimental Ebola virus infection in monkeys. The Journal of pathology 125, 131–138, doi: 10.1002/path.1711250303 (1978). [PubMed] [CrossRef] [Google Scholar]
Johnson E., Jaax N., White J. & Jahrling P. Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. International journal of experimental pathology 76, 227–236 (1995). [PMC free article] [PubMed] [Google Scholar]
Kortepeter M. G. et al.. Managing potential laboratory exposure to ebola virus by using a patient biocontainment care unit. Emerging infectious diseases 14, 881–887, doi: 10.3201/eid1406.071489 (2008). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Formenty P. et al.. Human infection due to Ebola virus, subtype Cote d'Ivoire: clinical and biologic presentation. The Journal of infectious diseases 179 Suppl 1, S48–S53, doi: 10.1086/514285 (1999). [PubMed] [CrossRef] [Google Scholar]
Emond R. T., Evans B., Bowen E. T. & Lloyd G. A case of Ebola virus infection. British medical journal 2, 541–544 (1977). [PMC free article] [PubMed] [Google Scholar]
Gunther S. et al.. Management of accidental exposure to Ebola virus in the biosafety level 4 laboratory, Hamburg, Germany. The Journal of infectious diseases 204 Suppl 3, S785–S790, doi: 10.1093/infdis/jir298 (2011). [PubMed] [CrossRef] [Google Scholar]
Weber D. J., Fischer W. A., Wohl D. A. & Rutala W. A. Protecting healthcare personnel from acquiring Ebola virus disease. Infection control and hospital epidemiology 36, 1229–1232, doi: 10.1017/ice.2015.205 (2015). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Cenciarelli O. et al.. Viral bioterrorism: Learning the lesson of Ebola virus in West Africa 2013-2015. Virus research 210, 318–326, doi: 10.1016/j.virusres.2015.09.002 (2015). [PubMed] [CrossRef] [Google Scholar]
Kuhn J. H. et al.. Reidentification of Ebola Virus E718 and ME as Ebola Virus/H.sapiens-tc/COD/1976/Yambuku-Ecran. Genome announcements 2, doi: 10.1128/genomeA.01178-14 (2014). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Dowall S. D. et al.. Elucidating variations in the nucleotide sequence of Ebola virus associated with increasing pathogenicity. Genome biology 15, 540, doi: 10.1186/PREACCEPT-1724277741482641 (2014). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Dowall S. et al.. Catheterized guinea pigs infected with Ebola Zaire virus allows safer sequential sampling to determine the pharmacokinetic profile of a phosphatidylserine-targeting monoclonal antibody. Antiviral research 97, 108–111, doi: 10.1016/j.antiviral.2012.11.003 (2013). [PubMed] [CrossRef] [Google Scholar]
Trombley A. R. et al.. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses. The American journal of tropical medicine and hygiene 82, 954–960, doi: 10.4269/ajtmh.2010.09-0636 (2010). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Articles from Scientific Reports are provided here courtesy of Nature Publishing Group
Tags: Ebola antigens


text:
His name was Jay Hanna "Dizzy" Dean
Posted on: October 14,2013
We had a visitor from St. Louis to your Mississippi Sports Hall of Fame and Museum today who was was quite interested in the Dizzy Dean collection, which is housed on our second floor and came to us from the old Dizzy Dean Museum.
Our visitor was perplexed when he saw a plaque that read: "Jerome Herman Dean."
"Who is Jerome Herman Dean?" the visitor wanted to know. "I've always known Dizzy as Jay Hanna "Dizzy" Dean."
Well that's a story worth retelling for those who don't know.
Dean was born — we think — on Dec. 10, 1910, in Lucas, Ark. His parents named him Jay Hannah Dean — Jay for Jay Gould, a 19th century railroad magnate, and Hanna for Mark Hanna, an Ohio political figure of the same era. That we know.
But Dean confused sports writers later on by adopting, unofficially and on occasion, the name Jerome Herman, the name of a former childhood playmate who died when Dizzy was only seven.
Dean added to the confusion shortly after joining the St. Louis Cardinals by telling three different sports writers three different birthdates and three different locations where he was born. All this, he did in a matter of hours.
When asked about why he kept making up stories, Dean explained, "I was helping the writers out….Them ain't lies, them's scoops."
Our Dizzy Dean collection was bequeathed to the City of Jackson by Dizzy's widow, the late Pat Dean, and became part your Mississippi Sports Hall of Fame and Museum when it opened on July 4, 1996.
Dizzy Dean is buried in a small country cemetery out from Bond, near Wiggins. You won't find it unless you are determined, and I was.
2 responses to "His name was Jay Hanna "Dizzy" Dean"
Danny Smith says:
Sara & I were at the museum yesterday also. We heard the guy asking about Dizzy's real name. Missed you at the Duff Dorrough Scholarship benefit Sunday evening at Hal & Mal's. The music was great. You must have been out out of town. 🙂
Van Burnham III says:
Used to watch Diz & Pee Wee on the "CBS Game of the Week" during the late '50s and '60s and was never bored listening to Ole Diz broadcast a game. He used words like "podna," "slud" and catch phrases such as "he's got a beat on it;" "he slud into second; picked himself up and headed for third;" and "that ball was tagged."
During rain delays, Diz would even do his own rendition of "The Wabash Cannonball." And sometimes you might even catch him snoring during a national telecast. His stories about himself and the other old timers like Frankie Frisch, Luke Appling and the old "Gas House Gang" in St. Louis were indeed legendary."
And as college football season approached, ole Diz would always give his favorite team, the Ole Miss Rebels, a plug or two, letting Pee Wee know exactly how bad the Rebels were going to beat up on Kentucky, who Reese followed religiously.
He was a great Mississippian and a great man who lived during the greatest of times in the Magnolia State. Dizzy Dean was the perfect goodwill ambassador for his favorite state, baseball and the human race. Wish he were still around!

redpajama_set_name: RedPajamaCommonCrawl


text:
Police: Stepmom admits giving antifreeze to kids
A detective says an Alabama woman told a fellow jail inmate she poisoned her two stepchildren with antifreeze in an attempt to reduce responsibilities for herself and her common-law husband.
MOBILE, Ala. – A detective says an Alabama woman told a fellow jail inmate she poisoned her two stepchildren with antifreeze in an attempt to reduce responsibilities for herself and her common-law husband.
Mobile Detective Angela Prine testified Monday that Heather Leavell-Keaton admitted the poisonings to another inmate while confined at the Mobile County jail.
She told the inmate that she and common-law husband John DeBlase wanted the children dead because "it would be better if they had less responsibilities," the detective said
At a previous hearing, investigators had said Leavell-Keaton told them DeBlase fed rat poison to the children.
Prine's testimony came in a preliminary hearing on capital murder charges against the 22-year-old stepmother.
Leavell-Keaton's attorney, Richard Horne, said she is functionally blind and not capable of some the things she's accused of doing. But Circuit Judge Rick Stout found probable cause to send the case to the grand jury.
The two children were reported missing in November. The body of 3-year-old Chase DeBlase was found near Vancleave, Miss., on Dec. 8 and the body of 4-year-old Natalie DeBlase was found near Citronelle three days later. Their father is jailed on murder charges.
Prine testified that jail inmate Roseanna Russell was assigned to help Leavell-Keaton because of her blindness and the defendant began talking to her about the case. The detective said Russell learned the couple first poisoned their dog with antifreeze "to find out how long it would take to kill a living thing." Then they started giving antifreeze to the children, who got progressively sicker.
Natalie died March 4, 2010, and they dumped her body, the detective said. They stopped poisoning Chase for a few months but started back because he began crying and asking for his sister in front of others, she testified.
Chase died June 20. On the way to dump his body in Mississippi, they stopped at a video game store to buy two games because they no longer had the responsibilities of the children, the detective said.
If convicted of capital murder, Leavell-Keaton could be executed or sentenced to life in prison without parole.
2 Rescued After Roof Collapses in Massachusetts During Snowstorm
Police: Standoff at Houston-area bank ends
Report Faults V.A. Secretary Shulkin Over Travel to Europe
Officials: 4 Kids, 2 Adults Die in Ohio House Fire
As economy gains strength, Fed weighs what's next
Grace to be replaced on TV show by Simpson judge


text:
Coronavirus metrics
Brown, seeking more in-person instruction, drops coronavirus metrics requirement
Gov. Kate Brown wants more in-person instruction by Feb. 15, and she is letting local officials make the call.
The governor sent a news release Wednesday announcing the coronavirus metrics will be advisory, not mandatory, as of Jan. 1.
The letter directs state agencies to partner with schools to increase in-person instruction and for the Oregon Department of Education and the Oregon Health Authority to update guidances by Jan. 19.
"Our school boards welcome the opportunity to reopen schools safely for our students, staff and communities," said Jim Green, OSBA executive director. "This next step will require close coordination with local health authorities. Above all we want to minimize risks as we return to in-person instruction."
The metrics in "Ready Schools, Safe Learners" set four case rate thresholds for in-person instruction that depend on a county's population. As of Monday, 28 counties were in distance-learning only.
The governor's change would give discretion to local health and education officials to decide the right instruction method for their communities. Some school boards have argued that although their county numbers may be high, they have had few cases nearby.
Medford School Board Chair Jeff Kinsella said the governor's letter is great news but it will put school boards under tremendous pressure to open. Earlier this month, the Medford board sent a letter to Brown asking her to loosen metrics and allow local leaders more say.
"We will follow science, period," Kinsella said.
In a letter to superintendents Wednesday, ODE Director Colt Gill asked schools to move carefully.
"I urge you to make sure your local decision-making process accounts for the fact that updated advisory Health Metrics for Returning to In-Person Instruction may be published later in January," he wrote.
School officials will have to weigh the needs of their students against the dangers of infection in their area.
School leaders have argued for more instruction, especially for student groups who would benefit the most from in-person instruction, including younger learners, students in special education, English language learners, students without adequate internet connectivity, and students who are falling behind.
The Legislature passed COVID-19 liability protection Monday, which will give schools more reassurance to reopen. Schools that follow the state's health and safety protocols will have some protection from lawsuits during Oregon's state of emergency.
Passions for and against in-person instruction are high. Everyone agrees most students learn better in a classroom, but some question whether it is worth the health risk not only to students but also to families and staff.
Students and families have associated increased mental health concerns, inadequate learning, inequitable opportunities and family child care stress with distance learning. On the other hand, students and families are concerned about students or their loved ones becoming sick if students associate too closely, too long and without masks. Studies have shown little increased COVID-19 spread associated with school openings that follow safety protocols.
District leaders must also work with teachers unions. Many teachers are insistent about the need to be face to face with their students, but many teachers are also in the highest risk groups for COVID-19 and fear for their health. Some districts have already faced contract negotiations over in-person instruction working conditions and other coronavirus protocols.
Schools that offer more in-person instruction could face a shortage of teachers. Schools around the state have had to shut down or move to distance learning because of teachers out sick or quarantined, with too few substitutes to replace them.
Brown said teachers should be among the essential workers vaccinated after health care workers, but they won't be required to be vaccinated. Health authorities don't know when those shots might be available, either.


text:
Андре́й Ри́мский-Ко́рсаков:
 Римский-Корсаков, Андрей Владимирович (1910―2002) ― акустик, доктор физико-математических наук.
 Римский-Корсаков, Андрей Николаевич (1878―1940) ― музыковед.
 Римский-Корсаков, Андрей Петрович (1784―1862) ― волынский гражданский губернатор.

redpajama_set_name: RedPajamaWikipedia


If I were to share tips and experiences of blogging with you I wouldn't know where to start.
I have been blogging for nearly 3 years, but have only really started taking it seriously just over a year ago.
There is so much to learn and when I think I have something figured out it changes.
That's sort of just life though isn't it? Nothing stays the same it's always moving.
I think about how much my blog has changed since I first started it. I had an idea, but I wasn't really sure where I wanted to go with it.
It's still changing as I write this. I am hoping to make a big change soon. More on that when I know more about that!
You have to want to do it. You will only get out of it what you put in.
Be prepared to change with new technology and to learn it.
Social media is your friend. Try them all, but stick with the few that really help you. Unless of course you have a team of people behind your blog that can help keep on top of all social media.
notes: This post was written for day 15 of the Blog Every Day in May blogging challenge with Rosalilium. #BEDM I was going to make a cake and post it today, but the sun distracted me and Grimm (I'm so behind on season 2) I googled "free images" and this was the first image so I used it for this post.


text:
Nnnism nmi history books
Prescott and a great selection of related books, art and collectibles available now at. He is a member of the lake worth first church of the nazarene where his father served as the founding pastor. Everyday low prices and free delivery on eligible orders. The purpose of this paper, abridged from the forthcoming new edition of slaying the dragon. Daily telegraphs 100 best novels, 1899 book awards. Nmi is the grassroots mobilizder of passion and prayer and financial support for the ends oftheearth outreach of the church of the nazarene. This column features articles about local history on the day that they occurred. In this exciting and provocative book, emer nolan looks at the relationship between modernism and nationalism, tracing the applicability of alternative notions of nationalism to the various phases of joyces work. Affiliate of the society for human resource management. Nmi is committed to helping local churches tell the missions story. History of the conquest of peru by prescott william h. Cheaney joel belz marvin olasky mindy belz the editors notebook. Abebooks is a global online marketplace for books, fine art, and collectables.
The project brought together specialists in the areas of church history and systematic theology to investigate questions of truth and tradition in light of their varying expertise. We provide resources and support for the local church as it informs and educates its members, not only in what other nazarenes are doing in missions, but how they can get involved, too. Desmond multiple formats at fifty famous stories retold, by james baldwin illustrated html at gateway to the classics filed under. The council has supported a the publication of several books, both under its ongoing micronesian authors initiative and through. We facilitate the sale of books, art, and collectables by connecting sellers with buyers all around the world. Money medicine lifestyle technology religion media health sports education politics history immigration law. The neurotic constitution 1921 edition open library. Welcome to the nmi museum of history and culture, containing one of the largest collections of the life and culture of the people of the island, both past and. Displayed items included candle holders, altar lamps, bells, basins, old books and illustrations, and an early twentieth century chair made of narra. The formation of the northern mariana islands, the prehistoric period, the spanish period, the german period, the japanese period, trust territory of the pacific period, united states commonwealth, the northern mariana islands and world war two.
Which two statements explain the basic tenets of christianity. Featured texts all books all texts latest this just in smithsonian. This collection of essays engages the basic themes of the fiveyear, joint research project goa the normativity of history. Then you can start reading kindle books on your smartphone, tablet, or computer no kindle device required. The human condition in legends of the black book minister. Much of their history is reflected in their craft production, such as textiles, beaten gold artefacts and above all funerary pottery, in which they used as many as seven colours, whose. The nbn sales force has years of experience and has honed this handson experience to gain access to the major retailers. The author assumes a fair amount of knowledge from the reader, from the four books to yin and yang.
Lyons talked about the national book network which acts as a middleman between publishers and booksellers. Nov 15, 2010 open library is an initiative of the internet archive, a 501c3 nonprofit, building a digital library of internet sites and other cultural artifacts in digital form. The nicu network neurobehavioral scales nnns is a tool for the comprehensive examination of normal fullterm infants, preterm infants, and infants at risk because of prenatal substance exposure. Report video issue javascript must be enabled in order to access cspan videos. Northern mariana islands history sources bibliography an annotated bibliography of german language sources on the mariana islands with links to many online sources, by dirk h. Rowling, the secret history by donna tartt, sex in the title. Northern mariana islands islands, pacific ocean britannica. It also has several of its publications on south australian history for sale. Brief history or story of the nazarene missions international nmi brief history of nmi. With fascinating accounts of the lives of seven prominent officials, he fashions a remarkably vivid portrayal of the court and the ruling class of late imperial china. David quinns career in the book business began as a sales clerk at a little professor book center 40 years ago. Yet in the seemingly unspectacular events of this ordinary year, ray huang finds exemplified the roots of chinas perennial inability to adapt to change.
Academic journal article scandinavian studies between sin and salvation. Apr 15, 2016 when you are planning work on a new musical, use the structural measuring sticks above to test your idea before it is fully formed. The nazca mystery by malatesta, parisina unesco courier. Farrells book has been widely used for years in the cnmis public schools and this version will bring its history up to the present day. The story of the greatest nations, from the dawn of history to the twentieth century. Sales services for publishers national book network. Show me the heroes that the youth of your country look up to, and i will tell you the future of your country. Help with reading books report a bad link suggest a new listing.
Search the history of over 424 billion web pages on the internet. Much of their history is reflected in their craft production, such as textiles, beaten gold artefacts and above all. Buy the naxalite movement in india book online at low prices. Conflict of northern and southern theories of man and.
Northern mariana islands history sources filed under. There isnt another gateway out there that has more integrations or a better support team. At the global level, the nmi holds an international convention in conjunction with the quadrennial once every four years nazarene general assembly. Flinders ranges research provides historical information and research services on both south australia and the northern territory. In politics, centrism is a political outlook or specific position that involves acceptance or support of a balance of a degree of social equality and a degree of social hierarchy, while opposing political changes which would result in a significant shift of society strongly to either the left or the right. History of the conquest of peru by prescott william h abebooks. If you would like to get a free pdf copy of the books, click here.
The postluciferian world by kerth barker when i was a young man i knew some members of the secret society that is now generally referred to as the illuminati. Selling is a special skill, and selling books requires a unique understanding of a distinctand constantly changingmarket. Christian books on nt church history apostolic period. Buy the naxalite movement in india book online at low. World war ii history books 1944 hachette book group.
Discover delightful childrens books with prime book box, a subscription that delivers new books every 1, 2, or 3 months new customers receive 15% off your. Free kindle book and epub digitized and proofread by project gutenberg. Although narcotics anonymous na was one of the earliest adaptations of the aa program, na remains less wellknown among addiction professionals. The history of addiction treatment and recovery in america, is to provide an overview of the. The knot that binds power and knowledge communication and society new ed by jansen, sue curry isbn. Christianity was founded by jesus and his followers. Find the best commentary on nt church history apostolic period. He was on the general council for nazarene missions international for 8. This day in nmi history northern marianas humanities council. In 1587, the year of the pig, nothing very special happened in china. Partnering with nmi has made my job easier, which has made me more efficient and led to increased revenue. In politics, centrism is a political outlook or specific position that involves acceptance or support of a balance of a degree of social equality and a degree of social hierarchy, while opposing political changes which would result in a significant shift of society strongly to either the left or the right both centreleft and centreright politics involve a general association with centrism.
History of the mission in the mariana islands 16671673. The nmi museum of history and culture, also known as the nmi museum, is a museum in. Introduction to nt christology paperback 18 aug 1994. District nmi president southern florida district church. The vast majority of nmi chamorros who were sent to guam to work for the. History of the northern mariana islands farrell, don a on. Tracy sahib, prelude and introduction 042020 by clyde park church of the nazarene. North korean leaders rewrite history at their own will.
If you need further assistance, please contact your sales rep text search. The political economy of communism by janos kornai and a great selection of related books, art and collectibles available now at. Sandra jowersbarber, an assistant professor of history and the director of the history program in the department of urban affairs. Used books may not include companion materials, some shelf wear, may contain highlightingnotes, may not include cdrom.
Nmi manufacturing supports the established semiconductor manufacturing and electronic systems manufacturing communities facilitating activity such as knowledge exchange, increased productivity, cost reduction, quality improvement and dedicated supplier forum. As a seller on abebooks you describe, store, and ship your items yourself. History of efforts to reunify the mariana islands guampedia. The basic text search looks for the terms specified within the title, isbn, subtitle, and description fields. Commonwealth of the northern mariana islands, northern marianas. Milestones in ninr history national institute of nursing. The term naxalites comes from naxalbari, a small village in west bengal where a section of the communist party of india marxist cpim led by charu majumdar, kanu sanyal, and jangal santhal initiated a violent uprising in 1967. Jul 31, 2011 the complicated history of deaf education. Buy the naxalite movement in india book online at best prices in india on. The fourmonthlong 1944 battle on italys coast, south of rome, was one of world war iis longest and bloodiest battles. When you are planning work on a new musical, use the structural measuring sticks above to test your idea before it is fully formed. New books continue to be added on a regular basis, so be sure to bookmark this site for your future browsing convenience.
The complete history of the commonwealth of the northern mariana islands. How every african can live the life of their dreams. Northern mariana islands history include extended shelves you can also browse an alphabetical list from this subject or from. Its basically the mechanism by which people with old money manipulate the world to their advantage. Nazarene missions international church of the nazarene. Organized in 1908, the denomination is now home to about 2. Nmi history book to get updates saipan news, headlines.
James joyce and nationalism comprehensively revises our understanding of joyce by reexamining his writing against irish nationalism. Free networking books download online networking ebooks. The story of the greatest nations, from the dawn of history. Forty history books for 40 or more evenings of edification. Dennis has served as the southern florida district missions president since 1986. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Sharethe second set of the history of the northern marianas islands by don farrell is in the works and should be published around summer of next year, according to education commissioner dr. Featured texts all books all texts latest this just in smithsonian libraries fedlink us genealogy lincoln collection. Common knowledge book awards daily telegraphs 100 best novels, 1899.
1152 456 767 1342 1492 1341 64 1346 296 47 906 812 631 1047 754 1016 442 1524 1075 1488 864 1623 426 789 1120 1054 584 1060 1014 1420 59 646 176 51 1418


I don't have much to say about this adventure. After a few hours of helping out at Susan Mains Gallery at the Spiceland Mall we ventured over to the Grand Anse beach to wind down. Drinking rum and watching the sunset is the perfect way to end a day.
Today I leave for Grenada and couldn't be more excited. For the next few months I will be exploring the country, eating chocolate, avoiding bugs, and making art. Follow me here and on Instagram (@amy_on_the_island) to see updates on what I am making and what this amazing country has to offer.


text:
Loose Lips Daily: Brown Needs to Transition Too Edition
by Alan Suderman November 9th, 2010 September 21st, 2020
As much local politics as humanly possible. Send your tips, releases, stories, events, etc. to lips@washingtoncitypaper.com. And get LL Daily sent straight to your inbox every morning!
IN CASE YOU MISSED IT:
Gray Camp: Now Hiring
Hey, You Should Run for Council
Good morning sweet readers! Boy, did it ever get dark early yesterday. LL feels like he's back in Alaska. News time:
A Team of Lobbyists: Almost Council Chairman Kwame Brown unveiled his boating club transition team yesterday to a pesky press corps who wanted to know why the team was so lobbyist-heavy, why a transition could cost $150,000 (which will come from private donors), and why Brown even needs a transition team in the first place. "Brown said Monday he will use his eight-week transition period to conduct a top-to-bottom review of council operations. Brown said he wants to 'improve the way the council does business' and to ensure that the government is more 'open and responsive to residents,'" reports the Post's Ann Marimow. Brown announced his transition would be led by Gregory McCarthy, vice president for government affairs for the Washington Nationals. McCarthy, who was a deputy chief of staff to former mayor Anthony Williams, said he is coming up with recommendations that will serve as a "blueprint not a dust collector" for Brown's tenure. … Brown said he would not accept public funds to cover the cost of the transition and instead plans to raise up to $150,000 in private donations. Brown said he would accept individual contributions of up to $25,000 and disclose the names of donors after the transition period. District law prohibits contributions of more than $2,000 to a political candidate, but the city's campaign finance regulations do not cover the transition period. Brown, an at-large council member first elected in 2004, said the money would pay for a Web site, research and travel. He said he plans to meet with the leaders of city councils in Los Angeles and Atlanta. Other transition team members include Walter Smith, head of the nonprofit public policy organization DC Appleseed; Jeffery Humber, past chairman of the board of the Washington Convention Center Authority; Rod Woodson, a lobbyist who was general counsel to the city's Housing Finance Agency; and Brown's campaign manager and chief of staff, Irma Esparza Diggs." As for who gets which committee assignment, Brown's only giving boilerplate "everything is on the table" answers for now, though just for fun he should put Marion Barry in charge of the finance committee, to see Jack Evans' reaction. See WAMU as well.
AFTER THE JUMP: More Budget Pain; No Answers on DC9; Special Election Madness Begins …
Here Comes the Pain Again: Props to WUSA9 for this headline: "DC, Get Ready for the Pain." In the accompanying story, Bruce Johnson, reveals that Almost Mayor Vince Gray and CFO Natwar Gandhi were in meetings yesterday trying to figure out how to close the city's $175 million budget gap.
Still No Answers: Protesters went to the Wilson Building yesterday to express their outrage over the dropped murder charges in death of Ali Mohammed outside DC9. "One of the protesters, Mohammed's cousin, Nunu Wako, told reporters, "We will never rest until we get justice." Later on, Wako held up a sign that displayed a photograph of a blood-spattered crosswalk that was taken the day her relative died. "Whose blood is this?" the sign asked. Originally, cops and prosecutors contended that the five DC9 men once scheduled to have a preliminary hearing today were guilty of murder. Cops said a witness told them the men punched and stomped Mohammed for vandalizing the club. The charges were eventually downgraded to aggravated assault, though, and have now been dropped all together," says WCP's Rend Smith. MyFoxDC has a round up of the whole saga.
Special Madness: Five folks have picked up the needed packets to be eligible to win Democratic State Committee's pick to replace Brown on the council for a few months before a special election is held, reports Mike DeBonis. "They are Sekou Biddle, the incumbent Ward 4 representative on the State Board of Education; Vincent Orange, the former Ward 5 council member whose campaign for the chairmanship fell short; Jacque Patterson, a Federal City Council staffer who also chairs the Ward 8 Democrats; and Kelvin Robinson, the former chief of staff to Mayor Anthony A. Williams who ran unsuccessfully for the Ward 6 seat this year. … Stanley Mayes, a Ward 1 lawyer, has also picked up petitions." DeBonis also reports that the general counsel of the Board of Elections and Ethics says Councilmember Michael A. Brown can't switch his party affiliation from an independent to a Democrat and automatically have Kwame Brown's seat.
It's So Hard, To Say Goodbye, To Yesterday: WBJ's Michael Neibauer has the news that Fenty's staffers have been given instructions on how to resign from their jobs. "It's traditional that one mayor's appointees and top hires resign ahead of the next mayor's arrival. But this news makes it so, well, real, that Mayor Adrian Fenty's brief reign is quickly coming to an end. Hundreds of employees will soon be out of a job. … The human resource documents, essentially walking papers, direct certain employees to submit resignation letters, at the request of the mayor, effective Dec. 31. They describe, said one recipient, what happens if the resignation is accepted, when the last check goes out, how long insurance benefits remain in place, options for continued medical coverage and what happens if the employee accepts a new government job after Jan. 1."
D.C. settles for $10 million for a sickening story of foster care abuse.
Teen in Brian Betts murder to plead guilty, that case led to DYRS overhaul.
Could a Circulator bus have a stop right outside your front door in the future?
Big deal, LL bikes 30.
What could be more American than the National Mall and Coca-Cola?
Council: Legislative meeting. Final vote schedule on wildlife protection act. 11 a.m.


text:
Business Bus drives success in Coffs Harbour and Woolgoolga
Media release | 2 May 2019
The NSW Government's Business Bus is visiting communities across NSW to provide advice to business owners looking to become more productive and profitable.
Small business owners in and around Coffs Harbour and Woolgoolga can hop on board the bus this week for trusted advice from a Business Connect advisor or a quick business health check.
Department of Industry Executive Director Trade and Investment NSW Kylie Bell said that the Business Bus had visited 100 communities across the state in the last year.
"The Business Bus is part of the NSW Government's multi-million dollar Business Connect program which supports small businesses to thrive in metropolitan and regional economies," Ms Bell said.
"The Bus travels all over NSW to let small business owners know about the NSW Government support available to help them start and grow.
"Visiting the Business Bus can help a business operator take the vital next step, whether they're just starting out, wanting to ensure their business is sustainable or aiming to go global.
Ms Bell said businesses can access guidance on everything from planning the next stage of your business to making the most of your marketing or using new digital tools.
"We are investing $30 million in Business Connect, with 70 per cent of services to be delivered to regional businesses," Ms Bell said
"There are 765,000 small businesses across NSW – and they're the lifeblood of our State's, regional and local economies," she said.
The Business Bus will visit the following locations between 9am and 3pm:
Coffs Harbour – City Square, Coffs Harbour (Between Harbour Drive and Park Ave) on Thursday 9 May
Woolgoolga – 56 Beach St, Woolgoolga (in front of NAB) on Friday 10 May
Visit www.industry.nsw.gov.au/businessbus to find out more and reserve your spot on the Business Bus or call 1300 134 359.
Page link: https://www.industry.nsw.gov.au/media/releases/2019/business-bus-drives-success-in-coffs-harbour-and-woolgoolga


This tour is guided in max. 3 languages.
The Seegrotte is not suitable for people with limited mobility.
On this bus tour you travel outside of Vienna to the southern part of the Vienna Woods, passing the former Roman spa town of Baden and driving through the romantic Helen's valley. Enjoy the ride in our comfortable coach while outside the beautiful landscape passes by on our way to Mayerling.
Our first stop on this bus trip is the memorial chapel built on the site of the former imperial hunting lodge of Mayerling. In 1889 the tragedy of crown prince Rudolf and his mistress Baroness Vetsera took place at the lodge. Learn more about these fateful events during a tour through the memorial chapel and the adjoining showroom.
Afterwards we visit the Abbey Heiligenkreuz, which was founded in 1133 and still today, it is an active monastery. On the tour we show you the medieval cloister and the burial place of the former Babenberger rulers. This dynasty was the oldest in Austria and shaped its history throughout many centuries.
Continuing on our trip we pass the Höldrichsmühle, once an old mill where the composer Franz Schubert created his music. Last stop on our bus tour is the Seegrotte Hinterbrühl, where Europe's biggest underground lake awaits you. By boat we travel through the mystically lighted galleries. The green shimmering water and the rough stonewalls add to the very special atmosphere.
At the end of our half-day trip to the Vienna woods we take you back to Vienna by bus. Final stop is at the Vienna State Opera.
96 % of reviewers liked this tour.
Nicely "packed" tour highlighted beautiful and famous sightseeing points.
I am satisfied with the tour program and the price.
Great tour with the guide giving us extra backstory on the sites. The places we visited were quiet, beautiful and had interesting history to them.
As a bonus, they also took us to a drive around the small village near Vienna. It was a real treat.
Really enjoyed this tour. All the places visited were interesting and Thomas explained everything in great detail. Know a lot more history than before. The little boat trip was a nice ending. I would definitely recommend this trip to others.
Need a little more time to see the museum in Mayerling and need to warn people that exact change is needed for the restrooms at the grotto.
Nessun rischio: tutto chiaro e semplice!
Buena opcion para ver cosas diferentes en viena.
It was very well organised!
Nice trip to Vienna woods, we have visited Mayerling and Heiligenkreuz abbey, which is beautiful, then had a visit to the former gypsum mine where an undeground lake has been formed. The boat trip is very short but quite nice. Good guide who fave us a lir of details about the region etc.


From a PR to a bad run.
On Saturday, I ran in a 5k race and ran my fastest 5k yet. It was a great run! Yesterday, I was supposed to run 12 miles. It was a hot day yesterday, and I didn't get out until after 10:00. I could tell as soon as I got through the first couple of miles… Continue reading From a PR to a bad run.
Yesterday I ran in our local Run for the Schools 5K. It's the first 5K race that I've done since last November. Although I run 5K's all of the time in my training programs, there's something a bit different when you run in an actual race, isn't there? I wasn't nervous at all. Since the… Continue reading New 5K PR!
I don't know about all of you, but I've always been the type of person to overthink and overanalyze things. When I first started eating a vegan diet, I drove myself crazy researching all of the combinations of food I would need to eat in order to get in all the vital nutrients and vitamins. … Continue reading Keep it simple.
I knew I was a runner when….
When did I know that I was a runner? Was it when I first decided to try to run instead of walk during one of my daily walking routines? Was it when I spent $100 on my first pair of running shoes? Was it when I was able to run my first mile without stopping?… Continue reading I knew I was a runner when….


text:
Vietnamese alumni: Poland will remain in our hearts forever
VNF - The Vietnam-Poland Friendship Association (VPFA) and the club of the Vietnamese alumni of Polish universities on November 4 organised a meeting to mark the 50th anniversary of their first day in Poland in 1968.
At the meeting in Hanoi.
Nearly 100 alumni from all over the country presenting in the gather recalled their meaningful time on the European country.
Polish Ambassador to Vietnam Wojciech Gerwel and representatives of the Polish Embassy in Vietnam; and a delegation of the AGH University of Science and Technology in Krakow (AGH UST) led by its rector, professor Tadeusz Słomka, also attended the meeting.
In a cozy atmosphere, Polish university graduates in the event had a nice time sharing their unforgettable memories of studying and working in Poland.
Opening the meeting, Pham Khoi Nguyen, VPFA chairman and former Minister of Natural Resources and Environment, said that Vietnam and Poland share a number of similarities in history, love for peace, and sacrifice for national independence and sovereignty.
Between 1960 and 1990, Poland trained over 4,000 Vietnamese students and scientists, and over 3,500 labourers, mostly in the coal and shipbuilding sectors. Many had came back home to build the nation, he said, adding that they have served as a bridge to promote the Vietnam-Poland friendship.
He highlighted the capability and skills of those who received training in Poland thanks to the European country's high-quality education system.
VPFA chairman, Pham Khoi Nguyen studied at AGH UST Faculty of Geology and Location (currently Faculty of Geology, Geophysics and Environmental Protection) between 1969 and 1974.
Currently, Poland grants some 20 scholarships to excellent Vietnamese students each year, most of which are in philosophy, health care, geology, technology, agriculture, information and technology, and graphics, among others, he underlined.
Polish Ambassador to Vietnam Wojciech Gerwel, for his part, thanked VPFA for their support for the Polish Embassy in successfully implementing many projects between Vietnam and Poland, contributing to the enhancement of mutual understanding, friendship and cooperation between the two countries.
He expressed his belief that the traditional cooperation with Vietnam will develop in a stronger manner, helping build a prosperous future in each country.
AGH UST Rector professor Tadeusz Słomka said he is pleased to meet Vietnamese alumni who have made great contributions to the bilateral friendship and hoped that they will continue to bring the Poland closer to the Vietnamese people.
Established in 1919, the Institute is one of the best and most renowned modern Polish universities. For many years it has been ranked at the top of the list of institutions of higher education. AGH UST is a leading Polish university in modern technologies, and belongs to a group of prestigious international educational centres.
Currently, the list of Vietnamese graduates of AGH UST includes approximately 300 names. In 2009, Hanoi-based AGH Alumni Club was founded and in 2015 AGH UST in cooperation with the Polish-Vietnamese Friendship Association released an official publication on Polish-Vietnamese friendship and Vietnamese graduates of AGH UST.
AGH UST is actively pursuing cooperation with Vietnam – so far the University has signed general agreements with nine Vietnamese research institutions, in areas such as biomedicine, mechatronics, IT, underground construction, ecology, geotourism, geodesy, mining as well as management and humanities.
Vietnamese graduates of AGH UST received the "golden school report".
Next year will mark the 100th Inauguration Ceremony of the Academic Year. On the occasion, AGH UST presented the "golden school report" to 55 Vietnamese students who graduated of the university 50 years ago./.
Translated by Chau Pham


text:
I Bewitched sono gruppo musicale heavy/black metal svedese fondato nel 1995 a Umeå.

Formazione

Formazione attuale
Kristoffer "Wrathyr" Olivius - voce, basso
A. Hellfire - chitarra
Marc Malice - batteria

Ex componenti
Marcus "Vargher" Norman - voce, chitarra
Fredrik Degerström - chitarra
Anders Nyström - voce, chitarra
Reaper - batteria
Stormlord - batteria

Discografia

Album in studio
1996 - Diabolical Desecration
1997 - Pentagram Prayer
1999 - At the Gates of Hell
2002 - Rise of the Antichrist
2006 - Spiritual Warfare

Live
1997 - Hell Comes to Essen

EP
1996 - Encyclopedia of Evil
2004 - Atrocities in A-Minor

Note

Collegamenti esterni


MUCH ADO ABOUT NOTHING – DRAGONS!!!
Yes you heard right! Tickets are on sale for the 2014 production of "Much Ado About Nothing/Dragons". Join us for our 7th annual Shakespeare at the Tanks Arts Centre. Follow the link and visit us in our dragon auction house.


SHINY Collection: Choose your color!
Our Shiny Collection is inspired by the colors of the Universe.
They shine like moon, planets and stars.
An iconic accessory that evokes the image of the halo: the shiny circle on head seen in many paintings.
Made in numerous sumptuous velvets, they are also available for bespoke hand dyeing. Contact us to discuss the details.


All the way from their first tour in 2011 until their Hiatus in 2015, I toured and performed all over the globe with the worldwide phenomenon.
The tours spanned the world's biggest Stadiums and Arenas such as Wembley Stadium and Madison Square Garden.
My time with them led me to perform on numerous international TV shows and Award nights including Saturday Night Live, The Late Late Show and the X Factor.
Here are just a few select video highlights from my time with One Direction.
Work began with the talented, soulful, singer-songwriter in Autumn 2017, joining him for his UK and European tours, together with a mass of festival and TV appearances.
My first venture into featured artistry. This stop motion Lego music video was created for Wheatus' song Only You featuring both One Direction drummer Josh Devine and myself.
Copyright Sandy Beales 2018. All Rights Reserved.


The D.C. Department of Health is seeking to initiate a multimillion-dollar, federally funded voucher program that would allow city residents to choose where they receive drug and alcohol recovery treatment.
The Access to Recovery program, approved by Congress last year, would give residents vouchers to receive treatment at any facility that meets federal and city standards, including faith-based centers.
Mr. Johnson said the Health Department is applying for funds through a $100 million federal grant included in President Bush's voucher-based substance-abuse treatment proposal.
City health officials say the District could receive $15 million over three years through the program.
Officials say the fact that the city already has its own locally funded vouchers-based treatment system will boost the District's chances of getting federal funding.
"The District is one of the few places in the country with a system already in place," said Wendy Salaam, chief of policy for addiction, prevention and recovery administration in the Health Department.
The D.C. Council passed the choice-in-drug-treatment legislation in 2000, but the city has had some difficulties running the program, according to Rob Fleming, publisher of Recovery Works, a newsletter for recovering substance abusers in the city.
Mr. Fleming said the District has a good chance of winning the federal grant because of the existing program. "We are better prepared to use the money than most places," he said.
However, Mr. Fleming said federal officials should make sure that the city spends the voucher money on substance-abuse programs. "My concern is that the money goes through the chief executive's offices, and I'm worried that they'll say here's this money for treatment from the federal government so therefore we can take money away from local sources," he said.
Leah Young, a spokeswoman for the federal Substance Abuse and Mental Health Services Administration, said the federal agency would award grants to states by Sept. 30.


text:
Valverde, Movistar and Spain receive WorldTour trophies
By Cycling News published 17 November 14
Cookson presents trophies in Madrid ceremony
Movistar manager Eusebio Unzue and UCI president Brian Cookson (Image credit: Movistar)
Brian Cookson hands Alejandro Valverde his award (Image credit: Movistar)
The winners, Eusebio Unzue, Alejandro Valverde and Jose Luis Lopez with UCI president Brian Cookson (Image credit: Movistar)
Nairo Quintana also made an appearance to collect the team prize (Image credit: Movistar)
Movistar, Alejandro Valverde and Spain have officially been awarded their trophies for their respective victories in the WorldTour. In a ceremony at the headquarters of team sponsor Telefonica in Madrid, UCI president Brian Cookson was on hand to dole out the awards to Valverde, Movistar boss Eusebio Unzué and Royal Spanish Cycling Federation chairman José Luis López. Giro d'Italia winner Nairo Quintana was also on hand to accept the plaudits.
Unzue: Quintana has the same strength of character as Hinault
Quintana happy to be to his best at Giro d'Italia
Valverde takes sixth medal in World Championships
Valverde, Movistar and Spain top final WorldTour rankings
It is the second year running that Movistar have claimed the top spot in the team rankings. It went down to the wire in 2013 with the Spanish outfit nicking the victory from Team Sky at the final WorldTour event of the year, the Tour of Beijing. Heading out to China this year, the team already had it wrapped up and ended the season with a 228-point advantage over their nearest competitor BMC Racing.
"It's a big recognition for all of us: not only the riders, a marvellous roster which allows us to claim such important victory and, most especially, to take cycling in our country back to the place it deserves, but also the staff, all people into our team whose efforts lead us to the top," Unzué said in a team press release.
Valverde beat his compatriot Alberto Contador for the rider's classification. The 34-year-old took the majority of the team's victories throughout 2014, including success at Roma Maxima, Fleche Wallonne and a third place finish at the Vuelta a España. It was his second place finish at Il Lombardia that boosted him into the lead with Contador failing to pick up any points.
"It's been undoubtedly a great season, and a proof of that is we got this prize that rewards the best throughout the season," Valverde said. "It could have been even better with minor improvements, like the Tour podium, but at the end, I gave everything I had on my body and you can't really ask for anything else. Though it's difficult to repeat this season's success, there are goals we didn't achieve yet and we will fight to conquer them in 2015."
Spain has dominated the nations' classification since the WorldTour was brought in for the 2009 season. Italy's victory in 2011 is the only time that they have not taken home the award.