G Model

JVAC 16556 1–9 ARTICLE IN PRESS

Vaccine xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

1 Review

2 Chikungunya virus vaccines: Current strategies and prospects for

3 developing plant-made vaccines
4 Q1 Jorge A. Salazar-González a , Carlos Angulo b , Sergio Rosales-Mendoza a,∗
a
5 Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Dr. Manuel Nava 6, San
6 Q2 Luis Potosí 78210, SLP, Mexico
b
7 Grupo de Inmunología y Vacunología, Centro de Investigaciones Biológicas del Noroeste, SC., Instituto Politécnico Nacional 195, Playa Palo de Santa Rita
8 Sur, La Paz, B.C.S., C.P. 23096 Mexico City, Mexico
9

10
27 a r t i c l e i n f o a b s t r a c t
11
12 Article history: Chikungunya virus is an emerging pathogen initially found in East Africa and currently spread into the
13 Received 3 May 2015 Indian Ocean Islands, many regions of South East Asia, and in the Americas. No licensed vaccines against
14 Received in revised form 25 May 2015 this eminent pathogen are available and thus intensive research in this field is a priority. This review
15 Accepted 28 May 2015
presents the current scenario on the developments of Chikungunya virus vaccines and identifies the
16 Available online xxx
use of genetic engineered plants to develop attractive vaccines. The possible avenues to develop plant-
17
made vaccines with distinct antigenic designs and expression modalities are identified and discussed
18 Keywords:
considering current trends in the field.
19 Oral vaccine
20 Vaccine cost © 2015 Published by Elsevier Ltd.
21 Transgenic plant
22 Transient expression
23 Transplastomic plant
24 Edible crop
25 E antigen
26 Virus like particles

28 1. Introduction the 5 end and polyadenylated at the 3 end. The genomic struc- 46

ture of CHIKV encodes for the following: one open reading frame 47

29Q4 Chikungunya virus (CHIKV) causes an infection typically charac- (5 ORF) yielding four non-structural proteins (nsP1–4) at the post- 48

30 terized with fever, skin rash, incapacitating arthralgia, and severe translational level which participate in genome replication, RNA 49

31 synovitis [1]. This virus is transmitted by the Aedes mosquitoes capping, polyprotein cleavage, and other functions required for 50

32 and its name derives from the Swahili or Makonde word Kun viral replication; and another ORF that yields three major struc- 51

33 qunwala that translates to “walk bent over”, which describes the tural proteins (Capsid, E1, and E2) and two small cleavage products 52

34 posture of infected persons experiencing severe joint pain. Chikun- (E3 and 6K) [3]. The mature virion is 70 nm in diameter and con- 53

35 gunya is easily confused with dengue as they share the same tains 240 heterodimers of E2/E1 arranged as trimeric spikes on 54

36 vectors, symptoms, and geographical distribution; but differs in its surface. These heterodimer spikes are inserted into the plasma 55

37 the absence of headache, and retro-orbital eye pain [2]. CHIKV, first membrane of infected cells after transported through the secretory 56

38 isolated in 1952 from a febrile patient during an outbreak in the pathway. Cytoplasmic nucleocapsids containing the genomic RNA 57

39 Makonde Plateau in the southern province of Tanzania (formerly and 240 copies of the capsid protein bud from the cell surface to 58

40 Tanganyika), is a prevalent pathogen in tropical and subtropical acquire the virion envelope and envelope protein spikes. The E1 59

41 regions of Africa, the Indian Ocean Islands, and south and southeast and E2 glycoproteins form heterodimers that associate as trimeric 60

42 Asia among the Makonde tribe [2]. spikes on the virion surface while E3 and 6K were demonstrated to 61

43 CHIKV is an enveloped alpha virus belonging to the family act as helper proteins in the budding and maturation process of the 62

44 Togaviridae, whose genome consists of a single-stranded positive- virion envelope [4,5]. 63

45 sense RNA of approximately 11.8 kb. The genome is capped at CHIKV is believed to be originated in Africa where two genet- 64

ically distinct lineages have been identified: one containing all 65

isolates from western Africa and the second comprising all south- 66

∗ Corresponding author. Tel.: +52 444 826 2440; fax: +52 444 826 2440. ern and East African strains, as well as isolates from Asia. West 67
Q3
E-mail address: rosales.s@fcq.uaslp.mx (S. Rosales-Mendoza). African lineages caused multiple CHIKV epidemics in East Africa, 68

http://dx.doi.org/10.1016/j.vaccine.2015.05.104
0264-410X/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
2 J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx

69 the Indian Ocean Islands, and many parts of South East Asia [6–11]. responses in mice models and aged NHPs promoted long-term virus 132

70 Before 2000 C.E., large outbreaks of CHIKV were rare but become persistence [35,36]. 133

71 more frequent afterwards. More recently new episodes of CHIKV

72 have been reported in the Americas, further broadening the geo-
73 graphical spread of the disease, which has been associated with 3. Production of vaccine candidates against CHIKV 134

74 an emerging genetic variability [12,13] leading to hypotheses on

75 possible mechanisms of evolutionary adaptation of the virus to the Based on the hypothesis that an efficacious CHIKV vaccine 135

76 mosquito vector [14,15]. should resemble the viral infection to provide accurately immuno- 136

77 The main preventive strategy against Chikungunya is mosquito protection against CHIKV disease [37], several promising vaccines 137

78 control, but this has proven to be difficult especially in poor coun- have been recently evaluated. For example the immunization with 138

79 tries; thus new strategies to fight the disease are needed. Currently virus-like particles (VLPs) in a monkey model elicited neutralizing 139

80 the only treatments for CHIKV-disease symptoms are non-steroidal antibodies against envelope proteins from different CHIKV strains, 140

81 anti-inflammatory drugs. The re-emergence of CHIKV has led to mediating protection against viremia when challenged with a high 141

82 the assessment of several potential treatments including ribavirin dose of CHIKV; moreover, the transfer of these antibodies into 142

83 [16,17], chloroquine [18,19], and CHIKV antibodies [20–23]. In immunodeficient mice conferred protection against a subsequent 143

84 addition the peptides Latarcin (LATA) and Thanatin (THAN) as lethal CHIKV challenge [38]. In addition, purifying human anti- 144

85 well as the protein PAP1, all of which having antiviral activ- CHIKV antibodies from patients in the convalescent phase exhibit 145

86 ity [24,25], have shown a promising potential to protect against a high in vitro CHIKV neutralizing activity and a powerful pro- 146

87 CHIKV [26]. Among the explored strategies, vaccination is con- phylactic and therapeutic efficacy against CHIKV infection in a 147

88 sidered the ideal intervention to prevent the CHIKV infection; mouse model, which correlates with the fact that infected individ- 148

89 however no licensed vaccines for human use are available yet. uals are in general protected against reinfections [20]. Therefore, 149

90 Despite the development of several animal models, few of them the protection against CHIKV disease is considered to be primarily 150

91 have met the requirement to be used in pre-clinical studies mediated by humoral responses. This knowledge have supported 151

92 to assess potential therapeutics. Recent epidemiological data the development of immunization approaches resembling the 152

93 showed the increasing importance of antibody-mediated pro- natural infection process as close as possible through the use atten- 153

94 tection against CHIKV [21–23], highlighting the feasibility of uated CHIKV strains or VLPs, which mimic the infection mechanism 154

95 using anti-CHIKV antibodies as a passive immunotherapy or and induce antibody-mediated. Among the technologies that have 155

96 as a prophylactic treatment. However, information about the been explored for the development of CHIKV vaccines stand-out: 156

97 exact target of the adaptive immune response either in human formalin-inactivated viral vaccines [39,40], live-attenuated viruses 157

98 or in animal models remains limited. In addition the cost for [41–43], alpha virus chimeras [44–46], consensus-based DNA vac- 158

99 immunotherapies produced under conventional platforms should cines [47–50], and recently virus-like particle (VLP) vaccines and 159

100 be considered, which is prohibitive for massive use in developing recombinant subunit vaccines. A detailed scenario on protein sub- 160

101 countries. unit vaccines development is provided below. 161

CHIKV structural proteins form enveloped VLPs (eVLPs) when 162

expressed alone in eukaryotic expression systems [51,52]. The 163

102 2. Immununopathogenesis of CHIKV infection and animal first CHIKV eVLP-based vaccine candidate was reported in 2010 164

103 models by researchers of the NIH and has become the most promising 165

VLP-based vaccine against CHIKV. DNA transfection of a plasmid 166

104 Deciphering CHIKV specific molecular features and how the comprising the full-length CHIKV structural coding region C-E3- 167

105 virus interacts with its host are key aspects to prevent, treat, E2-6K-E1 into human HEK293 cells successfully resulted in CHIKV 168

106 or cure the infection. However, the knowledge of human CHIKV VLPs assembly. The viral glycoproteins in the VLPs are organized 169

107 infection immunology is limited to small animal models (mouse) in 240 E1–E2 heterodimers, which form 80 spikes on the VLP sur- 170

108 [27] in which muscle and joint disease were recently achieved in face, resembling replication-competent alpha viruses. These eVLPs 171

109 C57BL/6 mice [28,29]. Although the mouse model is useful at pre- were isolated from the supernatant of transfected mammalian cells, 172

110 clinical level for vaccine development, CHIKV disease mice models purified, and used to immunize mice and nonhuman primates. Vac- 173

111 (young or immunodeficient mice) do not fully recapitulate human cination of rhesus macaques with 3 doses consisting of 20 ␮g of 174

112 disease patterns in terms of infectivity and immune responses. eVLPs at 0, 4, and 24 weeks induced an antibody response that was 175

113 Therefore, Labadie et al. [30] proposed a model for CHIKV infec- sufficient to confer protection upon a high-dose CHIKV challenge 176

114 tion in adult immunocompetent cynomolgus macaques (Macaca 15 weeks after the last boosting [53]. These results demonstrated 177

115 fascicularis). CHIKV pathogenesis using this animal model seems that immunization with these VLPs elicited neutralizing antibod- 178

116 to resemble the viral, clinical, and immunopathological features ies directed against envelope proteins and protected NHPs against a 179

117 observed in the human disease; and, interestingly, macrophages subsequent lethal CHIKV challenge, indicating a humoral-mediated 180

118 were identified as the main cellular reservoirs during the late stages mechanism of protection. The next developmental step for this 181

119 of CHIKV infection in vivo. Overall, the inflammatory response to vaccine consisted on performing a Phase I dose-escalation clinical 182

120 CHIKV infection in humans clearly contributes to virus elimina- trial under a 3-dose vaccination scheme (weeks 0, 4, and 20) of up 183

121 tion since the viral load has been associated to the serum levels to 40 ␮g of eVLPs per administration. This vaccine was safe, well 184

122 of proinflamatory mediators such as IFN-alpha, IFN-gamma, IL-1- tolerated and immunogenic [38]. Another VLPs-based promising 185

123 RA, IL-6, MCP-1/CCL-2, IL-12, IP-10/CXCL-10, IL-18, and IL-18BP vaccine development has consisted on a measles vaccine express- 186

124 [31,32]. However it is important to point out that proinflamatory ing CHIKV VLPs. A single immunization with this vaccine fully 187

125 mediators are orchestrated and depend largely on the stage of protected mice from a lethal CHIKV challenge [54]. This vaccine 188

126 the viral pathogenesis, as demonstrated in cynomolgus macaques induced high titers of neutralizing CHIKV antibodies although spe- 189

127 after CHIKV experimental infection [30]. Nevertheless, the bene- cific cellular immune responses were also elicited. 190

128 ficial or deleterious effects of inflammation on viral persistence CHIKV eVLPs have also been expressed in insect cells. Research 191

129 remain unclear even though CHIKV infection-associated mark- performed since 2011 demonstrated that the expression of the 192

130 ers have been described [33,34]. In general, T and B cells have structural coding regions C-E3-E2-6K-E1 in Sf21 insect cells led to 193

131 been associated to the clearance of CHIKV since reduced immune the assembly of VLPs [55–57]. Interestingly, these eVLPs displayed 194

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx 3

195 superior immunogenicity in subcutaneously immunized mice in antibodies and high levels of IFN-␥. A challenge assay with CHIKV 261

196 comparison with their subunit counterparts and effectively pro- was also conducted, observing complete protection in the vac- 262

197 tected against a CHIKV lethal challenge in both AG129 and WT cinated group whereas control mice exhibited high viral load in 263

198 mice models. Further analysis revealed an association between this muscles, brain, spleen, and blood [39]. 264

199 protective effect and the neutralizing antibody responses, which Epitope-based vaccines constitute the ideal vaccination 265

200 were induced even for a single immunization scheme (1 ␮g/mouse) approach as they can be designed to achieve proper immune 266

201 without the need of co-administered adjuvants [56,57]. This tech- responses in terms of breadth and neutralizing potential. How- 267

202 nology was adopted by the pharmaceutical company Merck that ever, this field has been rather narrowly explored in part due 268

203 conducted studies consisting on the immunization of Guinea pigs to the limited CHIKV B cell epitope mapping. Therefore, the 269

204 with two doses (on days 0 and 14) that ranged 0.01–10 ␮g along identification of a wider set of CHIKV neutralizing epitopes is 270

205 with the Adju-Phos aluminum adjuvant. The induction of spe- still a need for this field. One of the few investigations on this 271

206 cific IgG neutralizing antibodies in a dose-dependent manner was subject revealed 4 relevant epitopes in the E2 protein which were 272

207 observed [58]. identified by their reactivity against sera from immunized mice. 273

208 Similar findings have been found using live-attenuated CHIKV These epitopes include the previously reported E2EP3 epitope 274

209 vaccines, which have also been evaluated in mice models. For exam- (STKDNFNVYKATRPYLAH, aa 2800–2818 of the polyprotein) and 275

210 ple, a single administration of a live-attenuated CHIKV vaccine three new epitopes: KWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCR 276

211 (CHIKV/IRES) effectively activated T cells with a peak on day 10 (3033–3066 aa of the polyprotein), located in the acid-sensitive 277

212 post-immunization and elicited memory CD4+ and CD8+ T cells region (ASR); KKEVVLTVPTEGLEVTWGNNEPYKYW (3113–3138 aa 278

213 that produced IFN-␥, TNF-␣, and IL-2. However, only passive immu- of the polyprotein); and AGMCMCARRRCITPYELTPGATVPFL (3185 279

214 nization with anti-CHIKV/IRES immune serum provided protection to 3210 aa of the polyprotein). All these epitopes cluster at the 280

215 [59]. Another example is the CHIKV vaccine strain 181/clone25 C-terminus of the E2 glycoprotein [49]. In fact Kam et al. [21,22] 281

216 (181/25) developed by the United States Army Medical Research observed, using plasma samples from humans obtained during 282

217 Institute of Infectious Diseases (USAMRIID). It was demonstrated the early convalescent phase, that the naturally-acquired IgG 283

218 that a single intradermal footpad immunization of AG129 (defec- response is dominated by neutralizing IgG3 antibodies which are 284

219 tive in IFN-␣/␤ and IFN-␥ receptor signaling) or A129 (defective in mostly directed toward the single linear epitope E2EP3, a peptide 285

220 IFN-␣/␤ receptor signaling) mice with the attenuated CHIK 181/25 located at the N-terminus of the E2 glycoprotein and exposed on 286

221 vaccine resulted in different mortality rates. AG129 mice resulted the viral envelope. More recently, it was confirmed that epitopes 287

222 in rapid mortality within 3–4 days while A129 mice survived even on the exposed top-most and outer surfaces of the E2/E1 trimer 288

223 after wild type CHIKV-La Reunion challenge, with the associated structure may be useful for CHIKV neutralization by specific anti- 289

224 induction of significant levels of IFN-␥, IL-12, and specific anti- bodies, whereas epitopes facing the interior of the trimer are not 290

225 bodies. This vaccine was well-tolerated and highly immunogenic [64]. Overall, these findings pave the way for the development of 291

226 in phase I and II clinical trials [60]. Overall, these data highlight CHIKV-specific multi-epitopic- or VLP-based vaccination strategies 292

227 the importance of IFNs and neutralizing antibody responses on the that can be efficiently produced in plant-based platforms. 293

228 protection against CHIKV infection. As mentioned above, the implementation of advanced animal 294

229 Viral-vectored vaccines have also been developed against models to test vaccine candidates at the preclinical level is of rel- 295

230 CHIKV. A novel CHIKV vaccine candidate called MVA-CHIKV was evance, especially those that closely resemble the infection that 296

231 developed based on the highly attenuated modified vaccinia virus occurs in humans. Due to the close lineage relationship between 297

232 Ankara (MVA), which is a poxvirus vector. This vaccine candidate humans and macaques, macaque models of CHIKV infection have 298

233 relies on the expression of the CHIKV C, E3, E2, 6K, and E1 structural been developed [30,35,65,66]. These models allow comparing the 299

234 genes and has proven to induce robust innate immune responses adaptive immunity between humans and macaques. Therefore, 300

235 in human macrophages and monocyte-derived dendritic cells in sera from non-human primates infected with CHIKV could reveal 301

236 terms of the IFN-␤, proinflammatory cytokines, and chemokines new neutralizing epitopes. A remarkable research on mapping 302

237 production. After adjuvant-free intraperitoneal immunization of CHIKV epitopes drop a series of epitopes all along in the structural 303

238 C57BL/6 mice with the chimeric virus at 1 × 107 PFU doses, strong proteins in macaques and in humans, indicating that the E2EP3 304

239 CHIKV-specific CD8+ T cell responses were induced. CHIKV-specific and the 3025 to 3066 epitopes are recognized by both human and 305

240 CD8+ T cells were preferentially directed against the E1 and E2 pro- macaques; which is consistent with previous reports (Table 1 [67]). 306

241 teins. Neutralizing humoral responses were also induced at high This active research path leading to new epitopes is opening new 307

242 titers. Remarkably, mice treated with a single dose of the MVA- prospects for the development of epitope-based vaccines against 308

243 CHIKV vaccine were fully protected from a CHIKV challenge [61]. CHIKV. 309

244 Protein subunit vaccines based on portions of some viral pro-

245 teins have also been generated. A formulation based on two
246 components (the 254 aa C-terminus region of the E1 protein and 4. The plant-based vaccines scenario 310

247 the full-length E2 protein) was produced using Escherichia coli BL21
248 (DE3) as expression host. Subcutaneous immunization of BALB/c Current plant biotechnology tools make possible the use of 311

249 on days 0, 21, and 35 with 40 ␮g of either truncated E1 or E2 using plants as both efficient bio-factories and delivery vehicles of sub- 312

250 different adjuvants (Freund’s complete adjuvant, Alum, or Mon- unit vaccines [68]. A wide range of proteins of pharmaceutical 313

251 tanide ISA720) was performed. This subunit vaccine candidate was interest have been expressed in plants with several plant-made 314

252 able to induce broad and long lasting neutralizing antibodies and vaccine candidates being evaluated in clinical trials, including those 315

253 cell-mediated immune responses [62]. for swine influenza, rabies, and hepatitis B [69]. This technology 316

254 Interestingly, at least two CHIKV sub-domains (E2 domain A offers substantial advantages such as the absence of mammalian 317

255 and B) are associated with human protective immunity based on pathogens in the production process, low production cost, no 318

256 antibody-dependent neutralization [63]. Another subunit vaccine requirement for fermentation systems, and efficient synthesis of 319

257 candidate against CHIKV is based on the 364 aa ectodomain from complex proteins [70,71]. It is also possible to design oral vac- 320

258 E2 produced in the E. coli BL21 (DE3) strain. The intramuscular cines based on the induction of specific immune responses in the 321

259 administration of 50 ␮g of the recombinant protein, delivered gut associated lymphoid tissues (GALT) by orally administering 322

260 as liposomes (CadB), in BALB/c mice induced neutralizing IgG plant biomass from edible plant species. This oral administration 323

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
4 J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx

Table 1
Comparison of human and macaque CHIKV B-cell epitopes (taken from Kam et al. [67]).

CHIKV protein Identified B-cell epitope (human) Amino acid Identified B-cell epitope Amino acid
(macaque)

nsP1 AEEEREAELTREALPPLQ 497–514

nsP2
nsP3 GVNSVAIPLLSTGVYSGG 1433–1450 IQMRTQVELLDEHISIDC 1489–1506
FGASSETFPITFGDFNEGEIESLSSELLT 1801–1867 TVPVAPPRRRRGRNLTVT 1729–1746
FGDFLPGEVDDLTDSDWSTCSCSD
TDDELLDRAGGYIFS
nsP4 AAIIQRLKRGCRLYLMSETPKVPTYR 1937–1962
Capsid KDIVTKITPEGAEEW 2721–2735 PPKKKPAQKKKKPGRRERMCMKIEND 2561–2586
RPIFDNKGRVVAIVLGGA 2689–2706
E3 LLQASLTCSPHRQRR 2785–2799
E2 STKDNFNVYKATRPYLAHC 2800–2818 STKDNFNVYKATRPYLAHC 2800–2818
TDGTLKIQVSLQIGIKTDDSHDWTKLRY 2841–2882 ATTEEIEVHMPPDTPDRT 2961–2978
MDNHMPADAERAGL
LTTTDKVINNCKVDQCHAAVTNHKKW 3009–3034 GNVKITVNGQTVRYKCNC 2985–3002
HAAVTNHKKWQYNSPLVPRNAEL 3025–3058 HAAVTNHKKWQYNSPLVPRNAEL 3025–3066
GDRKGKIHIPFPLAN GDRKGKIHIPFPLANVTCR
PTVTYGKNQVIMLLYPDHPTLLSYRN 3073–3098
PTEGLEVTWGNNEPYKYWPQLSTNGT 3121–3146
LLSMVGMAAGMCMCARRRCITPY 3177–3210
ELTPGATVPFL
6K
E1

Regions of B cell epitopes found that are common to both human and macaque are bold.

324 approach offers additional attractive features including friendly subunit vaccines is aided by designs based on the B sub- 340

325 delivery, safer administration than parenteral vaccines, and the units of bacterial toxins [74,75], Virus Like Particles (VLPs) 341

326 avoidance of purification [72]. Thus these vaccines are highly [76], or immunoglobulin-based immune complexes [77]. These 342

327 attractive vaccines due to their low production cost which will approaches have allowed attaining high immunogenic activity for 343

328 allow attaining proper vaccination coverage in developing coun- several vaccines administered by the oral, intranasal, and par- 344

329 tries. The production of functional immunogens in the plant cell enteral routes. The main antigenic configurations explored in 345

330 has been reported thus far following either oral or parenteral plant-based vaccines are described below. 346

331 immunization schemes. The distinct expression modalities to pro-

332 duce antigenic proteins in the plant cell are described in Table 2. 4.1. LTB- and CTB-based chimeras 347
333 Until now, highly efficient transient expression systems have been
334 adopted by the industry to produce parenteral vaccines by purify- The B subunits of either the cholera toxin (from Vibrio cholerae) 348
335 ing the antigen from plant tissues [73]. Therefore the concept of or the heat labile toxin (from enterotoxigenic E. coli) called CTB 349
336 plant-based vaccines is in the transition from a vision to a reality, and LTB, respectively; have played a relevant role on vaccinol- 350
337 with several candidates under evaluation in clinical trials [69]. ogy since these toxin-derived molecules lack of toxic activity but 351
338 Table 3 lists the antigenic configurations successfully imple- retain the GM1 binding activity, show a high immunogenicity, and 352
339 mented in plant-based systems. The immunogenic activity of exert immunomodulatory effects toward co-administered or fused 353

Table 2
Characteristics of the expression modalities for plant-based vaccines.

Expression modality Advantages Limitations Possible CHIKV References

expression targets

Nuclear stable Stable transgene insertion Low levels of recombinant C, E3, E2, 6K, E1 [110]
transformation Post-translational protein nsP1–4
modifications Non-site specific transgene
Well established insertion
transformation methods Horizontal gene transfer is
possible
Susceptible to gene silencing
Nuclear transient Short production time Low reproducibility C, E3, E2, 6K, E1 [111]
expression Very high levels of Transient expression limit the
recombinant protein Purification of the antigen is
required
Chloroplast High levels of recombinant Lack of complex C, E3, 6K [112]
transformation protein post-translational nsP1–4
Polycistronic expression is modifications
viable Long time for generation of
Improved biosafety as transformants
transgene is inherited Limited transformation
maternally protocols
Site-specific insertion through
recombination
Non-susceptible to gene
silencing

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx 5

Table 3 or silencing by RNAi, the genes or transcripts coding for the target 396
Antigen configuration on plant-made vaccines.
glycosyltransferases, and/or introducing mammalian glycosylation 397

Antigen Advantages Disadvantages References genes [83,84]. Therefore, the precedents on VLPs production in 398
configuration plant systems for targeting other pathogens make these systems 399

CTB/LTB High mucosal May induce [113] a reliable path to develop attractive CHIKV vaccine candidates. 400
chimeras immunogenic- tolerance
ity 4.3. Immune complexes 401
VLPs High immuno- Limitations on [114]
genicity the size of
heterologous Plants can express complex recombinant proteins requiring 402

sequences in post-translational modifications, which are problematic to gen- 403

chimeric VLPs erate using other production platforms such as bacterial systems 404
Immune High immuno- Depend of a [93]
[85]. It has long been known that primary and secondary antibody 405
complexes genicity high-yield
protein responses to model antigens can be enhanced by immunization 406

expression with immune complexes (ICs) [86,87]. Importantly, it is well estab- 407
ELPylation High immuno- The longest ELP [97] lished that ICs can be cross-presented by antigen-presenting cells 408
genicity subunits the (APC) and can stimulate potent T cell responses via MHC class I as 409
Robust easiest to
well as class II molecules [88,89]. It has also been suggested that, 410
expression purify, but also
Easy achieves low through binding to Fc receptors and complement receptors, the ICs 411

purification expression localize on the surface of follicular dendritic cells (FDCs), which 412
levels play an important role in the selection and affinity maturation of 413

B cells, or the complexes might directly stimulate B cells via their 414

complement receptors [90]. 415

354 unrelated antigens [78]. In the field of plant-based vaccines, these However the conventional preparation of ICs is not feasible for 416
355 molecules have been extensively used as adjuvant carriers, espe- vaccine production at a commercial scale, since it relies on the 417
356 cially within the goal of developing oral vaccines since CTB/LTB use of either polyclonal antisera or expensive monoclonal anti- 418
357 serve as transmucosal carriers able to deliver the antigen at the sub bodies cocktails (mAbs) to achieve multimerization with a given 419
358 mucosa [79]; where they can be efficiently processed by dendritic antigen. To address these limitations, Chargelegue et al. described, 420
359 cells (DC) with the subsequent induction of robust mucosal and for the first time, the production of recombinant ICs in trans- 421
360 systemic Th1 immune responses. Several vaccination models based genic tobacco plants by expressing the tetanus toxin fragment C 422
361 on this approach have proven to be effective at the preclinical level fused to a mAb. The design of the recombinant IC fusion molecule 423
362 [80] and a clinical trial was performed with potatoes expressing LTB resulted in the expression of the antibody-antigen at a 1:2 ratio 424
363 with positive outcomes in terms of seroconversion [81]. Therefore, (an antigen molecule was fused to each antibody heavy chain). 425
364 fusing specific epitopes or domains from protective CHIKV antigens Moreover, it was demonstrated that serum antibody responses 426
365 to CTB/LTB will serve as an attractive approach in the development leading to protective immunity were induced without the use 427
366 of mucosal vaccines produced in plant tissues. The use of edible of additional adjuvants in subcutaneously immunized mice [91]. 428
367 plant species will greatly facilitate the development of low cost Using similar approaches, multiple antigens have been expressed 429
368 oral vaccine prototypes. in tobacco being able to induce both humoral and cellular immune 430

responses that protected against intracellular pathogens such as 431

369 4.2. Virus-like particles Mycobacterium tuberculosis. Intranasal immunization of mice with 432

ICs boosted the Bacillus Calmette–Guerin strain vaccine (BCG)- 433

370 Virus-Like Particles (VLPs) are self-assembled structures derived induced immunity and, importantly, conferred further protection 434

371 from viral antigens that mimic the native architecture of viruses but against M. tuberculosis infection [92]. Remarkably, these method- 435

372 lack the viral genome and thus are not infective. Several reports ologies have been applied to target viruses. For example, transient 436

373 on the expression of properly assembled VLPs at adequate lev- expression of Ebola-based immune complexes in plants has been 437

374 els have been published for Influenza virus, Human papillomavirus, achieved by fusing the Ebola GP1 glycoprotein subunit to a spe- 438

375 Human immunodeficiency virus, Norwalk virus, and Hepatitis B virus; cific humanized heavy chain of 6D8 IgG monoclonal antibody. Mice 439

376 among others (reviewed in [71]). Since recent reports on CHIKV subcutaneously immunized with Ebola ICs developed high titers of 440

377 eVLPs production in mammalian and bacterial expression systems Ebola-specific IgG antibodies [93]. Thus, these studies show that ICs 441

378 have proven to be efficacious (see above), the adoption of this might represent an attractive immunization strategy against viral 442

379 concept in the form of plant-derived vaccines is a possibility. The antigens and represent a promise for the development of highly 443

380 glycosylation patterns of VLP proteins have a major impact on immunogenic CHIKV vaccines. 444

381 their structure and function since these viral glycoproteins local-
382 ize, guide, and potentiate the process of enveloped virus assembly. 4.4. Elastin-like polypeptide fusions 445

383 Therefore, the design of plant-made vaccines based on the E1 and

384 E2 glycoproteins demands a platform with the capacity to perform The major cost in the production of subunit vaccines is related to 446

385 these complex post-translational modifications. The N-glycan syn- the separation and purification steps, which can account for up to 447

386 thesis in the endoplasmic reticulum is relatively well conserved in 90% of total production costs [94]. A benefit of plant-based expres- 448

387 eukaryotes [82], thus it is expected that plants will provide the func- sion systems is that they allow for alternative purification methods, 449

388 tional machinery to produce CHIKV eVLPs properly. In fact, some such as elastin-like polypeptide (ELP) fusion technology. The ELPs 450

389 differences on glycosylation are expected in plants in comparison ***Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly, Ala-Pro-Gly-Val-Gly-Val, 451

390 to mammal cells; however, this may account for the antigenicity of and Val-Pro-Gly-Xaa-Gly)n (where Xaa represents one random 452

391 the plant-made CHIKV antigen due to a better recognition by anti- amino acid excluding Pro) exhibit the unique characteristic of 453

392 gen presenting cells through pattern-recognition receptors. If the reversible phase transition, meaning that they can be precipi- 454

393 hypothesis of higher antigenicity due to plant glycosylation fails, tated out of solution and re-suspended again through temperature 455

394 an alternative path consists on using the glycoengineering strate- manipulation. The temperature-dependent, reversible aggrega- 456

395 gies that have been implemented in plants through knocking out tion/precipitation properties of ELPs provides an alternative to the 457

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
6 J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx

Fig. 1. Schematic representation of CHIKV immunopathology and the implications of plant-based vaccines in preventing the CHIKV infection. (A) It is known that CHIKV
activates immune mechanisms that include the recruitment of macrophages as well as lymphocytes, early secretion of IgG3, and secretion of IFN␣/␤. (B) Plant-based vaccines
are proposed as a convenient approach to produce antigenic proteins in the form of immune complexes, multiepitope vaccines, or Virus like particles. These systems will
allow for the evaluation of prime-boost immunization schemes against CHIKV, where plant-made antigens can be purified and used for parenteral priming while minimally
processed plant material can be used for oral boosting.

458 cost-intensive affinity chromatography and allows to separate the recent identification and characterization of linear CHIKV B cell- 488

459 protein of interest from host proteins via centrifugation of crude epitopes will allow designing innovative vaccines. Multiepitope 489

460 extracts [95]. The efforts on plant-derived vaccines against human vaccines against CHIKV are also a possibility when producing plant- 490

461 diseases using this approach are limited. One of the few studies based vaccines since multiepitope chimeric proteins have been 491

462 comprises the M. tuberculosis antigens Ag85B and ESAT-6, which produced in plants leading to promising findings [98,99]. Our group 492

463 were fused to ELP and expressed in transgenic tobacco [96]. The has developed plant-made vaccines against HIV, which were based 493

464 subcutaneous immunization of mice with either 5 ␮g or 10 ␮g of on multiepitope polypeptides and against Taenia solium, which 494

465 ICs on days 0, 14, and 28; induced long-lasting humoral immune were based on several peptides produced through the 2A-based 495

466 responses after 84 days. Interestingly the ELP was not removed ribosomal skip mechanism [100]. Other groups have developed 496

467 from the antigen and it was found that the ELP had no effect on multicomponent vaccines against enteric pathogens based on 497

468 the immune response against the target antigens in mice. Another chimeric proteins [101]. 498

469 interesting approach consisted on the production of ELP fused to an Since some targets have been identified as protective anti- 499

470 established soluble trimer-forming H5N1 HA (ELPylated H5 HA, or gens against CHIKV, the plants producing those antigens along 500

471 H5-ELP) in tobacco. These recombinant proteins were easily puri- with convenient carriers could be evaluated as CHIKV vaccine 501

472 fied by the inverse transition cycling technique. The subcutaneous candidates. The vaccine developmental steps would comprise: (i) 502

473 administration in BL6 mice induced neutralizing antibodies after designing immunogens based on proteins or epitopes associated 503

474 two doses administered on days 0 and 14. It was also found that with immunoprotection against CHIKV and developing transgenic 504

475 ELPylation does not interfere with the immunogenicity of the vac- plants carrying the corresponding genes; (ii) characterizing the 505

476 cine candidate [97]. Therefore, this precedent of positive outcomes plant-made antigen in terms of yields and antigenic activity; (iii) 506

477 on targeting an enveloped virus through the ELP technology repre- proving the immunogenic activity at the preclinical level through 507

478 sents a positive prospect for proposing CHIKV vaccine candidates test animals immunization under distinct administration routes, 508

479 under this configuration. and (iv) planning clinical trials once efficacy and safety in test ani- 509

mals have been proven. 510

Therefore, this scenario indicates that a new avenue on devel- 511

480 5. Prospects for using plant-based CHIKV immunization oping anti-CHIKV vaccines will be viable to implement with the 512

481 approaches experience gained in the field working with other pathogens over 513

the last two decades. This will constitute a relevant research field, 514

482 Plant-based technologies offer a myriad of possibilities to allowing for the development of low cost and efficient vaccines. 515

483 develop new vaccines against CHIKV thanks to the improvements However it should be recognized that the progress in the field of 516

484 of the technology achieved during the last two decades. Since sev- plant-made vaccines has been focused to parenteral formulations 517

485 eral subunit vaccines against CHIKV have been explored using using transient expression systems [71], while oral vaccination 518

486 other expression systems, there is sufficient knowledge to sup- approaches still require optimization to address proper immuno- 519

487 port a straightforward design of plant-based vaccines (Fig. 1). The genicity, a better control on dosage as well as antigen degradation, 520

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx 7

521 and finding a proper regulatory framework [102,103]. Regarding [9] Renault P, Solet JL, Sissoko D, Balleydier E, Larrieu S, et al. A major epidemic 585

522 oral vaccines, a recently proposed idea is based on the inducible of Chikungunya virus infection on Reunion Island, France, 2005–2006. Am J 586
Trop Med Hyg 2007;77:727–31. 587
523 expression of antigens mediated by viral vectors in edible plant [10] Duong V, Andries AC, Ngan C, Sok T, Richner B, et al. Reemergence of Chikun- 588
524 species; which will allow acquiring high expression levels avoid- gunya virus in Cambodia. Emerg Infect Dis 2012;18:2066–9. 589
525 ing the purification steps. Therefore, plant biomass expressing high [11] Ansumana R, Jacobsen KH, Leski TA, Covington AL, Bangura U, et al. 590
Reemergence of Chikungunya virus in Bo, Sierra Leone. Emerg Infect Dis 591
526 levels of the antigen might be used for the formulation of low cost 2013;19:1108–10. 592
527 oral vaccines consisting of freeze-dried plant material delivered in [12] Leparc-Goffart I, Nougairede A, Cassadou S, Prat C, de Lamballerie X. Chikun- 593
528 gelatin capsules [71]. Overall a convenient possibility for immu- gunya in the Americas. Lancet 2014;383:514. 594
[13] Fischer M, Staples E. Update on emerging infections: news from the Cen- 595
529 nization schemes against CHIKV infection comprises parenteral
ters for Disease Control and Prevention. Notes from the field: Chikungunya 596
530 priming with purified plant-made antigens and subsequent oral virus spreads in the Americas-Caribbean and South America, 2013–2014. Ann 597
531 boosts with freeze-dried plant material expressing the candidate Emerg Med 2014;64(5):552–3. 598
[14] Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation 599
532 subunit vaccine (Fig. 1), as it has been proposed for the case of
in Chikungunya virus affects vector specificity and epidemic potential. PLoS 600
533 Hepatitis B Virus infection [104]. Pathog 2007;3:1895–906. 601
[15] Schuffenecker I, Iteman I, Michault A, et al. Genome microevolution of Chikun- 602
gunya viruses causing the Indian Ocean outbreak. PLoS Med 2006;3:1058–70. 603
534 6. Concluding remarks [16] Ravichandran R, Manian M. Ribavirin therapy for Chikungunya arthritis. J Infect 604
Dev Ctries 2008;2:140–2. 605

535 Considering that no vaccines against CHIKV approved for human [17] Briolant S, Garin D, Scaramozzino N, Jouan A, Crance JM. In vitro inhibition of 606
Chikungunya and Semliki Forest viruses replication by antiviral compounds: 607
536 use are available and that the disease is already present in the synergistic effect of interferon-alpha and ribavirin combination. Antiviral Res 608
537 Americas, the need for advancing in this research field is great. 2004;61:111–7. 609
538 The following years will be critical to envision the real poten- [18] De Lamballerie X, Boisson V, Reynier JC, Enault S, Charrel RN, Flahault A, et al. 610
On chikungunya acute infection and chloroquine treatment. Vector Borne 611
539 tial to address this issue. Considering that promising candidates Zoonotic Dis 2008;8:837–9. 612
540 are in preclinical trials, the implementation in parallel of low cost [19] Khan M, Santhosh SR, Tiwari M, Lakshmana Rao PV, Parida M. Assessment of 613
541 production platforms will be determinant as CHKV is mainly affect- in vitro prophylactic and therapeutic efficacy of chloroquine against Chikun- 614
gunya virus in vero cells. J Med Virol 2010;82:817–24. 615
542 ing developing countries where access to vaccine is limited due
[20] Couderc T, Khandoudi N, Grandadam M, Visse C, Gangneux N, Bagot S, 616
543 to the high cost of conventional vaccines. In this review, plant- et al. Prophylaxis and therapy for Chikungunya virus infection. J Infect Dis 617
544 based platforms have been identified as promising approaches to 2009;200:516–23. 618
[21] Kam YW, Lum FM, Teo TH, Lee WW, Simarmata D, Harjanto S, et al. Early 619
545 fight CHIKV. The maturation that this technology achieved dur-
neutralizing IgG response to Chikungunya virus in infected patients tar- 620
546 ing the last decade currently allows for the production of several gets a dominant linear epitope on the E2 glycoprotein. EMBO Mol Med 621
547 vaccine types ranging from oral formulations, which will required 2012;4:330–43. 622

548 long-term research to be assessed and optimized, to parenteral [22] Kam YW, Lee WW, Simarmata D, Harjanto S, Teng TS, Tolou H, et al. Longitudi- 623
nal analysis of the human antibody response to Chikungunya virus infection: 624
549 vaccines produced under GMP-compliant transient expression sys- implications for serodiagnosis and vaccine development. J Virol 2012;86(Dec 625
550 tems [105,106]. The latter has the best potential to become a reality (23)):13005–15. 626

551 in the near future. Based on previous experiences, such as the devel- [23] Lum FM, Teo TH, Lee WW, Kam YW, Rénia L, Ng LF. An essential role 627
of antibodies in the control of Chikungunya virus infection. J Immunol 628
552 opments on influenza and other viral pathogens vaccines [107], 2013;190:6295–302. 629
553 VLPs are the most efficacious approach to render immunogenic [24] Aron GM, Irvin JD. Inhibition of herpes simplex virus multiplication by the 630
554 formulations even at low doses [108]. For example, influenza plant- pokeweed antiviral protein. Antimicrob Agents Chemother 1980;17:1032–3. 631
[25] Ishag HZ, Li C, Huang L, Sun MX, Ni B, Guo CX, et al. Inhibition of Japanese 632
555 based vaccines are in an advanced developmental stage, being encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein. 633
556 evaluated in clinical trials [109]. Virus Res 2013;171:89–96. 634
557 In conclusion, the concept of plant-based vaccines constitutes a [26] Rothan HA, Bahrani H, Shankar EM, Rahman NA, Yusof R. Inhibitory effects of 635
a peptide-fusion protein (Latarcin-PAP1-Thanatin) against Chikungunya virus. 636
558 relevant path for the development of CHIV vaccines with attractive
Antiviral Res 2014;108:173–80. 637
559 features. [27] Couderc T, Chretien F, Schilte C, Disson O, Brigitte M, et al. A mouse model for 638
Chikungunya: young age and inefficient Type-I interferon signaling are risk 639
factors for severe disease. PLoS Pathog 2008;4:e29. 640
560 Acknowledgements [28] Gardner J, Anraku I, Le TT, Larcher T, Major L, et al. Chikungunya virus arthritis 641
in adult wild-type mice. J Virol 2010;84:8021–32. 642

561 Current investigations from the group are supported by [29] Morrison TE, Oko L, Montgomery SA, Whitmore AC, Lotstein AR, et al. A mouse 643
model of Chikungunya virus-induced musculoskeletal inflammatory disease: 644
Q5 CONACYT/México (grant CB-2008-01, 102109to SRM and grant CB-
562
evidence of arthritis, tenosynovitis, myositis, and persistence. Am J Pathol 645
563 2010-01, 151818 to CA) and FAI/UASLP/2015 to SRM. 2011;178:32–40. 646
[30] Labadie K, Larcher T, Joubert C, Mannioui A, Delache B, Brochard P, et al. 647
Chikungunya disease in nonhuman primates involves long-term viral per- 648
564 References sistence in macrophages. J Clin Invest 2010;120:894–906. 649
[31] Chow A, Her Z, Ong EK, Chen JM, Dimatatac F, et al. Persistent arthralgia 650
565 [1] Brighton SW, Simson IW. A destructive arthropathy following Chikungunya induced by Chikungunya virus infection is associated with interleukin- 651
566 virus arthritis—a possible association. Clin Rheumatol 1984;3(2):253–8. 6 and granulocyte macrophage colony-stimulating factor. J Infect Dis 652
567 [2] Ross RW. The Newala epidemic. III. The virus: isolation, pathogenic properties 2011;203:149–57. 653
568 and relationship to the epidemic. J Hyg (London) 1956;54(2):177–91. [32] Chirathaworn C, Rianthavorn P, Wuttirattanakowit N, Poovorawan Y. Serum 654
569 [3] Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and IL-18 and IL-18BP levels in patients with Chikungunya virus infection. Viral 655
570 evolution. Microbiol Rev 1994;58:491–562. Immunol 2010;23:113–7. 656
571 [4] Kuhn RJ. Togaviridae: the viruses and their replication. In: Knipe DM, How- [33] Hoarau JJ, Jaffar Bandjee MC, Krejbich Trotot P, Das T, Li-Pat-Yuen G, Dassa B, 657
572 ley PM, editors. Fields’ virology. 5th ed. NY, USA: Lippincott, Williams and et al. Persistent chronic inflammation and infection by Chikungunya arthri- 658
573 Wilkins; 2007. p. 1001–22. togenic alphavirus in spite of a robust host immune response. J Immunol 659
574 [5] Voss JE, Vaney MC, Duquerroy S, Vonrhein C, Girard-Blanc C, et al. Gly- 2010;184:5914–27. 660
575 coprotein organization of Chikungunya virus particles revealed by X-ray [34] Dupuis-Maguiraga L, Noret M, Brun S, Le Grand R, Gras G, Roques P. Chikun- 661
576 crystallography. Nature 2010;468:709–12. gunya disease: infection-associated markers from the acute to the chronic 662
577 [6] Munasinghe DR, Amarasekera PJ, Fernando CF. An epidemic of dengue-like phase of arbovirus-induced arthralgia. PLoS Negl Trop Dis 2012;6:e1446. 663
578 fever in Ceylon (chikungunya—a clinical and haematological study). Ceylon [35] Messaoudi I, Vomaske J, Totonchy T, Kreklywich CN, Haberthur K, Springgay 664
579 Med J 1966;11:129–42. L, et al. Chikungunya virus infection results in higher and persistent viral repli- 665
580 [7] Pavri K. Disappearance of Chikungunya virus from India and South East Asia. cation in aged rhesus macaques due to defects in anti-viral immunity. PLoS 666
581 Trans R Soc Trop Med Hyg 1986;80(491). Negl Trop Dis 2013;7:e2343. 667
582 [8] Lam SK, Chua KB, Hooi PS, Rahimah MA, Kumari S, et al. Chikungunya [36] Hawman DW, Stoermer KA, Montgomery SA, Pal P, Oko L, Diamond MS, et al. 668
583 infection—an emerging disease in Malaysia. Southeast Asian J Trop Med Public Chronic joint disease caused by persistent Chikungunya virus infection is con- 669
584 Health 2001;32:447–51. trolled by the adaptive immune response. J Virol 2013;87:13878–88. 670

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
8 J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx

671 [37] Weaver SC, Osorio JE, Livengood JA, Chen R, Stinchcomb DT. Chikungunya virus [64] Fong RH, Banik SS, Mattia K, Barnes T, Tucker D, Liss N, et al. Exposure of 756
672 and prospects for a vaccine. Expert Rev Vaccines 2010;11:1087–101. epitope residues on the outer face of the Chikungunya virus envelope trimer 757
673 [38] Akahata W, Yang ZY, Andersen H, Sun S, Holdaway HA, Kong WP, et al. A determines antibody neutralizing efficacy. J Virol 2014;88:14364–79. 758
674 virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman [65] Chen CI, Clark DC, Pesavento P, Lerche NW, Luciw PA, Reisen WK, et al. 759
675 primates against infection. Nat Med 2010;16:334–8. Comparative pathogenesis of epidemic and enzootic Chikungunya virus 760
676 [39] White A, Berman S, Lowenthal JP. Comparative immunogenicities of Chikun- in a pregnant Rhesus macaque model. Am J Trop Med Hyg 2010;83: 761
677 gunya vaccines propagated in monkey kidney monolayers and chick embryo 1249–58. 762
678 suspension cultures. Appl Microbiol 1972;23:951–2. [66] Higgs S, Ziegler SA. A nonhuman primate model of Chikungunya disease. J 763
679 [40] Kumar M, Sudeep AB, Arankalle VA. Evaluation of recombinant E2 protein- Clin Invest 2010;120:657–60. 764
680 based and whole-virus inactivated candidate vaccines against Chikungunya [67] Kam YW, Lee WW, Simarmata D, Le Grand R, Tolou H, Merits A, et al. Unique 765
681 virus. Vaccine 2012;30:6142–9. epitopes recognized by antibodies induced in Chikungunya virus-infected 766
682 [41] Levitt NH, Ramsburg HH, Hasty SE, Repik PM, Cole Jr FE, Lupton HW. Devel- non-human primates: implications for the study of immunopathology and 767
683 opment of an attenuated strain of Chikungunya virus for use in vaccine vaccine development. PLoS ONE 2014;9:e95647. 768
684 production. Vaccine 1986;4:157–62. [68] Govea-Alonso DO, Rybicki E, Rosales-Mendoza S. Plant-based vaccines as a 769
685 [42] McClain DJ, Pittman PR, Ramsburg HH, Nelson GO, Rossi CA, Mangiafico JA, global vaccination approach: current perspectives. In: Genetically engineered 770
686 et al. Immunologic interference from sequential administration of live atten- plants as a source of vaccines against wide spread diseases diseases—an inte- 771
687 uated alphavirus vaccines. J Infect Dis 1998;177:634–41. grated view. New York, NY: Springer; 2014. p. 265–80. 772
688 [43] Edelman R, Tacket CO, Wasserman SS, Bodison SA, Perry JG, Mangiafico JA. [69] Yusibov V, Streatfield SJ, Kushnir N. Clinical development of plant-produced 773
689 Phase II safety and immunogenicity study of live Chikungunya virus vaccine recombinant pharmaceuticals: vaccines, antibodies and beyond. Hum Vaccin 774
690 TSI-GSD-218. Am J Trop Med Hyg 2000;62:681–5. 2011;7:313–21. 775
691 [44] Wang E, Volkova E, Adams AP, Forrester N, Xiao SY, Frolov I, et al. Chimeric [70] Govea-Alonso DO, Cardineau GA, Rosales-Mendoza S. Principles of plant- 776
692 alphavirus vaccine candidates for chikungunya. Vaccine 2008;26:5030–9. based vaccines. In: Genetically engineered plants as a source of vaccines 777
693 [45] Wang D, Suhrbier A, Penn-Nicholson A, Woraratanadharm J, Gardner J, Luo against wide spread diseases—an integrated view. New York, NY: Springer; 778
694 M, et al. A complex adenovirus vaccine against Chikungunya virus provides 2014. p. 1–14. 779
695 complete protection against viraemia and arthritis. Vaccine 2011;29:2803–9. [71] Salazar-González JA, Bañuelos-Hernández B, Rosales-Mendoza S. Current sta- 780
696 [46] Darwin JR, Kenney JL, Weaver SC. Transmission potential of two chimeric tus of viral expression systems in plants and perspectives for oral vaccines 781
697 Chikungunya vaccine candidates in the urban mosquito vectors, Aedes aegypti development. Plant Mol Biol 2015;87:203–17. 782
698 and Ae. albopictus. Am J Trop Med Hyg 2011;84(6):1012–5. [72] García-Hernández AL, Rubio-Infante N, Moreno-Fierros L. Mucosal immunol- 783
699 [47] Muthumani K, Lankaraman KM, Laddy DJ, Sundaram SG, Chung CW, Sako E, ogy and oral vaccination. In: Genetically engineered plants as a source of 784
700 et al. Immunogenicity of novel consensus-based DNA vaccines against Chikun- vaccines against wide spread diseases diseases—an integrated view. New 785
701 gunya virus. Vaccine 2008;26:5128–34. York, NY: Springer; 2014. p. 15–42. 786
702 [48] Mallilankaraman K, Shedlock DJ, Bao H, Kawalekar OU, Fagone P, Ramanathan [73] Gleba Y, Klimyuk V, Marillonnet S. Magnifection—a new platform for express- 787
703 AA, et al. A DNA vaccine against Chikungunya virus is protective in mice and ing recombinant vaccines in plants. Vaccine 2005;23:2042–8. 788
704 induces neutralizing antibodies in mice and nonhuman primates. PLoS Negl [74] Granell A, Fernández del-Carmen A, Orzáez D. In planta production 789
705 Trop Dis 2011;5(1):e928. of plant-derived and non-plant-derived adjuvants. Expert Rev Vaccines 790
706 [49] Hallengärd D, Lum FM, Kümmerer BM, Lulla A, Lulla V, García-Arriaza J, 2010;9:843–58. 791
707 et al. Prime-boost immunization strategies against Chikungunya virus. J Virol [75] Kwon KC, Verma D, Singh ND, Herzog R, Daniell H. Oral delivery of human 792
708 2014;88:13333–43. biopharmaceuticals, autoantigens and vaccine antigens bioencapsulated in 793
709 [50] Hallengärd D, Kakoulidou M, Lulla A, Kümmerer BM, Johansson DX, Mutso plant cells. Adv Drug Delivery Rev 2013;Jun (65):782–99. 794
710 M, et al. Novel attenuated Chikungunya vaccine candidates elicit protective [76] Scotti N, Rybicki EP. Virus-like particles produced in plants as potential vac- 795
711 immunity in C57BL/6 mice. J Virol 2014;88:2858–66. cines. Expert Rev Vaccines 2013;12:211–24. 796
712 [51] Bredenbeek PJ, Rice CM. Animal RNA virus expression systems. Sem Virol [77] Kim MY, Reljic R, Kilbourne J, Ceballos-Olvera I, Yang MS, Reyes- 797
713 1992;3:297–310. Del Valle J, et al. Novel vaccination approach for dengue infec- 798
714 [52] Frolov I, Hoffman TA, Prágai BM, Dryga SA, Huang HV, Schlesinger S, et al. tion based on recombinant immune complex universal platform. Vac- 799
715 Alphavirus-based expression vectors: strategies and applications. Proc Natl cine 2015, http://dx.doi.org/10.1016/j.vaccine.2015.02.036 (pii: S0264- 800
716 Acad Sci USA 1996;93:11371–7. 410X(15)00225-X). 801
717 [53] Chang LJ, Dowd KA, Mendoza FH, Saunders JG, Sitar S, Plummer SH, et al. [78] Langridge W, Dénes B, Fodor I. Cholera toxin B subunit modulation of mucosal 802
718 Safety and tolerability of Chikungunya virus-like particle vaccine in healthy vaccines for infectious and autoimmune diseases. Curr Opin Investig Drugs 803
719 adults: a phase 1 dose-escalation trial. Lancet 2014;384:2046–52. 2010;11:919–28. 804
720 [54] Brandler S, Ruffié C, Combredet C, Brault JB, Najburg V, Prevost MC, et al. A [79] Kohli N, Westerveld DR, Ayache AC, Verma A, Shil P, Prasad T, et al. Oral 805
721 recombinant measles vaccine expressing Chikungunya virus-like particles is delivery of bioencapsulated proteins across blood–brain and blood–retinal 806
722 strongly immunogenic and protects mice from lethal challenge with Chikun- barriers. Mol Ther 2014;22:535–46. 807
723 gunya virus. Vaccine 2013;31:3718–25. [80] Daniell H, Singh ND, Mason H, Streatfield SJ. Plant-made vaccine antigens and 808
724 [55] Metz SW, Geertsema C, Martina BE, Andrade P, Heldens JG, van Oers MM, biopharmaceuticals. Trends Plant Sci 2009;14:669–79. 809
725 et al. Functional processing and secretion of Chikungunya virus E1 and E2 [81] Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ. 810
726 glycoproteins in insect cells. Virol J 2011;8:353. Immunogenicity in humans of a recombinant bacterial antigen delivered in a 811
727 [56] Metz SW, Martina BE, van den Doel P, Geertsema C, Osterhaus AD, Vlak JM, transgenic potato. Nat Med 1998;4:607–9. 812
728 Pijlman GP. Chikungunya virus-like particles are more immunogenic in a lethal [82] Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson 813
729 AG129 mouse model compared to glycoprotein E1 or E2 subunits. Vaccine C, Michaud D, et al. Plant-specific glycosylation patterns in the context of 814
730 2013;31:6092–6. therapeutic protein production. Plant Biotechnol J 2010;8:564–87. 815
731 [57] Metz SW, Gardner J, Geertsema C, Le TT, Goh L, Vlak JM, et al. Effective Chikun- [83] Strasser R, Stadlmann J, Svoboda B, Altmann F, Glossl J, Mach L. Molecular 816
732 gunya virus-like particle vaccine produced in insect cells. PLoS Negl Trop Dis basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana 817
733 2013;7:e2124. plants lacking complex N-glycans. Biochem J 2005;387:385–91. 818
734 [58] Wagner JM, Pajerowski JD, Daniels CL, McHugh PM, Flynn JA, Balliet JW, et al. [84] Schähs M, Strasser R, Stadlmann J, Kunert R, Rademacher T, Steinkellner 819
735 Enhanced production of Chikungunya virus-like particles using a high-pH H. Production of a monoclonalantibody in plants with a humanized N- 820
736 adapted spodoptera frugiperda insect cell line. PLoS ONE 2014;9:e94401. glycosylation pattern. Plant Biotechnol J 2007;5:657–63. 821
737 [59] Chu H, Das SC, Fuchs JF, Suresh M, Weaver SC, Stinchcomb DT, et al. Deci- [85] Gomord V, Faye L. Posttranslational modification of therapeutic proteins in 822
738 phering the protective role of adaptive immunity to CHIKV/IRES a novel plants. Curr Opin Plant Biol 2004;7:171–81. 823
739 candidate vaccine against Chikungunya in the A129 mouse model. Vaccine [86] Laissue J, Cottier H, Hess MW, Stoner RD. Early and enhanced germinal center 824
740 2013;31:3353–60. formation and antibody responses in mice after primary stimulation with 825
741 [60] Partidos CD, Weger J, Brewoo J, Seymour R, Borland EM, Ledermann JP, et al. antigen-isologous antibody complexes as compared with antigen alone. J 826
742 Probing the attenuation and protective efficacy of a candidate Chikungunya Immunol 1971;107:822–31. 827
743 virus vaccine in mice with compromised interferon (IFN) signaling. Vaccine [87] Nie X, Basu S, Cerny J. Immunization with immune complex alters the reper- 828
744 2011;29:3067–73. toire of antigen-reactive B cells in the germinal centers. Eur J Immunol 829
745 [61] García-Arriaza J, Cepeda V, Hallengärd D, Sorzano CÓ, Kümmerer BM, Lil- 1997;27:3517–25. 830
746 jeström P, et al. A novel poxvirus-based vaccine, MVA-CHIKV, is highly [88] Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, et al. Fc gamma 831
747 immunogenic and protects mice against Chikungunya infection. J Virol receptor-mediated induction of dendritic cell maturation and major histo- 832
748 2014;88:3527–47. compatibility complex class I-restricted antigen presentation after immune 833
749 [62] Khan M, Dhanwani R, Rao PV, Parida M. Subunit vaccine formulations complex internalization. J Exp Med 1999;189:371–80. 834
750 based on recombinant envelope proteins of Chikungunya virus elicit bal- [89] Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, van Schip JJ, Sedlik C, 835
751 anced Th1/Th2 response and virus-neutralizing antibodies in mice. Virus Res et al. Antigen-antibody immune complexes empower dendritic cells to effi- 836
752 2012;167:236–46. ciently prime specific CD8+ CTL responses in vivo. J Immunol 2002;168: 837
753 [63] Lee CH, Kam YW, Fric J, Malleret B, Koh E, Prakash C, et al. Chikungunya virus 2240–6. 838
754 neutralization antigens and direct cell-to-cell transmission are revealed by [90] Heyman B. The immune complex: possible ways of regulating the antibody 839
755 human antibody-escape mutants. PLoS Pathog 2011;7:e1002390. response. Immunol Today 1990;11:310–3. 840

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104
 G Model
JVAC 16556 1–9 ARTICLE IN PRESS
J.A. Salazar-González et al. / Vaccine xxx (2015) xxx–xxx 9

841 [91] Chargelegue D, Drake PM, Obregon P, Prada A, Fairweather N, et al. Highly [103] Rosales-Mendoza S, Salazar-González JA. Immunological aspects of using 876
842 immunogenic and protective recombinant vaccine candidate expressed in plant cells as delivery vehicles for oral vaccines. Expert Rev Vaccines 877
843 transgenic plants. Infect Immun 2005;73:5915–22. 2014;13:737–49. 878
844 [92] Pepponi I, Diogo GR, Stylianou E, van Dolleweerd CJ, Drake PMW, [104] Pniewski T. Is an oral plant-based vaccine against hepatitis B virus possible. 879
845 Paul MJ, et al. Plant-derived recombinant immune complexes as self- Curr Pharm Biotechnol 2012;13:2692–704. 880
846 adjuvanting TB immunogens for mucosal boosting of BCG. Plant Biotechnol J [105] Shamloul M, Trusa J, Mett V, Yusibov V. Optimization and utilization of 881
847 2014;12:840–50. Agrobacterium-mediated transient protein production in Nicotiana. J Vis Exp Q8 882
848 [93] Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, et al. Expression of 2014;19(Apr (86)). 883
849 an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant [106] Wirz H, Sauer-Budge AF, Briggs J, Sharpe A, Shu S, Sharon A. Automated 884
850 Biotechnol J 2011;9:807–16. production of plant-based vaccines and pharmaceuticals. J Lab Autom 885
851 [94] Cunha T, Aires-Barros R. Large-scale extraction of proteins. Mol Biotechnol 2012;17(Dec (6)):449–57. 886
852 2002;20:29–40. [107] Yusibov V, Kushnir N, Streatfield SJ. Advances and challenges in the develop- 887
853 [95] Floss DM, Schallau K, Rose-John S, Conrad U, Scheller J. Elastin-like polypep- ment and production of effective plant-based influenza vaccines. Expert Rev 888
854 tides revolutionize protein expression and their biomedical application. Vaccines 2015;14(4):519–35. 889
855 Trends Biotechnol 2010;28:37–45. [108] Shoji Y, Prokhnevsky A, Leffet B, Vetter N, Tottey S, Satinover S, et al. 890
856 [96] Floss DM, Mockey M, Zanello G, Brosson D, Diogon M, Frutos R, et al. Immunogenicity of H1N1 influenza virus-like particles produced in Nicotiana 891
857 Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens benthamiana. Hum Vaccin Immunother 2015;11:118–23. 892
Q6
858 produced in transgenic plants by elastin-like peptide fusion strategy. Biomed [109] Landry N, Pillet S, Favre D, Poulin JF, Trépanier S, Yassine-Diab B, et al. 893
859 Biotechnol 2010:2010. Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit 894
860 [97] Phan HT, Pohl J, Floss DM, Rabenstein F, Veits J, Le BT, et al. ELPylated durable, poly-functional and cross-reactive T cell responses to influenza HA 895
861 haemagglutinins produced in tobacco plants induce potentially neutralizing antigens. Clin Immunol 2014;154:164–77. 896
862 antibodies against H5N1 viruses in mice. Plant Biotechnol J 2013;11:582–93. [110] Finer JJ. Plant nuclear transformation. In: Kempken F, Jung C, editors. Genetic 897
863 [98] Rubio-Infante N, Govea-Alonso DO, Romero-Maldonado A, García-Hernández modification of plants, biotechnology in agriculture and forestry, vol. 64. 898
864 AL, Ilhuicatzi-Alvarado D, Salazar-González JA, et al. A plant-derived multi- Berlin Heidelberg: Springer-Verlag; 2010. p. 3–21. 899
Q7
865 HIV antigen induces broad immune responses in orally immunized mice. Mol [111] Sheludko YV. Agrobacterium-mediated transient expression as an approach 900
866 Biotechnol 2015. to production of recombinant proteins in plants. Recent Pat Biotechnol 901
867 [99] Govea-Alonso DO, Rubio-Infante N, García-Hernández AL, Varona-Santos JT, 2008;2(3):198–208. 902
868 Korban SS, Moreno-Fierros L, et al. Immunogenic properties of a lettuce- [112] Verma D, Daniell H. Chloroplast vector systems for biotechnology applica- 903
869 derived C4(V3)6 multiepitopic HIV protein. Planta 2013;238:785–92. tions. Plant Physiol 2007;145:1129–43. 904
870 [100] Monreal-Escalante E, Bañuelos-Hernández B, Hernández M, Fragoso G, Garate [113] Matoba N, Kajiura H, Cherni I, Doran JD, Bomsel M, Fujiyama K, et al. Bio- 905
871 T, Sciutto E, et al. Expression of multiple Taenia solium immunogens in plant chemical and immunological characterization of the plant-derived candidate 906
872 cells through a ribosomal skip mechanism. Mol Biotechnol 2015. human immunodeficiency virus type 1 mucosal vaccine CTB-MPR. Plant 907
873 [101] Yu J, Langridge WH. A plant-based multicomponent vaccine protects mice Biotechnol J 2009;7:129–45. 908
874 from enteric diseases. Nat Biotechnol 2001;19:548–52. [114] Rybicki EP. Plant-based vaccines against viruses. Virol J 2014;11:205. 909
875 [102] Lamichhane A, Azegamia T, Kiyonoa H. The mucosal immune system for vac-
cine development. Vaccine 2014;32(Nov (49)):6711–23.

Please cite this article in press as: Salazar-González JA, et al. Chikungunya virus vaccines: Current strategies and prospects for developing
plant-made vaccines. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.05.104