Accepted Manuscript Development of therapeutics for treatment of Ebola virus infection Haoyang Li , Tianlei Ying , Fei Yu , Lu Lu , Shibo Jiang PII:

S1286-4579(14)00311-6

DOI:

10.1016/j.micinf.2014.11.012

Reference:

MICINF 4228

To appear in:

Microbes and Infection

Received Date: 12 November 2014 Revised Date:

25 November 2014

Accepted Date: 28 November 2014

Please cite this article as: H. Li, T. Ying, F. Yu, L. Lu, S. Jiang, Development of therapeutics for treatment of Ebola virus infection, Microbes and Infection (2015), doi: 10.1016/j.micinf.2014.11.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Development of therapeutics for treatment of Ebola virus infection

1 2

Haoyang Li a, Tianlei Ying a, Fei Yu a, Lu Lu a,*, Shibo Jiang a,b,*

3

RI PT

4 5

a

6

Fudan University, 130 Dong An Rd., Xuhui District, Shanghai 200032, China;

7

b

8

10065, USA.

Key Lab of Medical Molecular Virology of MOE/MOH, Shanghai Medical College,

M AN U

SC

Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY

9

*Corresponding author: Key Laboratory of Medical Molecular Virology of Ministries

11

of Education and Health, Shanghai Medical College, Fudan University, Shanghai

12

200032, China. Tel.: +86 21 54237673; fax: +86 21 54237465.

15

16

E-mail address: [email protected] (S. Jiang),

EP

14

[email protected] (L. Lu)

AC C

13

TE D

10

ACCEPTED MANUSCRIPT 17

Abstract Ebola virus infection can cause Ebola virus disease (EVD). Patients usually show

19

severe symptoms, and the fatality rate can reach up to 90%. No licensed medicine is

20

available. In this review, development of therapeutics for treatment of Ebola virus

21

infection and EVD will be discussed.

SC

22

RI PT

18

Keywords

24

Ebola virus; Ebola virus disease; therapeutics; treatment; antibody; small-molecule

25

compounds

M AN U

23

AC C

EP

TE D

26

ACCEPTED MANUSCRIPT 27

1. Introduction Ebola virus disease (EVD), which is caused by infection with Ebola viruses

29

(EBOVs), has existed as an endemic infectious disease sporadically occurring in

30

Central Africa since it first appeared in 1976 [1, 2]. At present, people along the West

31

African coast, especially in Guinea, Liberia, and Sierra Leone, are going through the

32

largest, most severe, and most complex Ebola outbreak [3-6]. Indeed, the United

33

States and European countries have reported domestic infection cases, and the

34

epidemic situation may last into next year and spread to other countries, according to

35

estimates of WHO [7] and a computational epidemic prediction from Northeastern

36

University in the USA [8]. As of November 2, 2014, more than 13000 people have

37

been confirmed with, or suspected of, contracting the disease in the present epidemic,

38

out of which about 4818 have died [9]. Ebola viruses transmit through direct contact

39

with infectious bodily fluids, such as blood, sweat, saliva, and tears, from EVD

40

patients or wild animal carriers, such as nonhuman primates (NHPs) [10, 11], and the

41

incubation period is 2 to 21 days [10, 12]. In the early stages, EVD patients usually

42

show symptoms like fever, intense weakness, muscle pain, and headache, while both

43

internal and external bleeding, as well as kidney and liver dysfunction, will arise as

44

the course of EVD progresses [10, 12]. The fatality rate of EVD is 40-90%, according

45

to the historical analyses of Ebola outbreaks [10]. Although EVD is considered a

46

potential public health threat, no licensed drug or vaccine is currently available

47

[13-15]. The most efficient measure for controlling disease propagation is insolation

48

of patients and strict barrier nursing procedures to protect healthcare workers.

AC C

EP

TE D

M AN U

SC

RI PT

28

ACCEPTED MANUSCRIPT Meanwhile, symptomatic and supportive treatment is the sole choice for patients

50

suffering from EVD [10, 16]. However, based on the fundamental research of EBOVs

51

and EVD, several promising drugs and vaccine candidates [6, 17] are under

52

development. These therapeutic treatments will be compared and discussed in this

53

review article.

RI PT

49

Ebola viruses, which belong to the family Filoviridae, are classified into five

55

species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Bundibugyo

56

ebolavirus (BEBOV), Tai Forest ebolavirus (also known as Cote d’Ivoire ebolavirus,

57

CIEBOV), and Reston ebolavirus (REBOV) [2, 18]. ZEBOV and SEBOV are

58

predominant and more pathogenic than the others, as they have been historically

59

associated with about 90% of EVD outbreaks and higher mortality [2, 10]. The

60

causative agent of the current outbreak is also a variant strain of ZEBOV [19, 20].

TE D

M AN U

SC

54

EBOVs form a threadlike shape, with a uniform diameter of 80 nm [21], and the

62

typical length of a virion with peak infectivity is about 1,200 nm [22]. One Ebola

63

virion consists of a nonsegmented, single-stranded negative-sense RNA genome, and

64

seven kinds of viral proteins serve as structural or multifunctional proteins [2]. Major

65

nucleoprotein (NP) and virion protein 30 (VP30, minor nucleoprotein) are associated

66

with the RNA genome and are required for RNA encapsidation [23, 24], while VP30

67

is also a viral transcription activator [25]. Like phosphoproteins of other

68

minus-stranded RNA viruses, virion protein 35 (VP35) links NPs with the viral

69

RNA-dependent RNA polymerase (RdRP, polymerase L) to construct the viral RNA

70

synthesis complex for transcription and genome replication [26, 27]. The matrix

AC C

EP

61

ACCEPTED MANUSCRIPT proteins, virion protein 40 (VP40) and virion protein 24 (VP24), which have specific

72

affinity for membranes, play essential roles in the process of virus assembly and

73

budding [28]. Glycoprotein (GP) spikes, which embed on the virion surface, mediate

74

virus entry [29] (Fig. 1). The GP gene also encodes soluble GP (sGP) and small

75

soluble GP (ssGP), which are secreted from the host cell [2]. After being synthesized,

76

the glycoprotein precursor (GP0) is cleaved by furin enzyme into GP1 and GP2 (GP2

77

has a transmembrane domain), which are further modified to form a heterodimer in

78

the Golgi apparatus, and three of these dimers constitute a functional GP tripolymer

79

spike. GP1 contains an excessively O-linked glycosylated mucin-like domain and a

80

heavily N-linked glycosylated glycan cap domain, and these exterior domains are

81

responsible for binding with a variety of host cell surface factors, as well as covering

82

the receptor binding domain (RBD) under them [30]. The specific or nonspecific

83

interactions between GP1 and cell surface host factors, such as T-cell immunoglobulin

84

mucin domain-1 (TIM-1), facilitate virus attachment and endocytosis without

85

changing the conformation of GP trimers [31]. While the whole virion is endocytosed

86

and transported into mature endosome, GP1 is cleaved by endosomal proteases

87

Cathepsin L and B (CatL/CatB) to remove the hyperglycosylated region [32, 33].

88

Then the exposed RBD interacts with endosomal lumen receptor Niemann-Pick C1

89

(NPC1) to transform the conformation of GP1 and GP2 at low pH [29]. Meanwhile,

90

three fusion peptides on the N terminal of GP2 trimer insert into endosomal

91

membrane, launching the six-helix bundle (6-HB) formation between the N- and

92

C-terminal heptad repeats (NHR and CHR, respectively) and viral-host cell

AC C

EP

TE D

M AN U

SC

RI PT

71

ACCEPTED MANUSCRIPT membrane fusion, in a manner similar to that mediated by other type I viral membrane

94

proteins [34, 35] (Fig. 2). Sequentially, the genome and RNA synthesis machinery is

95

released into cytoplasm for another cycle of transcription, protein translation, genome

96

replication, and virion assembly [2] (Fig. 3).

RI PT

93

Notwithstanding that Ebola viruses have the replication tropism of a large range

98

of cell types like hepatocytes, kidney cells and other epithelial cells, it is believed that

99

EBOVs prefer to use mononuclear cells in the early stage of infection, such as tissue

100

macrophages, monocytes and dendritic cells, for rapid virus replication [2, 36]. This

101

kind of massively unchecked replication is mainly because of the viral proteins that

102

antagonize the host interferon response. VP24 interferes with the expression of

103

IFN-stimulated genes by preventing dimerization of STAT, while VP35 keeps the viral

104

dsRNA away from RIG-I and Dicer and inhibits the activation of other anti-viral

105

responsors in host cell, such as IRF-3, IRF-7 and dsRNA-dependent PKR [37]. At the

106

same time, cytokines released from infected cells recruit more mononuclear cells to

107

the initial infection site, in turn amplifying infection and apoptosis of mononuclear

108

cells. At the same time, virions are systemically spread through blood circulation. The

109

quantitative and functional loss of dendritic cells and macrophages causes acute

110

lymphocytic apoptosis, although Ebola viruses cannot infect lymphocytes

111

productively. During the middle or advanced stage of EVD, inflammatory

112

molecule-caused vasodilatation results in both internal and external bleeding. Worse

113

still, since hepatocyte infection leads to liver damage, the coagulation system

114

becomes disordered [2, 38, 39]. Body injury and viral spread in blood circulation and

AC C

EP

TE D

M AN U

SC

97

ACCEPTED MANUSCRIPT 115

organs lead to a vicious downward spiral. If viral spread cannot be controlled, patients

116

can succumb to organ failure or secondary bacterial infection [2, 10]. However, before the advanced stage of EVD, the use of efficacious treatments

118

might limit virus replication to the extent necessary to allow successful mounting of

119

adaptive immune response. The glycoprotein and RNA synthesis machinery, which

120

play important roles in viral entry and RNA replication, respectively, are promising

121

drug targets for Ebola therapies. Ebola investigators have developed several research

122

models at both cell culture [40-43] and animal model levels [44-47]. EBOVs can be

123

cultured with the Vero E6 cell line, and this model provides the entire virus replication

124

cycle for drug research [42, 48]. However, since EBOVs are biosafety level 4

125

pathogens, the facility limitation restricts the development of antivirals. To screen for

126

antivirals that inhibit viral entry, Ebola pseudotyped systems based on either lentivirus

127

backbone [40] or vesicular stomatitis virus (VSV) backbone [41], which is conjugated

128

with luciferase reporter gene, can be performed in BSL-2 laboratories. Mini-genome

129

replicon and partial reverse genetics systems, which can also be safely handled in

130

BSL-2 laboratories, have been developed for screening chemical inhibitors to counter

131

RNA transcription or replication [43, 49]. For EVD symptoms caused by infection of

132

EBOVs in primates, lethal challenge studies in NHPs are the gold standard for testing

133

the effectiveness and safety of antivirals [47, 50]. To facilitate drug research, either a

134

mouse-adapted strain or an immunodeficient mouse model has been used for

135

preliminary testing [48, 51]. A similar strategy has also been applied in guinea pigs

136

[45] and Syrian hamsters [46]. Normal test indicators for Ebola animal models are

AC C

EP

TE D

M AN U

SC

RI PT

117

ACCEPTED MANUSCRIPT 137

survival rate, weight loss, body temperature, viremia, alanine aminotransferase (ALT)

138

and aspartate aminotransferase (AST) index [48].

140

2. Antivirals targeting viral entry step

141

2.1. Antibody-based therapy

RI PT

139

Neutralizing antibody-based therapies are commonly used to treat post-exposure

143

infection because they can directly target the virion and cut off virus replication at the

144

very early stage of viral entry. Throughout the history of Ebola outbreaks,

145

antibody-based EVD therapies have been studied from convalescent serum to

146

monoclonal antibody and from single antibody treatment to antibody cocktail.

M AN U

SC

142

In 1995, during the ZEBOV outbreak in Kikwit, DRC, eight patients had

148

symptoms of EVD, two of whom had even been in severe coma, and all of them were

149

administered with convalescent sera from recovered EVD patients. This emergency

150

treatment led to the survival of seven of these patients, showing a significantly

151

improved survival rate, compared with the average mortality of 80% during that

152

epidemic [52]. However, these patients also received supportive treatment, and the

153

sample size was too small to elucidate whether the sera transfusion was the crucial

154

factor for patients’ survival.

AC C

EP

TE D

147

155

Please be aware that these so called “convalescent sera” are actually

156

“convalescent plasma” since the anticoagulant was used in collection of the donated

157

blood [52]. The blood donors and recipients must have the same or matched blood

ACCEPTED MANUSCRIPT group and the donor-recipient compatibility for blood plasma is the converse of that

159

of red blood cells. For example, plasma from a type AB blood donor can be transfused

160

to individuals of any blood group, while individuals of blood group O can receive

161

plasma from any blood group (http://www.bloodbook.com/compat.html). Although no

162

blood group antigens are presented in the donated plasma, the ABO blood group

163

antibodies in the donated plasma are able to bind with the recipient’s red blood cells,

164

thus causing agglutination reactions.

SC

RI PT

158

Later, polyclonal IgG was obtained from the sera of EBOV-hyperimmunized

166

horses, and this equine IgG was transfused to EBOV-infected cynomolgous macaques.

167

IgG-treated macaques showed a delay in viremia and the appearance of symptoms,

168

but the survival rate was not increased [53], possibly because of IgG depletion caused

169

by neutralizing consumption and host immune clearance of these heterologous IgG.

TE D

M AN U

165

Homogeneous polyclonal IgG purified from Ebola vaccine immunized NHPs was

171

also used to treat Ebola infected NHPs. A primary dose of 80 mg/kg of IgG at 48

172

hours, and additional doses at 4 days and 8 days postinfection were provided to

173

challenged NHPs. All the animals in treatment group survived, while animals in

174

control group were dead [54].

AC C

175

EP

170

However, serum-based therapy may cause some toxicity-related problems, such

176

as transmission of the contaminated pathogen(s) transmission and transfusion

177

reactions mentioned above. Although purified IgG can lower these risks, lot-to-lot

178

variation is still a problem. Besides, the potential antibody-dependent enhancement of

179

EBOV infection was reported previously [55]. To solve these problems, researchers

ACCEPTED MANUSCRIPT 180

tended to obtain the highly purified polyclonal antibodies or monoclonal antibodies

181

specifically target the main neutralizing epitopes on EBOV envelope glycolproteins

182

for clinical use. The antibody KZ52, derived from a survivor of the Kikwit ZEBOV outbreak in

184

1995, showed potent neutralizing activity at the cell culture level and was protective

185

in a small animal challenge test under post-exposure conditions [56, 57]. That some of

186

the above-mentioned animals survived despite the display of high-level viremia is

187

puzzling [58]. Two administrations of KZ52 failed to protect NHPs from ZEBOV

188

challenge, with the first dose given before viral infection [59]. The crystal structure of

189

KZ52 and Ebola GP trimer shows that KZ52 recognizes the pre-fusion conformation

190

of GP trimer by clamping regions of the pre-fusion GP2 and part of GP1 together [30].

191

Although the KZ52 protection test on NHPs was unsuccessful, experiments on this

192

antibody have led to the conclusion that the GP1 and GP2 interface, rather than the

193

hyperglycosylated head of GP trimer, could be a target for neutralizing antibody

194

design. Electron microscope images show that the whole, long virions are thickly

195

coated by the GP trimers [21], indicating that a group of antibodies might be used

196

compositionally to efficiently inhibit all GP trimers.

SC

M AN U

TE D

EP

AC C

197

RI PT

183

During past years, researchers have developed three generations of antibody

198

cocktail formulations for EVD therapy. The first one was based on the combination of

199

two human-mouse chimeric mAbs, ch133 and ch226, which presented strong

200

neutralizing activity against ZEBOV in vitro. In the NHPs challenge assay, this

201

antibody cocktail was administered to three rhesus macaques at 24 hours before and

ACCEPTED MANUSCRIPT 24 and 72 hours after infection. One of them survived with low-level viremia and no

203

obvious symptoms. However, the other two died with typical EVD symptoms,

204

apparently caused by significant reduction of circulating mAbs titer and the resulting

205

increase of blood virus titer [60]. This finding indicates the necessity for prolonging

206

the half-life of antibodies and choosing antibodies with greater neutralization potency.

207

As the second generation of anti-Ebola antibody cocktail formulas, ZMAb and

208

MB-003 each consist of three completely different neutralizing mAbs derived from

209

ZEBOV GP trimer antigen-immunized mice, respectively [61, 62]. ZMAb, containing

210

mAbs 1H3, 2G4 and 4G7, showed 100% protection of cynomolgus macaques, with

211

the first dose being given 24 hours post-exposure followed by two additional doses

212

every 3 days (25 mg/kg per dose). When the first treatment was administered 48 hours

213

post-infection, 50% of the cynomolgus macaques survived [61]. The surviving

214

animals demonstrated potent humoral and cellular immune responses against ZEBOV,

215

and all of them survived in the later rechallenge test [63]. The treatment window

216

could also be extended to 72 hours post-infection with the combined utilization of

217

adenovirus-vectored human interferon-α [64]. The MB-003 cocktail, including

218

antibodies of c13C6, h-13F6, and c6D8, showed 67% protection in NHPs when

219

treatment was initiated at 48 hours post-challenge with two additional doses (50

220

mg/kg per dose) [62]. This evidence strongly hints that rational design of antibody

221

utilization, such as optimization of cocktail composition or immunofocusing to

222

determine the strongest epitopes for neutralization antibodies, may help to limit virus

223

replication more effectively and further amplify the treatment window.

AC C

EP

TE D

M AN U

SC

RI PT

202

ACCEPTED MANUSCRIPT The latest study tested different combinations of antibodies from MB-003 and

225

ZMAb in NHPs challenge experiment, and the selected formulation with the best

226

preventive effect, termed ZMapp, was composed of c13C6 from MB-003 and two

227

antibodies, 2G4 and 4G7, from ZMAb. All three mAbs recognized conformational

228

epitopes located on GP2 or the stem region of GP trimer, while the remaining three

229

antibodies from MB-003 and ZMAb were bound to the trimer head. Three doses of

230

ZMapp were given to challenged rhesus macaques at 5, 8 and 11days (50 mg/kg per

231

dose), and 100% of them survived. Animals showed EVD symptoms and detectable

232

viremia at 5 dpi before treatment with ZMapp, but viral load could not be detected in

233

blood 21 days post-infection [50]. This experimental scenario is similar to the current

234

situation in West Africa where people usually receive diagnosis and treatment only

235

after they have shown EVD symptoms. This study is also significant in that people

236

may be rescued by antibody-based drug, even when EVD symptoms have appeared.

237

Furthermore, even though NHPs in the experiment were challenged with the Kikwit

238

strain of ZEBOV, ZMapp showed inhibitory activity against the epidemic strain in

239

cell culture [50], while it was recently reported that some patients administered with

240

ZMapp had recovered from EVD. ZMapp is now the most promising antibody-based

241

drug against Ebola virus, and it may soon be available after clinical trials. To increase

242

the production of ZMapp, the compositional antibodies are currently produced from N.

243

Benthamiana [50]. By applying a novel technology in plant protein expression system,

244

named magnifection, 500 mg of full-size monoclonal antibody can be purified from

245

per kg fresh leaf biomass [65]. This is an easier and cheaper approach to scale up the

AC C

EP

TE D

M AN U

SC

RI PT

224

ACCEPTED MANUSCRIPT production of ZMapp, especially for developing countries. However, this technology

247

may be insufficiently robust to scale up the production of ZMapp for combating the

248

current Ebola outbreak in West Africa

249

2.2. Peptide-based viral entry inhibitor

RI PT

246

Since the first CHR-peptide-based HIV entry inhibitor discovered in 1992 [66],

251

this potential treatment strategy has been applied against many enveloped viruses,

252

including EBOVs [67, 68]. The working principle of this kind of inhibitors is based

253

on that added a CHR (or HR2) sequence-containing peptide (C-peptide) can interact

254

with NHR (or HR1) region in the viral GP2 and block the 6-HB formation between

255

the viral GP2 NHR and CHR domains, resulting in inhibition of viral-host cell

256

membrane fusion [35]. Unlike HIV, however, Ebola viruses enter the target cell

257

mainly through the endocytic pathway [29, 30]; thus, the peptide inhibitors must exist

258

in the endosome where the 6-HB is formed.

TE D

M AN U

SC

250

Hence, researchers added a HIV-1 Tat protein segment, which is known to target

260

host endosome [69], to the N-terminal of EBOV CHR (610-633 aa of GP) with a short

261

linker, and this synthesized peptide was called Tat-Ebo [67]. Tat-Ebo was shown to

262

enrich in endosome, and this location property proved to be associated with efficient

263

entry inhibition of Ebola pseudovirus and authentic Ebola virus at cell culture level.

264

For inhibition testing of authentic Ebola virus, when multiplicity of infection (MOI)

265

was 1, at least 50 µM of Tat-Ebo must be used to protect 90% of cells from infection.

266

Tat-Ebo showed inhibition activity against three EBOV species, including ZEBOV,

267

SEBOV, REBOV, and even Marburg virus [67].

AC C

EP

259

ACCEPTED MANUSCRIPT In another approach, CHR of GP2 was conjugated with cholesterol and a Tat

269

analog to form α-helical conformation. In the Ebola pseudovirus assay, infection

270

could be reduced by 1,000-fold with this designed peptide at a concentration of 40

271

µM. On the other hand, the peptide could also inhibit the entry of control virus (VSV),

272

suggesting its lack of specificity [68]. Tat-Ebo showed better specificity, but less

273

potency, to cholesterol-conjugated CHR-peptide. Neither of them has been tested in

274

an animal model. Moreover, peptide entry inhibitors of EBOVs need to maximize the

275

treatment window and minimize dosage.

276

2.3. Other approaches to interrupt the entry step

M AN U

SC

RI PT

268

Several small-molecule compounds can also block cell entry of EBOVs.

278

A high-throughput screen (HTS) of small-compound libraries was carried out

279

based on a pseudotyped system, and a benzodiazepine derivative, termed compound 7,

280

was identified to block the entry step of Ebola virus and Marburg virus with an IC50

281

value of 10 µM and 12 µM, respectively. Binding assay and computer calculation

282

suggested that compound 7 seemingly bound to the hydrophobic pocket near the GP1

283

and GP2 interface [70], but further study is needed to confirm this.

EP

AC C

284

TE D

277

A kind of benzylpiperazine adamantane diamide-derived compound could inhibit

285

the entry of EBOVs by preventing viral glycoproteins from binding NPC1 [71].

286

Similarly, inhibitors of CatL or CatB may also be antiviral candidates [72].

287

Since drug candidates generally require several years of testing before approval

288

for use in human, one research group screened licensed drugs to identify whether any

ACCEPTED MANUSCRIPT of them would have anti-EBOV effects. They finally chose three clinically approved

290

ion channel inhibitors, amiodarone (an anti-arrhythmic drug), dronedarone and

291

verapamil, all of which were previously shown to inhibit the entry of EBOVs based

292

on pseudovirus assay results. Importantly, the concentration of amiodarone required to

293

block EBOV entry was equal to serum concentration in humans (1.5-2.5 µg/ml) in

294

routed anti-arrhythmic therapy. Although the mechanism for blocking virus entry was

295

unclear, it was thought that the drugs worked by interference with cell signaling

296

pathways which coordinated viral entry [73].

M AN U

SC

RI PT

289

297

3. Antivirals targeting viral RNA synthesis or translation steps

299

3.1. Small-molecule compounds that interfere with RNA synthesis

TE D

298

Early studies showed that the commonly used anti-RNA virus drug ribavirin

301

could not limit the replication of EBOVs and failed to protect animals from lethal

302

challenge [74, 75]. Recently, however, one pyrazinecarboxamide derivative named

303

favipiravir (T-705), which showed potent antiviral activity against numerous negative-

304

or positive-strand RNA viruses [76-80], was demonstrated as an anti-EBOV drug in a

305

mouse

306

favipiravir-ribofuranosyl-5’-triphosphate

307

phosphoribosylation by cellular enzymes in vivo. Favipiravir-RTP is considered to be

308

a nucleotide analog that occupies the catalytic center of viral RdRP or incorporates

309

into the newly synthesized viral RNA to cause lethal mutagenesis [82-84]. Favipiravir

AC C

EP

300

model

[48,

81].

Favipiravir

transforms

into

its

(favipiravir-RTP),

active

form, through

ACCEPTED MANUSCRIPT inhibited RNA replication of ZEBOV in a cell culture model with an IC90 of 110 µM.

311

Animal experiments were carried out on two kinds of type I IFN receptor knockout

312

mice (IFNAR-/-): IFNAR-/- C57BL/6 and IFNAR-/- 129/Sv mice. Daily favipiravir

313

administration was given from 6 days to 13 days post-infection (300 mg/Kg each day).

314

Consequently, symptoms that had already appeared, such as AST/ALT level or viral

315

titer increase in blood, were rapidly cleared, and all mice survived [48]. Moreover,

316

favipiravir could be administered through the oral route [48, 81], which could prevent

317

potential risks during drug injection. More importantly, phase III clinical trials of

318

favipiravir for influenza treatment had been completed [76], making it possible for its

319

quick use in EVD therapy, as long as its anti-Ebola activity could be proved in the

320

NHPs model. Recently, favipiravir has been used to treat an Ebola-infected nurse

321

worked for the Doctors Without Borders [85].,.

TE D

M AN U

SC

RI PT

310

A prodrug of cidofovir, named brincidofovir (CMX001), showed potent

323

anti-Ebola activity in cell culture level, and had been used in EVD patients treatment

324

[86]. Brincidofovir has been studied as the drug against several DNA viruses infection

325

[87], and is currently in Phase III clinical testing against cytomegalovirus and

326

adenovirus [86]. Brincidofovir inhibits viral replication by inhibiting viral DNA

327

polymerases [88], so it may interfere the RNA polymerase of EBOVs. Although the

328

mechanism of anti-Ebola activity is unclear, a new phase II clinical trials of

329

brincidofovir is launched for testing its potential safety and antiviral activity in EVD

330

patients [86].

331

AC C

EP

322

A novel adenosine analogue, BCX4430, interferes with the function of RNA

ACCEPTED MANUSCRIPT 332

polymerase of Ebola virus, and confers protecion to Ebola-challenged rodent animals

333

[89].

334

3.2. Oligonucleotides-based antivirals

RI PT

335

The technology of siRNAs was introduced to the anti-Ebola field. The siRNAs

337

specifically recognizing the RNA sequences of RdRP (EK-1), VP24 (VP-24-1160),

338

and

339

polyethylenimine (PEI) or lipid particles for in vivo delivery. The drug formulation

340

was named “LNP/siRNA: TKM-Ebola” [90]. Seven doses (2 mg/kg per dose) were

341

given to EBOV-challenged NHPs intravenously, and all animals survived, albeit with

342

a moderately increasing blood AST level [91].

were

identified,

and

they were

packaged

with

M AN U

(VP-35-855)

Another

TE D

343

VP35

SC

336

antisense

oligonucleotides-based

technology,

termed

phosphorodiamidate morpholino oligomers (PMOs), was also applied for EVD

345

therapy. This kind of DNA oligomer recognizes specific single-stranded RNA or DNA

346

of viruses to form stable complexes in order to block viral replication [92]. The

347

Ebola-specific PMO drug AVI-6002, a mixture of positively charged PMOs targeting

348

mRNA sequences of VP24 and VP35, protected five of eight rhesus monkeys from

349

ZEBOV challenge with daily treatment up to 14 days (40mg/kg per dose), and the

350

first dose was given 30 to 60 minutes after viral infection [93].

AC C

EP

344

351

Although AVI-6002 and LNP/siRNA:TKM-Ebola are under phase I clinical trials

352

[94], two important issues affecting both approaches must be considered. For RNA

ACCEPTED MANUSCRIPT viruses, the mutation rate at nucleic acid level is usually higher than the antigenic drift

354

rate at protein level; therefore, antisense oligonucleotides-based drugs may face more

355

problems in genetic variation of virus than other antivirals which target viral proteins

356

like antibodies. Also both PMOs and siRNAs should be delivered into cytoplasm

357

more efficiently to reduce the dosage and frequency of the drugs.

RI PT

353

SC

358

4. Other treatment strategies

360

4.1. Drugs to modulate symptoms without directly targeting EBOVs

M AN U

359

Since EBOVs antagonize the functions of type I interferons [37, 95, 96],

362

exogenous interferon-α or interferon-β could delay the occurrence of viremia or

363

prolong survival time, but not rescue NHPs from lethal infection [97, 98]. The

364

coagulation disorders caused by EBOV infection are also an important factor in the

365

development of EVD [99]. Two licensed drugs, the recombinant nematode

366

anticoagulant protein c2 (rNAPc2) and the recombinant human activated protein C

367

(rhAPC), originally used for anticoagulation, were approved to partially protect NHPs

368

from ZEBOV lethal challenge, and the survival rates were 33% and 18%, respectively

369

[100, 101]. However, these two approaches are unsuitable for use alone by their

370

relatively low efficacy, while they can be used as part of a cocktail treatment, and

371

these drugs are much safer than newly discovered antivirals.

372

4.2. Post-exposure vaccine

373

AC C

EP

TE D

361

One of the EBOV prophylactic vaccines, an attenuated recombinant VSV

ACCEPTED MANUSCRIPT expressing and displaying the GP of ZEBOV (VSV-Ebola GP), has been studied as a

375

therapeutic vaccine for EVD. By administering it 20-30 minutes after infection,

376

VSV-Ebola GP protected 50% of NHPs from ZEBOV lethal challenge [102]. This

377

vaccine has not been approved for clinical trials, but in 2009, it was applied in human

378

under an intractable situation. One researcher was injured by a syringe containing

379

concentrated ZEBOV during an animal experiment. She was administered with

380

VSV-Ebola GP and then survived with no detectable EVD symptoms [103]. Although

381

no evidence was postulated to judge whether she had been accidentally infected or

382

protected by the vaccine, the use of therapeutic vaccine is a potential treatment for

383

post-exposure.

M AN U

SC

RI PT

374

385

5. Future prospect

TE D

384

EBOVs are members of the so-called "viruses below rocks", which have their

387

own reservoirs and susceptible wildlife in the natural world. Human beings are

388

susceptible, but only by intrusion into the life cycle of viral ecology in nature,

389

meaning that this kind of virus cannot become extinct, like smallpox. For EBOVs,

390

fruit bats and apes in Africa are considered as the reservoirs and susceptible hosts,

391

respectively [104, 105]. By their culture, Africans normally come into contact with

392

infected wildlife, and because of the underdeveloped system of public health in Africa,

393

such high-risk contacts may develop into new outbreaks. Even worse, an animal

394

challenge experiment indicated that inhaling aerosolized virus led to EBOV infections

395

in NHPs [106]. This means that EBOV may adapt to the respiratory transmission

AC C

EP

386

ACCEPTED MANUSCRIPT 396

route and cause larger epidemics, or even pandemics [107]. Therefore, the ideal plan would include rapid diagnostic methods, patient

398

management, prophylactic vaccines, and post-exposure treatments in a combined

399

effort to combat the disease. Once suspected cases have been confirmed, those

400

infected and their close contacts must be isolated. Then, the local medical staff and

401

inhabitants should be immunized with prophylactic vaccines immediately in order to

402

prevent hospital-acquired infections and help break the chain of dissemination.

403

Actually, several viral vector-based or virus-like particle-based prophylactic vaccines

404

have exhibited 100% protection in EBOV challenge tests with NHPs [13, 14]. Since

405

infected individuals may be discovered only after they have shown EVD symptoms,

406

drugs with a relatively long treatment window, like ZMapp, are needed, and broad

407

spectrum antivirals, like favipiravir, should be used jointly, since species-specific

408

drugs may demonstrate low efficiency against variant strains or rare species of EBOV.

409

However, almost all antivirals should be used at a high level of working concentration

410

to provide complete protection, as mentioned above, and could result in the demand

411

for drugs exceeding the supply. Salutary lessons should be drawn from the anti-Rabies

412

strategy, i.e., using therapeutic vaccines in order to activate acquired immune

413

response against EBOVs. The utilization of both therapeutic vaccine and prophylactic

414

vaccine would save drug resources in areas affected by Ebola.

AC C

EP

TE D

M AN U

SC

RI PT

397

415

416

6. Conclusion

ACCEPTED MANUSCRIPT To summarize, multiple drug candidates against EVD are under development.

418

The most promising antivirals are ZMapp and favipiravir, which represent two

419

mainstream anti-Ebola approaches that target the viral entry step and RNA replication

420

step, respectively. However, to win the war against Ebola, we need powerful drugs

421

and vaccines, as well as continuing basic research.

422

RI PT

417

Acknowledgements

424

This study was supported by the National Natural Science Foundation of China

425

(81102476 and 81173098), and Shanghai Municipal Commission of Health and

426

Family Planning (2013QLG009 and 2013QLG010).

M AN U

SC

423

427

References

429

[1] Ebola haemorrhagic fever in Zaire, 1976. Bull World Health Organ 1978;56:271-93.

430 431

[2] Knipe DM, Howley PM. Fields virology, Wolters Kluwer/Lippincott Williams & Wilkins Health,

432 433

[3] Butler D, Morello L. Ebola by the numbers: The size, spread and cost of an outbreak. Nature

434 435

[4] Boozary AS, Farmer PE, Jha AK. The Ebola Outbreak, Fragile Health Systems, and Quality as a

436 437 438

[5] Incident Management System Ebola Epidemiology Team CDC, Ministries of Health of Guinea

439 440 441

[6] To K, Chan J, Tsang A, Cheng V, Yuen KY. Ebola virus disease: a highly fatal and

442

[7] Gulland A. Fifteen countries are at risk of Ebola outbreak, says WHO. BMJ 2014;349:g6305.

443 444

[8] Poletto C, Gomes M, Pastore YPA, Rossi L, Bioglio L, Chao D, et al. Assessing the impact of

AC C

2014;514:284-5.

EP

Philadelphia, PA, 2013.

TE D

428

Cure. JAMA 2014.

SLLN, Senegal, Viral Special Pathogens Branch NCfE, Zoonotic Infectious Diseases CDC. Ebola virus disease outbreak - west Africa, september 2014. MMWR Morb Mortal Wkly Rep 2014;63:865-6.

panic-generating infectious disease reemerging in West Africa. Microbes and Infection / Institut Pasteur 2015. [Epub ahead of print].

travel restrictions on international spread of the 2014 West African Ebola epidemic. Euro Surveill

ACCEPTED MANUSCRIPT 445

2014;19.

446 447

[9] Situation

448

[10] Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet 2011;377:849-62.

449

[11] Bray M. Defense against filoviruses used as biological weapons. Antiviral Res 2003;57:53-60.

450 451

[12] Kortepeter MG, Bausch DG, Bray M. Basic clinical and laboratory features of filoviral

452 453

[13] Hoenen T, Groseth A, Feldmann H. Current ebola vaccines. Expert Opin Biol Ther

454 455

[14] Marzi A, Feldmann H. Ebola virus vaccines: an overview of current approaches. Expert Rev

456

[15] Mullard A. Experimental Ebola drugs enter the limelight. Lancet 2014;384:649.

457 458

[16] Clark DV, Jahrling PB, Lawler JV. Clinical management of filovirus-infected patients. Viruses

459 460

[17] Ye L, Yang C. Development of vaccines for prevention of Ebola virus infection. Microbes and

461

[18] Li YH, Chen SP. Evolutionary history of Ebola virus. Epidemiol Infect 2014;142:1138-45.

462 463

[19] Baize S, Pannetier D, Oestereich L, Rieger T, Koivogui L, Magassouba N, et al. Emergence of

464

[20] Gatherer D. The 2014 Ebola virus disease outbreak in West Africa. J Gen Virol 2014;95:1619-24.

465 466 467

[21] Bharat TA, Noda T, Riches JD, Kraehling V, Kolesnikova L, Becker S, et al. Structural dissection

468 469

[22] Geisbert TW, Jahrling PB. Differentiation of filoviruses by electron microscopy. Virus Res

470 471 472

[23] Huang Y, Xu L, Sun Y, Nabel GJ. The assembly of Ebola virus nucleocapsid requires

473 474

[24] John SP, Wang T, Steffen S, Longhi S, Schmaljohn CS, Jonsson CB. Ebola virus VP30 is an RNA

475 476

[25] Modrof J, Becker S, Muhlberger E. Ebola virus transcription activator VP30 is a zinc-binding

477 478

[26] Noda T, Kolesnikova L, Becker S, Kawaoka Y. The importance of the NP: VP35 ratio in Ebola

479 480 481

[27] Trunschke M, Conrad D, Enterlein S, Olejnik J, Brauburger K, Muhlberger E. The L-VP35 and

reports:

Ebola

response

roadmap.

World

Health

Organization,

RI PT

http://www.who.int/csr/disease/ebola/situation-reports/en/ (accessed 14.11.06).

hemorrhagic fever. J Infect Dis 2011;204 Suppl 3:S810-6.

2012;12:859-72.

SC

Vaccines 2014;13:521-31.

M AN U

2012;4:1668-86.

infection / Institut Pasteur 2015. [Epub ahead of print].

TE D

Zaire Ebola virus disease in Guinea. N Engl J Med 2014;371:1418-25.

of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci U S

AC C

1995;39:129-50.

EP

A 2012;109:4275-80.

virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol Cell 2002;10:307-16.

binding protein. J Virol 2007;81:8967-76.

protein. J Virol 2003;77:3334-8.

virus nucleocapsid formation. J Infect Dis 2011;204 Suppl 3:S878-83.

L-L interaction domains reside in the amino terminus of the Ebola virus L protein and are potential targets for antivirals. Virology 2013;441:135-45.

ACCEPTED MANUSCRIPT 482

[28] Hartlieb B, Weissenhorn W. Filovirus assembly and budding. Virology 2006;344:64-70.

483 484

[29] Hunt CL, Lennemann NJ, Maury W. Filovirus entry: a novelty in the viral fusion world. Viruses

485 486

[30] Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO. Structure of the Ebola virus

487 488

[31] Moller-Tank S, Albritton LM, Rennert PD, Maury W. Characterizing functional domains for

489 490 491

[32] Hood CL, Abraham J, Boyington JC, Leung K, Kwong PD, Nabel GJ. Biochemical and structural

492 493

[33] Chandran K, Sullivan NJ, Felbor U, Whelan SP, Cunningham JM. Endosomal proteolysis of the

494 495

[34] Harrison JS, Higgins CD, Chandran K, Lai JR. Designed protein mimics of the Ebola virus

496 497

[35] Jiang S, Zhao Q, Debnath AK. Peptide and non-peptide HIV fusion inhibitors. Curr Pharm Des

498 499

[36] Olejnik J, Ryabchikova E, Corley RB, Muhlberger E. Intracellular events and cell fate in filovirus

500 501

[37] Wong G, Kobinger GP, Qiu X. Characterization of host immune responses in Ebola virus

502 503

[38] Paessler S, Walker DH. Pathogenesis of the viral hemorrhagic fevers. Annu Rev Pathol

504 505

[39] Hensley LE, Jones SM, Feldmann H, Jahrling PB, Geisbert TW. Ebola and Marburg viruses:

506 507

[40] Chan SY, Speck RF, Ma MC, Goldsmith MA. Distinct mechanisms of entry by envelope

508 509

[41] Ito H, Watanabe S, Takada A, Kawaoka Y. Ebola virus glycoprotein: proteolytic processing,

510 511 512

[42] Volchkova VA, Dolnik O, Martinez MJ, Reynard O, Volchkov VE. Genomic RNA editing and its

513 514 515

[43] Uebelhoer LS, Albarino CG, McMullan LK, Chakrabarti AK, Vincent JP, Nichol ST, et al.

516 517

[44] Bradfute SB, Warfield KL, Bray M. Mouse models for filovirus infections. Viruses

518 519

[45] Connolly BM, Steele KE, Davis KJ, Geisbert TW, Kell WM, Jaax NK, et al. Pathogenesis of

2012;4:258-75.

TIM-mediated enveloped virus entry. J Virol 2014;88:6702-13.

RI PT

glycoprotein bound to an antibody from a human survivor. Nature 2008;454:177-82.

characterization of cathepsin L-processed Ebola virus glycoprotein: implications for viral entry and immunogenicity. J Virol 2010;84:2972-82.

SC

Ebola virus glycoprotein is necessary for infection. Science 2005;308:1643-5.

2002;8:563-80.

infection. Viruses 2011;3:1501-31.

M AN U

glycoprotein GP2 alpha-helical bundle: stability and pH effects. Protein Sci 2011;20:1587-96.

TE D

infections. Expert Rev Clin Immunol 2014;10:781-90.

2013;8:411-40.

EP

pathogenesis and development of countermeasures. Curr Mol Med 2005;5:761-72.

AC C

glycoproteins of Marburg and Ebola (Zaire) viruses. J Virol 2000;74:4933-7.

acylation, cell tropism, and detection of neutralizing antibodies. J Virol 2001;75:1576-80.

impact on Ebola virus adaptation during serial passages in cell culture and infection of guinea pigs. J Infect Dis 2011;204 Suppl 3:S941-6.

High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses. Antiviral Res 2014;106:86-94.

2012;4:1477-508.

experimental Ebola virus infection in guinea pigs. J Infect Dis 1999;179 Suppl 1:S203-17.

ACCEPTED MANUSCRIPT 520 521

[46] Ebihara H, Zivcec M, Gardner D, Falzarano D, LaCasse R, Rosenke R, et al. A Syrian golden

522 523

[47] Twenhafel NA, Mattix ME, Johnson JC, Robinson CG, Pratt WD, Cashman KA, et al. Pathology

524 525 526

[48] Oestereich L, Ludtke A, Wurr S, Rieger T, Munoz-Fontela C, Gunther S. Successful treatment of

527 528

[49] Hoenen T, Feldmann H. Reverse genetics systems as tools for the development of novel therapies

529 530

[50] Qiu X, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, et al. Reversion of advanced Ebola

531 532

[51] Bray M, Davis K, Geisbert T, Schmaljohn C, Huggins J. A mouse model for evaluation of

533 534 535

[52] Mupapa K, Massamba M, Kibadi K, Kuvula K, Bwaka A, Kipasa M, et al. Treatment of Ebola

536 537 538

[53] Jahrling PB, Geisbert J, Swearengen JR, Jaax GP, Lewis T, Huggins JW, et al. Passive

539 540 541

[54] Dye JM, Herbert AS, Kuehne AI, Barth JF, Muhammad MA, Zak SE, et al. Postexposure antibody

542 543

[55] Takada A, Feldmann H, Ksiazek TG, Kawaoka Y. Antibody-dependent enhancement of Ebola

544 545

[56] Maruyama T, Rodriguez LL, Jahrling PB, Sanchez A, Khan AS, Nichol ST, et al. Ebola virus can

546 547 548

[57] Shedlock DJ, Bailey MA, Popernack PM, Cunningham JM, Burton DR, Sullivan NJ.

549 550 551

[58] Parren PW, Geisbert TW, Maruyama T, Jahrling PB, Burton DR. Pre- and postexposure

552 553

[59] Oswald WB, Geisbert TW, Davis KJ, Geisbert JB, Sullivan NJ, Jahrling PB, et al. Neutralizing

554 555 556

[60] Marzi A, Yoshida R, Miyamoto H, Ishijima M, Suzuki Y, Higuchi M, et al. Protective efficacy of

557 558

[61] Qiu X, Audet J, Wong G, Pillet S, Bello A, Cabral T, et al. Successful treatment of ebola

hamster model recapitulating ebola hemorrhagic fever. J Infect Dis 2013;207:306-18.

of experimental aerosol Zaire ebolavirus infection in rhesus macaques. Vet Pathol 2013;50:514-29.

advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral Res

against filoviruses. Expert Rev Anti Infect Ther 2014;12:1253-63.

SC

virus disease in nonhuman primates with ZMapp. Nature 2014;514:47-53.

RI PT

2014;105:17-21.

M AN U

prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 1999;179 Suppl 1:S248-58.

hemorrhagic fever with blood transfusions from convalescent patients. International Scientific and Technical Committee. J Infect Dis 1999;179 Suppl 1:S18-23.

immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses. Arch Virol Suppl 1996;11:135-40.

2012;109:5034-9.

TE D

prophylaxis protects nonhuman primates from filovirus disease. Proc Natl Acad Sci U S A

EP

virus infection. J Virol 2003;77:7539-44.

be effectively neutralized by antibody produced in natural human infection. J Virol 1999;73:6024-30.

AC C

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms. Virology 2010;401:228-35.

prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody. J Virol 2002;76:6408-12.

antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathog 2007;3:e9.

neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever. PLoS One 2012;7:e36192.

virus-infected cynomolgus macaques with monoclonal antibodies. Sci Transl Med 2012;4:138ra81.

ACCEPTED MANUSCRIPT 559 560 561

[62] Olinger GG, Jr., Pettitt J, Kim D, Working C, Bohorov O, Bratcher B, et al. Delayed treatment of

562 563

[63] Qiu X, Audet J, Wong G, Fernando L, Bello A, Pillet S, et al. Sustained protection against Ebola

564 565 566

[64] Qiu X, Wong G, Fernando L, Audet J, Bello A, Strong J, et al. mAbs and Ad-vectored IFN-alpha

567 568 569

[65] Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, et al. Rapid high-yield

570

[66] Jiang S, Lin K, Strick N, Neurath AR. HIV-1 inhibition by a peptide. Nature 1993;365:113.

571 572

[67] Miller EH, Harrison JS, Radoshitzky SR, Higgins CD, Chi X, Dong L, et al. Inhibition of Ebola

573 574 575

[68] Higgins CD, Koellhoffer JF, Chandran K, Lai JR. C-peptide inhibitors of Ebola virus

576 577

[69] Gump JM, Dowdy SF. TAT transduction: the molecular mechanism and therapeutic prospects.

578 579

[70] Basu A, Li B, Mills DM, Panchal RG, Cardinale SC, Butler MM, et al. Identification of a

580 581

[71] Cote M, Misasi J, Ren T, Bruchez A, Lee K, Filone CM, et al. Small molecule inhibitors reveal

582 583 584

[72] Shah PP, Wang T, Kaletsky RL, Myers MC, Purvis JE, Jing H, et al. A small-molecule

585 586 587

[73] Gehring G, Rohrmann K, Atenchong N, Mittler E, Becker S, Dahlmann F, et al. The clinically

588 589

[74] Canonico PG, Kende M, Luscri BJ, Huggins JW. In-vivo activity of antivirals against exotic RNA

590 591

[75] Huggins JW. Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum

592 593

[76] Furuta Y, Gowen BB, Takahashi K, Shiraki K, Smee DF, Barnard DL. Favipiravir (T-705), a novel

594 595 596

[77] Gowen BB, Wong MH, Jung KH, Sanders AB, Mendenhall M, Bailey KW, et al. In vitro and in

Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc Natl Acad Sci U S A 2012;109:18030-5.

virus infection following treatment of infected nonhuman primates with ZMAb. Sci Rep 2013;3:3365.

RI PT

therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Sci Transl Med 2013;5:207ra143.

expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors. Proc Natl

SC

Acad Sci U S A 2006;103:14701-6.

M AN U

virus entry by a C-peptide targeted to endosomes. J Biol Chem 2011;286:15854-61.

glycoprotein-mediated cell entry: effects of conjugation to cholesterol and side chain-side chain crosslinking. Bioorg Med Chem Lett 2013;23:5356-60.

Trends Mol Med 2007;13:443-8.

TE D

small-molecule entry inhibitor for filoviruses. J Virol 2011;85:3106-19.

Niemann-Pick C1 is essential for Ebola virus infection. Nature 2011;477:344-8.

EP

oxocarbazate inhibitor of human cathepsin L blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293T cells. Mol Pharmacol 2010;78:319-24.

AC C

approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry. J Antimicrob Chemother 2014;69:2123-31.

viral infections. J Antimicrob Chemother 1984;14 Suppl A:27-41.

antiviral drug. Rev Infect Dis 1989;11 Suppl 4:S750-61.

viral RNA polymerase inhibitor. Antiviral Res 2013;100:446-54.

vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother 2007;51:3168-76.

ACCEPTED MANUSCRIPT 597 598 599

[78] Safronetz D, Falzarano D, Scott DP, Furuta Y, Feldmann H, Gowen BB. Antiviral efficacy of

600 601

[79] Rocha-Pereira J, Jochmans D, Dallmeier K, Leyssen P, Nascimento MS, Neyts J. Favipiravir

602 603 604

[80] Morrey JD, Taro BS, Siddharthan V, Wang H, Smee DF, Christensen AJ, et al. Efficacy of orally

605 606 607

[81] Smither SJ, Eastaugh LS, Steward JA, Nelson M, Lenk RP, Lever MS. Post-exposure efficacy of

608 609

[82] Furuta Y, Takahashi K, Kuno-Maekawa M, Sangawa H, Uehara S, Kozaki K, et al. Mechanism of

610 611

[83] Baranovich T, Wong SS, Armstrong J, Marjuki H, Webby RJ, Webster RG, et al. T-705

612 613 614

[84] Jin Z, Smith LK, Rajwanshi VK, Kim B, Deval J. The ambiguous base-pairing and high substrate

615 616 617

[85] French

618 619 620

[86] Brincidofovir

621 622

[87] Florescu DF, Keck MA. Development of CMX001 (Brincidofovir) for the treatment of serious

623 624

[88] Price NB, Prichard MN. Progress in the development of new therapies for herpesvirus infections.

625 626 627

[89] Warren TK, Wells J, Panchal RG, Stuthman KS, Garza NL, Van Tongeren SA, et al. Protection

628 629 630

[90] Geisbert TW, Hensley LE, Kagan E, Yu EZ, Geisbert JB, Daddario-DiCaprio K, et al.

631 632 633

[91] Geisbert TW, Lee AC, Robbins M, Geisbert JB, Honko AN, Sood V, et al. Postexposure protection

634 635

[92] Warren TK, Shurtleff AC, Bavari S. Advanced morpholino oligomers: a novel approach to

favipiravir against two prominent etiological agents of hantavirus pulmonary syndrome. Antimicrob Agents Chemother 2013;57:4673-80.

(T-705) inhibits in vitro norovirus replication. Biochem Biophys Res Commun 2012;424:777-80.

RI PT

administered T-705 pyrazine analog on lethal West Nile virus infection in rodents. Antiviral Res 2008;80:377-9.

oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model. Antiviral Res

SC

2014;104:153-5.

action of T-705 against influenza virus. Antimicrob Agents Chemother 2005;49:981-6.

M AN U

(favipiravir) induces lethal mutagenesis in influenza A H1N1 viruses in vitro. J Virol 2013;87:3741-51.

efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate towards influenza A virus polymerase. PLoS One 2013;8:e68347. nurse

cured

of

Ebola

contracted

in

Liberia.

Agence

France

Presse,

http://www.msn.com/en-gb/news/world/french-nurse-cured-of-ebola-contracted-in-liberia/ar-BB7r1UJ

TE D

(accessed 14.11.22). for

Ebola.

Chimerix,

http://www.chimerix.com/discovery-clinical-trials/brincidofovir/brincidofovir-for-ebola/ 14.11.22).

Inc, (accessed

EP

diseases or conditions caused by dsDNA viruses. Expert Rev Anti Infect Ther 2014;12:1171-8.

AC C

Curr Opin Virol 2011;1:548-54.

against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature 2014;508:402-5.

Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference. J Infect Dis 2006;193:1650-7.

of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet 2010;375:1896-905.

antiviral therapy. Antiviral Res 2012;94:80-8.

ACCEPTED MANUSCRIPT 636 637 638

[93] Warren TK, Warfield KL, Wells J, Swenson DL, Donner KS, Van Tongeren SA, et al. Advanced

639 640

[94] Choi JH, Croyle MA. Emerging targets and novel approaches to Ebola virus prophylaxis and

641 642

[95] Cardenas WB. Evasion of the interferon-mediated antiviral response by filoviruses. Viruses

643 644

[96] Kuhl A, Pohlmann S. How Ebola virus counters the interferon system. Zoonoses Public Health

645 646 647

[97] Jahrling PB, Geisbert TW, Geisbert JB, Swearengen JR, Bray M, Jaax NK, et al. Evaluation of

648 649 650

[98] Smith LM, Hensley LE, Geisbert TW, Johnson J, Stossel A, Honko A, et al. Interferon-beta

651

[99] Ansari AA. Clinical features and pathobiology of Ebolavirus infection. J Autoimmun 2014.

652 653 654

[100]

655 656 657

[101]

658 659

[102]

660 661 662

[103]

663 664

[104]

665 666

[105]

667 668 669

[106]

670 671

[107]

672

673

antisense therapies for postexposure protection against lethal filovirus infections. Nat Med 2010;16:991-4.

treatment. BioDrugs 2013;27:565-83.

RI PT

2010;2:262-82.

2012;59 Suppl 2:116-31.

infections. J Infect Dis 1999;179 Suppl 1:S224-34.

SC

immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus

Dis 2013;208:310-8.

M AN U

therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever. J Infect

Geisbert TW, Hensley LE, Jahrling PB, Larsen T, Geisbert JB, Paragas J, et al. Treatment of

Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet 2003;362:1953-8.

Hensley LE, Stevens EL, Yan SB, Geisbert JB, Macias WL, Larsen T, et al. Recombinant

2007;196 Suppl 2:S390-9.

TE D

human activated protein C for the postexposure treatment of Ebola hemorrhagic fever. J Infect Dis

Feldmann H, Jones SM, Daddario-DiCaprio KM, Geisbert JB, Stroher U, Grolla A, et al.

Effective post-exposure treatment of Ebola infection. PLoS Pathog 2007;3:e2.

EP

Gunther S, Feldmann H, Geisbert TW, Hensley LE, Rollin PE, Nichol ST, et al. Management

of accidental exposure to Ebola virus in the biosafety level 4 laboratory, Hamburg, Germany. J Infect Dis 2011;204 Suppl 3:S785-90.

AC C

Groseth A, Feldmann H, Strong JE. The ecology of Ebola virus. Trends Microbiol

2007;15:408-16.

Pourrut X, Kumulungui B, Wittmann T, Moussavou G, Delicat A, Yaba P, et al. The natural

history of Ebola virus in Africa. Microbes Infect 2005;7:1005-14. Reed DS, Lackemeyer MG, Garza NL, Sullivan LJ, Nichols DK. Aerosol exposure to Zaire

ebolavirus in three nonhuman primate species: differences in disease course and clinical pathology. Microbes Infect 2011;13:930-6. Lu L, Liu Q, Jiang S. Biosecurity and biosafety in research on emerging pathogens.

Emerging microbes and infections 2012. [letter to the editor].

ACCEPTED MANUSCRIPT Figure legend

676

Figure 1. The organization of genome of Ebola virus (A) and schematic

677

representation of Ebola virion (B). Different building blocks are represented with

678

different colors: nucleoproteins (red), phosphoprotein (pink), polymerase (dark blue),

679

matrix proteins (green), glycoprotein (yellow), genome RNA (blue helix).

RI PT

674 675

680

Figure 2. Features of the glycoprotein of Ebola virus (A) and the viral-host membrane

682

fusion process in endosome (B). The glycoprotein precursor is cleaved into GP1 and

683

GP2 by furin protease. GP1 and GP2 are linked with a disulfide bond. In endosome,

684

mucin-like domain and glycan cap domain are removed by cathepsin L and B; then

685

the NPC1 interacts with the exposed RBD, and this interaction results in the

686

conformational change of RBD. Sequentially, fusion peptides insert into the

687

endosomal membrane, and then NHR and CHR hexamer is formed to induce

688

viral-host membrane fusion. RBD, receptor binding domain; NHR, N-terminal heptad

689

repeat; CHR, C-terminal heptad repeat; TM, transmembrane domain; Cat L/B,

690

cathepsin L and B; TIM-1, T-cell immunoglobulin mucin domain-1; NPC1,

691

Niemann-Pick C1.

M AN U

TE D

EP

AC C

692

SC

681

693

Figure 3. The life cycle of Ebola virus. Antiviral candidates are classified into groups

694

based on different drug targets. TIM-1, T-cell immunoglobulin mucin domain-1;

695

NPC1, Niemann-Pick C1; Cat L/B, cathepsin L and B; PMOs, phosphorodiamidate

696

morpholino oligomers.

ACCEPTED MANUSCRIPT

A VP35 VP40

VP30 VP24

GP

RI PT

NP

5'

M AN U

SC

3'

L

AC C

EP

TE D

B

Figure 1

ACCEPTED MANUSCRIPT

A 32

Signal peptide

54

GP2

202

RBD

501

305

Glycan cap

Mucin-like domain

524

539 554

595 615

RI PT

1

GP1 Fusion peptide

NHR

634 651

CHR

671 676

TM

B

NPC1

NPC1

AC C

Cat L/B cleavage

Cytosol Endosomal membrane

EP

TE D

TIM-1

M AN U

S

S

SC

Furin cleavage site

RBD conformational change

Viral membrane

Figure 2

ACCEPTED MANUSCRIPT

Attachment

Peptide entry inhibitors Cat L/B inhibitors Compound 7 NPC1 binding compounds Ion channel inhibitors Genome RNA

Membrane fusion

?

RNA replication

SC

TIM-1

Polymerase inhibitors (e.g. favipiravir) PMOs siRNAs

RI PT

Neutralizing antibodies (e.g. ZMapp)

M AN U

NPC1

mRNA

Endocytosis

Cat L/B

Translation Assembly

TE D

TIM-1

AC C

EP

Nucleus

Other treatments: Type I interferons Coagulation modulators (e.g.rNAPc2) Post-exposure vaccines

Budding

PMOs siRNAs

Progeny virions

Figure 3