Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

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Contents lists available at ScienceDirect

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

Review

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Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

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Jorge A. Salazar-González a , Carlos Angulo b , Sergio Rosales-Mendoza a,∗ Laboratorio de Biofarmacéuticos Recombinantes, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, Av. Dr. Manuel Nava 6, San

Q2 Luis Potosí 78210, SLP, Mexico b

Grupo de Inmunología y Vacunología, Centro de Investigaciones Biológicas del Noroeste, SC., Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, B.C.S., C.P. 23096 Mexico City, Mexico

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a r t i c l e

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a b s t r a c t

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Article history: Received 3 May 2015 Received in revised form 25 May 2015 Accepted 28 May 2015 Available online xxx

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Keywords: Oral vaccine Vaccine cost Transgenic plant Transient expression Transplastomic plant Edible crop E antigen Virus like particles

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1. Introduction

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Chikungunya virus is an emerging pathogen initially found in East Africa and currently spread into the Indian Ocean Islands, many regions of South East Asia, and in the Americas. No licensed vaccines against this eminent pathogen are available and thus intensive research in this field is a priority. This review presents the current scenario on the developments of Chikungunya virus vaccines and identifies the use of genetic engineered plants to develop attractive vaccines. The possible avenues to develop plantmade vaccines with distinct antigenic designs and expression modalities are identified and discussed considering current trends in the field. © 2015 Published by Elsevier Ltd.

Chikungunya virus (CHIKV) causes an infection typically characterized with fever, skin rash, incapacitating arthralgia, and severe synovitis [1]. This virus is transmitted by the Aedes mosquitoes and its name derives from the Swahili or Makonde word Kun qunwala that translates to “walk bent over”, which describes the posture of infected persons experiencing severe joint pain. Chikungunya is easily confused with dengue as they share the same vectors, symptoms, and geographical distribution; but differs in the absence of headache, and retro-orbital eye pain [2]. CHIKV, first isolated in 1952 from a febrile patient during an outbreak in the Makonde Plateau in the southern province of Tanzania (formerly Tanganyika), is a prevalent pathogen in tropical and subtropical regions of Africa, the Indian Ocean Islands, and south and southeast Asia among the Makonde tribe [2]. CHIKV is an enveloped alpha virus belonging to the family Togaviridae, whose genome consists of a single-stranded positivesense RNA of approximately 11.8 kb. The genome is capped at

∗ Corresponding author. Tel.: +52 444 826 2440; fax: +52 444 826 2440. E-mail address: [email protected] (S. Rosales-Mendoza).

the 5 end and polyadenylated at the 3 end. The genomic structure of CHIKV encodes for the following: one open reading frame (5 ORF) yielding four non-structural proteins (nsP1–4) at the posttranslational level which participate in genome replication, RNA capping, polyprotein cleavage, and other functions required for viral replication; and another ORF that yields three major structural proteins (Capsid, E1, and E2) and two small cleavage products (E3 and 6K) [3]. The mature virion is 70 nm in diameter and contains 240 heterodimers of E2/E1 arranged as trimeric spikes on its surface. These heterodimer spikes are inserted into the plasma membrane of infected cells after transported through the secretory pathway. Cytoplasmic nucleocapsids containing the genomic RNA and 240 copies of the capsid protein bud from the cell surface to acquire the virion envelope and envelope protein spikes. The E1 and E2 glycoproteins form heterodimers that associate as trimeric spikes on the virion surface while E3 and 6K were demonstrated to act as helper proteins in the budding and maturation process of the virion envelope [4,5]. CHIKV is believed to be originated in Africa where two genetically distinct lineages have been identified: one containing all isolates from western Africa and the second comprising all southern and East African strains, as well as isolates from Asia. West African lineages caused multiple CHIKV epidemics in East Africa,

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

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the Indian Ocean Islands, and many parts of South East Asia [6–11]. Before 2000 C.E., large outbreaks of CHIKV were rare but become more frequent afterwards. More recently new episodes of CHIKV have been reported in the Americas, further broadening the geographical spread of the disease, which has been associated with an emerging genetic variability [12,13] leading to hypotheses on possible mechanisms of evolutionary adaptation of the virus to the mosquito vector [14,15]. The main preventive strategy against Chikungunya is mosquito control, but this has proven to be difficult especially in poor countries; thus new strategies to fight the disease are needed. Currently the only treatments for CHIKV-disease symptoms are non-steroidal anti-inflammatory drugs. The re-emergence of CHIKV has led to the assessment of several potential treatments including ribavirin [16,17], chloroquine [18,19], and CHIKV antibodies [20–23]. In addition the peptides Latarcin (LATA) and Thanatin (THAN) as well as the protein PAP1, all of which having antiviral activity [24,25], have shown a promising potential to protect against CHIKV [26]. Among the explored strategies, vaccination is considered the ideal intervention to prevent the CHIKV infection; however no licensed vaccines for human use are available yet. Despite the development of several animal models, few of them have met the requirement to be used in pre-clinical studies to assess potential therapeutics. Recent epidemiological data showed the increasing importance of antibody-mediated protection against CHIKV [21–23], highlighting the feasibility of using anti-CHIKV antibodies as a passive immunotherapy or as a prophylactic treatment. However, information about the exact target of the adaptive immune response either in human or in animal models remains limited. In addition the cost for immunotherapies produced under conventional platforms should be considered, which is prohibitive for massive use in developing countries.

2. Immununopathogenesis of CHIKV infection and animal models Deciphering CHIKV specific molecular features and how the virus interacts with its host are key aspects to prevent, treat, or cure the infection. However, the knowledge of human CHIKV infection immunology is limited to small animal models (mouse) [27] in which muscle and joint disease were recently achieved in C57BL/6 mice [28,29]. Although the mouse model is useful at preclinical level for vaccine development, CHIKV disease mice models (young or immunodeficient mice) do not fully recapitulate human disease patterns in terms of infectivity and immune responses. Therefore, Labadie et al. [30] proposed a model for CHIKV infection in adult immunocompetent cynomolgus macaques (Macaca fascicularis). CHIKV pathogenesis using this animal model seems to resemble the viral, clinical, and immunopathological features observed in the human disease; and, interestingly, macrophages were identified as the main cellular reservoirs during the late stages of CHIKV infection in vivo. Overall, the inflammatory response to CHIKV infection in humans clearly contributes to virus elimination since the viral load has been associated to the serum levels of proinflamatory mediators such as IFN-alpha, IFN-gamma, IL-1RA, IL-6, MCP-1/CCL-2, IL-12, IP-10/CXCL-10, IL-18, and IL-18BP [31,32]. However it is important to point out that proinflamatory mediators are orchestrated and depend largely on the stage of the viral pathogenesis, as demonstrated in cynomolgus macaques after CHIKV experimental infection [30]. Nevertheless, the beneficial or deleterious effects of inflammation on viral persistence remain unclear even though CHIKV infection-associated markers have been described [33,34]. In general, T and B cells have been associated to the clearance of CHIKV since reduced immune

responses in mice models and aged NHPs promoted long-term virus persistence [35,36].

3. Production of vaccine candidates against CHIKV Based on the hypothesis that an efficacious CHIKV vaccine should resemble the viral infection to provide accurately immunoprotection against CHIKV disease [37], several promising vaccines have been recently evaluated. For example the immunization with virus-like particles (VLPs) in a monkey model elicited neutralizing antibodies against envelope proteins from different CHIKV strains, mediating protection against viremia when challenged with a high dose of CHIKV; moreover, the transfer of these antibodies into immunodeficient mice conferred protection against a subsequent lethal CHIKV challenge [38]. In addition, purifying human antiCHIKV antibodies from patients in the convalescent phase exhibit a high in vitro CHIKV neutralizing activity and a powerful prophylactic and therapeutic efficacy against CHIKV infection in a mouse model, which correlates with the fact that infected individuals are in general protected against reinfections [20]. Therefore, the protection against CHIKV disease is considered to be primarily mediated by humoral responses. This knowledge have supported the development of immunization approaches resembling the natural infection process as close as possible through the use attenuated CHIKV strains or VLPs, which mimic the infection mechanism and induce antibody-mediated. Among the technologies that have been explored for the development of CHIKV vaccines stand-out: formalin-inactivated viral vaccines [39,40], live-attenuated viruses [41–43], alpha virus chimeras [44–46], consensus-based DNA vaccines [47–50], and recently virus-like particle (VLP) vaccines and recombinant subunit vaccines. A detailed scenario on protein subunit vaccines development is provided below. CHIKV structural proteins form enveloped VLPs (eVLPs) when expressed alone in eukaryotic expression systems [51,52]. The first CHIKV eVLP-based vaccine candidate was reported in 2010 by researchers of the NIH and has become the most promising VLP-based vaccine against CHIKV. DNA transfection of a plasmid comprising the full-length CHIKV structural coding region C-E3E2-6K-E1 into human HEK293 cells successfully resulted in CHIKV VLPs assembly. The viral glycoproteins in the VLPs are organized in 240 E1–E2 heterodimers, which form 80 spikes on the VLP surface, resembling replication-competent alpha viruses. These eVLPs were isolated from the supernatant of transfected mammalian cells, purified, and used to immunize mice and nonhuman primates. Vaccination of rhesus macaques with 3 doses consisting of 20 ␮g of eVLPs at 0, 4, and 24 weeks induced an antibody response that was sufficient to confer protection upon a high-dose CHIKV challenge 15 weeks after the last boosting [53]. These results demonstrated that immunization with these VLPs elicited neutralizing antibodies directed against envelope proteins and protected NHPs against a subsequent lethal CHIKV challenge, indicating a humoral-mediated mechanism of protection. The next developmental step for this vaccine consisted on performing a Phase I dose-escalation clinical trial under a 3-dose vaccination scheme (weeks 0, 4, and 20) of up to 40 ␮g of eVLPs per administration. This vaccine was safe, well tolerated and immunogenic [38]. Another VLPs-based promising vaccine development has consisted on a measles vaccine expressing CHIKV VLPs. A single immunization with this vaccine fully protected mice from a lethal CHIKV challenge [54]. This vaccine induced high titers of neutralizing CHIKV antibodies although specific cellular immune responses were also elicited. CHIKV eVLPs have also been expressed in insect cells. Research performed since 2011 demonstrated that the expression of the structural coding regions C-E3-E2-6K-E1 in Sf21 insect cells led to the assembly of VLPs [55–57]. Interestingly, these eVLPs displayed

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

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superior immunogenicity in subcutaneously immunized mice in comparison with their subunit counterparts and effectively protected against a CHIKV lethal challenge in both AG129 and WT mice models. Further analysis revealed an association between this protective effect and the neutralizing antibody responses, which were induced even for a single immunization scheme (1 ␮g/mouse) without the need of co-administered adjuvants [56,57]. This technology was adopted by the pharmaceutical company Merck that conducted studies consisting on the immunization of Guinea pigs with two doses (on days 0 and 14) that ranged 0.01–10 ␮g along with the Adju-Phos aluminum adjuvant. The induction of specific IgG neutralizing antibodies in a dose-dependent manner was observed [58]. Similar findings have been found using live-attenuated CHIKV vaccines, which have also been evaluated in mice models. For example, a single administration of a live-attenuated CHIKV vaccine (CHIKV/IRES) effectively activated T cells with a peak on day 10 post-immunization and elicited memory CD4+ and CD8+ T cells that produced IFN-␥, TNF-␣, and IL-2. However, only passive immunization with anti-CHIKV/IRES immune serum provided protection [59]. Another example is the CHIKV vaccine strain 181/clone25 (181/25) developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). It was demonstrated that a single intradermal footpad immunization of AG129 (defective in IFN-␣/␤ and IFN-␥ receptor signaling) or A129 (defective in IFN-␣/␤ receptor signaling) mice with the attenuated CHIK 181/25 vaccine resulted in different mortality rates. AG129 mice resulted in rapid mortality within 3–4 days while A129 mice survived even after wild type CHIKV-La Reunion challenge, with the associated induction of significant levels of IFN-␥, IL-12, and specific antibodies. This vaccine was well-tolerated and highly immunogenic in phase I and II clinical trials [60]. Overall, these data highlight the importance of IFNs and neutralizing antibody responses on the protection against CHIKV infection. Viral-vectored vaccines have also been developed against CHIKV. A novel CHIKV vaccine candidate called MVA-CHIKV was developed based on the highly attenuated modified vaccinia virus Ankara (MVA), which is a poxvirus vector. This vaccine candidate relies on the expression of the CHIKV C, E3, E2, 6K, and E1 structural genes and has proven to induce robust innate immune responses in human macrophages and monocyte-derived dendritic cells in terms of the IFN-␤, proinflammatory cytokines, and chemokines production. After adjuvant-free intraperitoneal immunization of C57BL/6 mice with the chimeric virus at 1 × 107 PFU doses, strong CHIKV-specific CD8+ T cell responses were induced. CHIKV-specific CD8+ T cells were preferentially directed against the E1 and E2 proteins. Neutralizing humoral responses were also induced at high titers. Remarkably, mice treated with a single dose of the MVACHIKV vaccine were fully protected from a CHIKV challenge [61]. Protein subunit vaccines based on portions of some viral proteins have also been generated. A formulation based on two components (the 254 aa C-terminus region of the E1 protein and the full-length E2 protein) was produced using Escherichia coli BL21 (DE3) as expression host. Subcutaneous immunization of BALB/c on days 0, 21, and 35 with 40 ␮g of either truncated E1 or E2 using different adjuvants (Freund’s complete adjuvant, Alum, or Montanide ISA720) was performed. This subunit vaccine candidate was able to induce broad and long lasting neutralizing antibodies and cell-mediated immune responses [62]. Interestingly, at least two CHIKV sub-domains (E2 domain A and B) are associated with human protective immunity based on antibody-dependent neutralization [63]. Another subunit vaccine candidate against CHIKV is based on the 364 aa ectodomain from E2 produced in the E. coli BL21 (DE3) strain. The intramuscular administration of 50 ␮g of the recombinant protein, delivered as liposomes (CadB), in BALB/c mice induced neutralizing IgG

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antibodies and high levels of IFN-␥. A challenge assay with CHIKV was also conducted, observing complete protection in the vaccinated group whereas control mice exhibited high viral load in muscles, brain, spleen, and blood [39]. Epitope-based vaccines constitute the ideal vaccination approach as they can be designed to achieve proper immune responses in terms of breadth and neutralizing potential. However, this field has been rather narrowly explored in part due to the limited CHIKV B cell epitope mapping. Therefore, the identification of a wider set of CHIKV neutralizing epitopes is still a need for this field. One of the few investigations on this subject revealed 4 relevant epitopes in the E2 protein which were identified by their reactivity against sera from immunized mice. These epitopes include the previously reported E2EP3 epitope (STKDNFNVYKATRPYLAH, aa 2800–2818 of the polyprotein) and three new epitopes: KWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCR (3033–3066 aa of the polyprotein), located in the acid-sensitive region (ASR); KKEVVLTVPTEGLEVTWGNNEPYKYW (3113–3138 aa of the polyprotein); and AGMCMCARRRCITPYELTPGATVPFL (3185 to 3210 aa of the polyprotein). All these epitopes cluster at the C-terminus of the E2 glycoprotein [49]. In fact Kam et al. [21,22] observed, using plasma samples from humans obtained during the early convalescent phase, that the naturally-acquired IgG response is dominated by neutralizing IgG3 antibodies which are mostly directed toward the single linear epitope E2EP3, a peptide located at the N-terminus of the E2 glycoprotein and exposed on the viral envelope. More recently, it was confirmed that epitopes on the exposed top-most and outer surfaces of the E2/E1 trimer structure may be useful for CHIKV neutralization by specific antibodies, whereas epitopes facing the interior of the trimer are not [64]. Overall, these findings pave the way for the development of CHIKV-specific multi-epitopic- or VLP-based vaccination strategies that can be efficiently produced in plant-based platforms. As mentioned above, the implementation of advanced animal models to test vaccine candidates at the preclinical level is of relevance, especially those that closely resemble the infection that occurs in humans. Due to the close lineage relationship between humans and macaques, macaque models of CHIKV infection have been developed [30,35,65,66]. These models allow comparing the adaptive immunity between humans and macaques. Therefore, sera from non-human primates infected with CHIKV could reveal new neutralizing epitopes. A remarkable research on mapping CHIKV epitopes drop a series of epitopes all along in the structural proteins in macaques and in humans, indicating that the E2EP3 and the 3025 to 3066 epitopes are recognized by both human and macaques; which is consistent with previous reports (Table 1 [67]). This active research path leading to new epitopes is opening new prospects for the development of epitope-based vaccines against CHIKV.

4. The plant-based vaccines scenario Current plant biotechnology tools make possible the use of plants as both efficient bio-factories and delivery vehicles of subunit vaccines [68]. A wide range of proteins of pharmaceutical interest have been expressed in plants with several plant-made vaccine candidates being evaluated in clinical trials, including those for swine influenza, rabies, and hepatitis B [69]. This technology offers substantial advantages such as the absence of mammalian pathogens in the production process, low production cost, no requirement for fermentation systems, and efficient synthesis of complex proteins [70,71]. It is also possible to design oral vaccines based on the induction of specific immune responses in the gut associated lymphoid tissues (GALT) by orally administering plant biomass from edible plant species. This oral administration

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

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Table 1 Comparison of human and macaque CHIKV B-cell epitopes (taken from Kam et al. [67]). CHIKV protein nsP1 nsP2 nsP3

nsP4 Capsid E3 E2

Identified B-cell epitope (human)

Amino acid

Identified B-cell epitope (macaque)

Amino acid

AEEEREAELTREALPPLQ

497–514

GVNSVAIPLLSTGVYSGG FGASSETFPITFGDFNEGEIESLSSELLT FGDFLPGEVDDLTDSDWSTCSCSD TDDELLDRAGGYIFS

1433–1450 1801–1867

IQMRTQVELLDEHISIDC TVPVAPPRRRRGRNLTVT

1489–1506 1729–1746

KDIVTKITPEGAEEW

2721–2735

AAIIQRLKRGCRLYLMSETPKVPTYR PPKKKPAQKKKKPGRRERMCMKIEND RPIFDNKGRVVAIVLGGA

1937–1962 2561–2586 2689–2706

LLQASLTCSPHRQRR STKDNFNVYKATRPYLAHC TDGTLKIQVSLQIGIKTDDSHDWTKLRY MDNHMPADAERAGL LTTTDKVINNCKVDQCHAAVTNHKKW HAAVTNHKKWQYNSPLVPRNAEL GDRKGKIHIPFPLAN PTVTYGKNQVIMLLYPDHPTLLSYRN PTEGLEVTWGNNEPYKYWPQLSTNGT LLSMVGMAAGMCMCARRRCITPY ELTPGATVPFL

2785–2799 2800–2818 2841–2882

STKDNFNVYKATRPYLAHC ATTEEIEVHMPPDTPDRT

2800–2818 2961–2978

GNVKITVNGQTVRYKCNC HAAVTNHKKWQYNSPLVPRNAEL GDRKGKIHIPFPLANVTCR

2985–3002 3025–3066

3009–3034 3025–3058 3073–3098 3121–3146 3177–3210

6K E1 Regions of B cell epitopes found that are common to both human and macaque are bold.

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approach offers additional attractive features including friendly delivery, safer administration than parenteral vaccines, and the avoidance of purification [72]. Thus these vaccines are highly attractive vaccines due to their low production cost which will allow attaining proper vaccination coverage in developing countries. The production of functional immunogens in the plant cell has been reported thus far following either oral or parenteral immunization schemes. The distinct expression modalities to produce antigenic proteins in the plant cell are described in Table 2. Until now, highly efficient transient expression systems have been adopted by the industry to produce parenteral vaccines by purifying the antigen from plant tissues [73]. Therefore the concept of plant-based vaccines is in the transition from a vision to a reality, with several candidates under evaluation in clinical trials [69]. Table 3 lists the antigenic configurations successfully implemented in plant-based systems. The immunogenic activity of

subunit vaccines is aided by designs based on the B subunits of bacterial toxins [74,75], Virus Like Particles (VLPs) [76], or immunoglobulin-based immune complexes [77]. These approaches have allowed attaining high immunogenic activity for several vaccines administered by the oral, intranasal, and parenteral routes. The main antigenic configurations explored in plant-based vaccines are described below. 4.1. LTB- and CTB-based chimeras

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The B subunits of either the cholera toxin (from Vibrio cholerae) or the heat labile toxin (from enterotoxigenic E. coli) called CTB and LTB, respectively; have played a relevant role on vaccinology since these toxin-derived molecules lack of toxic activity but retain the GM1 binding activity, show a high immunogenicity, and exert immunomodulatory effects toward co-administered or fused

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

Advantages

Limitations

Possible CHIKV expression targets

References

Nuclear stable transformation

Stable transgene insertion Post-translational modifications Well established transformation methods

C, E3, E2, 6K, E1 nsP1–4

[110]

Nuclear transient expression

Short production time Very high levels of recombinant protein

C, E3, E2, 6K, E1

[111]

Chloroplast transformation

High levels of recombinant protein Polycistronic expression is viable Improved biosafety as transgene is inherited maternally Site-specific insertion through recombination Non-susceptible to gene silencing

Low levels of recombinant protein Non-site specific transgene insertion Horizontal gene transfer is possible Susceptible to gene silencing Low reproducibility Transient expression limit the Purification of the antigen is required Lack of complex post-translational modifications Long time for generation of transformants Limited transformation protocols

C, E3, 6K nsP1–4

[112]

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

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Advantages

Disadvantages

References

CTB/LTB chimeras

High mucosal immunogenicity High immunogenicity

May induce tolerance

[113]

Limitations on the size of heterologous sequences in chimeric VLPs Depend of a high-yield protein expression The longest ELP subunits the easiest to purify, but also achieves low expression levels

[114]

VLPs

Immune complexes

ELPylation

High immunogenicity

High immunogenicity Robust expression Easy purification

[93]

[97]

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unrelated antigens [78]. In the field of plant-based vaccines, these molecules have been extensively used as adjuvant carriers, especially within the goal of developing oral vaccines since CTB/LTB serve as transmucosal carriers able to deliver the antigen at the sub mucosa [79]; where they can be efficiently processed by dendritic cells (DC) with the subsequent induction of robust mucosal and systemic Th1 immune responses. Several vaccination models based on this approach have proven to be effective at the preclinical level [80] and a clinical trial was performed with potatoes expressing LTB with positive outcomes in terms of seroconversion [81]. Therefore, fusing specific epitopes or domains from protective CHIKV antigens to CTB/LTB will serve as an attractive approach in the development of mucosal vaccines produced in plant tissues. The use of edible plant species will greatly facilitate the development of low cost oral vaccine prototypes.

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4.2. Virus-like particles

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Virus-Like Particles (VLPs) are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome and thus are not infective. Several reports on the expression of properly assembled VLPs at adequate levels have been published for Influenza virus, Human papillomavirus, Human immunodeficiency virus, Norwalk virus, and Hepatitis B virus; among others (reviewed in [71]). Since recent reports on CHIKV eVLPs production in mammalian and bacterial expression systems have proven to be efficacious (see above), the adoption of this concept in the form of plant-derived vaccines is a possibility. The glycosylation patterns of VLP proteins have a major impact on their structure and function since these viral glycoproteins localize, guide, and potentiate the process of enveloped virus assembly. Therefore, the design of plant-made vaccines based on the E1 and E2 glycoproteins demands a platform with the capacity to perform these complex post-translational modifications. The N-glycan synthesis in the endoplasmic reticulum is relatively well conserved in eukaryotes [82], thus it is expected that plants will provide the functional machinery to produce CHIKV eVLPs properly. In fact, some differences on glycosylation are expected in plants in comparison to mammal cells; however, this may account for the antigenicity of the plant-made CHIKV antigen due to a better recognition by antigen presenting cells through pattern-recognition receptors. If the hypothesis of higher antigenicity due to plant glycosylation fails, an alternative path consists on using the glycoengineering strategies that have been implemented in plants through knocking out

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or silencing by RNAi, the genes or transcripts coding for the target glycosyltransferases, and/or introducing mammalian glycosylation genes [83,84]. Therefore, the precedents on VLPs production in plant systems for targeting other pathogens make these systems a reliable path to develop attractive CHIKV vaccine candidates. 4.3. Immune complexes Plants can express complex recombinant proteins requiring post-translational modifications, which are problematic to generate using other production platforms such as bacterial systems [85]. It has long been known that primary and secondary antibody responses to model antigens can be enhanced by immunization with immune complexes (ICs) [86,87]. Importantly, it is well established that ICs can be cross-presented by antigen-presenting cells (APC) and can stimulate potent T cell responses via MHC class I as well as class II molecules [88,89]. It has also been suggested that, through binding to Fc receptors and complement receptors, the ICs localize on the surface of follicular dendritic cells (FDCs), which play an important role in the selection and affinity maturation of B cells, or the complexes might directly stimulate B cells via their complement receptors [90]. However the conventional preparation of ICs is not feasible for vaccine production at a commercial scale, since it relies on the use of either polyclonal antisera or expensive monoclonal antibodies cocktails (mAbs) to achieve multimerization with a given antigen. To address these limitations, Chargelegue et al. described, for the first time, the production of recombinant ICs in transgenic tobacco plants by expressing the tetanus toxin fragment C fused to a mAb. The design of the recombinant IC fusion molecule resulted in the expression of the antibody-antigen at a 1:2 ratio (an antigen molecule was fused to each antibody heavy chain). Moreover, it was demonstrated that serum antibody responses leading to protective immunity were induced without the use of additional adjuvants in subcutaneously immunized mice [91]. Using similar approaches, multiple antigens have been expressed in tobacco being able to induce both humoral and cellular immune responses that protected against intracellular pathogens such as Mycobacterium tuberculosis. Intranasal immunization of mice with ICs boosted the Bacillus Calmette–Guerin strain vaccine (BCG)induced immunity and, importantly, conferred further protection against M. tuberculosis infection [92]. Remarkably, these methodologies have been applied to target viruses. For example, transient expression of Ebola-based immune complexes in plants has been achieved by fusing the Ebola GP1 glycoprotein subunit to a specific humanized heavy chain of 6D8 IgG monoclonal antibody. Mice subcutaneously immunized with Ebola ICs developed high titers of Ebola-specific IgG antibodies [93]. Thus, these studies show that ICs might represent an attractive immunization strategy against viral antigens and represent a promise for the development of highly immunogenic CHIKV vaccines. 4.4. Elastin-like polypeptide fusions The major cost in the production of subunit vaccines is related to the separation and purification steps, which can account for up to 90% of total production costs [94]. A benefit of plant-based expression systems is that they allow for alternative purification methods, such as elastin-like polypeptide (ELP) fusion technology. The ELPs ***Val-Pro-Gly-Gly, Val-Pro-Gly-Val-Gly, Ala-Pro-Gly-Val-Gly-Val, and Val-Pro-Gly-Xaa-Gly)n (where Xaa represents one random amino acid excluding Pro) exhibit the unique characteristic of reversible phase transition, meaning that they can be precipitated out of solution and re-suspended again through temperature manipulation. The temperature-dependent, reversible aggregation/precipitation properties of ELPs provides an alternative to the

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

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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.

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cost-intensive affinity chromatography and allows to separate the protein of interest from host proteins via centrifugation of crude extracts [95]. The efforts on plant-derived vaccines against human diseases using this approach are limited. One of the few studies comprises the M. tuberculosis antigens Ag85B and ESAT-6, which were fused to ELP and expressed in transgenic tobacco [96]. The subcutaneous immunization of mice with either 5 ␮g or 10 ␮g of ICs on days 0, 14, and 28; induced long-lasting humoral immune responses after 84 days. Interestingly the ELP was not removed from the antigen and it was found that the ELP had no effect on the immune response against the target antigens in mice. Another interesting approach consisted on the production of ELP fused to an established soluble trimer-forming H5N1 HA (ELPylated H5 HA, or H5-ELP) in tobacco. These recombinant proteins were easily purified by the inverse transition cycling technique. The subcutaneous administration in BL6 mice induced neutralizing antibodies after two doses administered on days 0 and 14. It was also found that ELPylation does not interfere with the immunogenicity of the vaccine candidate [97]. Therefore, this precedent of positive outcomes on targeting an enveloped virus through the ELP technology represents a positive prospect for proposing CHIKV vaccine candidates under this configuration.

5. Prospects for using plant-based CHIKV immunization approaches Plant-based technologies offer a myriad of possibilities to develop new vaccines against CHIKV thanks to the improvements of the technology achieved during the last two decades. Since several subunit vaccines against CHIKV have been explored using other expression systems, there is sufficient knowledge to support a straightforward design of plant-based vaccines (Fig. 1). The

recent identification and characterization of linear CHIKV B cellepitopes will allow designing innovative vaccines. Multiepitope vaccines against CHIKV are also a possibility when producing plantbased vaccines since multiepitope chimeric proteins have been produced in plants leading to promising findings [98,99]. Our group has developed plant-made vaccines against HIV, which were based on multiepitope polypeptides and against Taenia solium, which were based on several peptides produced through the 2A-based ribosomal skip mechanism [100]. Other groups have developed multicomponent vaccines against enteric pathogens based on chimeric proteins [101]. Since some targets have been identified as protective antigens against CHIKV, the plants producing those antigens along with convenient carriers could be evaluated as CHIKV vaccine candidates. The vaccine developmental steps would comprise: (i) designing immunogens based on proteins or epitopes associated with immunoprotection against CHIKV and developing transgenic plants carrying the corresponding genes; (ii) characterizing the plant-made antigen in terms of yields and antigenic activity; (iii) proving the immunogenic activity at the preclinical level through test animals immunization under distinct administration routes, and (iv) planning clinical trials once efficacy and safety in test animals have been proven. Therefore, this scenario indicates that a new avenue on developing anti-CHIKV vaccines will be viable to implement with the experience gained in the field working with other pathogens over the last two decades. This will constitute a relevant research field, allowing for the development of low cost and efficient vaccines. However it should be recognized that the progress in the field of plant-made vaccines has been focused to parenteral formulations using transient expression systems [71], while oral vaccination approaches still require optimization to address proper immunogenicity, a better control on dosage as well as antigen degradation,

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

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and finding a proper regulatory framework [102,103]. Regarding oral vaccines, a recently proposed idea is based on the inducible expression of antigens mediated by viral vectors in edible plant species; which will allow acquiring high expression levels avoiding the purification steps. Therefore, plant biomass expressing high levels of the antigen might be used for the formulation of low cost oral vaccines consisting of freeze-dried plant material delivered in gelatin capsules [71]. Overall a convenient possibility for immunization schemes against CHIKV infection comprises parenteral priming with purified plant-made antigens and subsequent oral boosts with freeze-dried plant material expressing the candidate subunit vaccine (Fig. 1), as it has been proposed for the case of Hepatitis B Virus infection [104].

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Considering that no vaccines against CHIKV approved for human use are available and that the disease is already present in the Americas, the need for advancing in this research field is great. The following years will be critical to envision the real potential to address this issue. Considering that promising candidates are in preclinical trials, the implementation in parallel of low cost production platforms will be determinant as CHKV is mainly affecting developing countries where access to vaccine is limited due to the high cost of conventional vaccines. In this review, plantbased platforms have been identified as promising approaches to fight CHIKV. The maturation that this technology achieved during the last decade currently allows for the production of several vaccine types ranging from oral formulations, which will required long-term research to be assessed and optimized, to parenteral vaccines produced under GMP-compliant transient expression systems [105,106]. The latter has the best potential to become a reality in the near future. Based on previous experiences, such as the developments on influenza and other viral pathogens vaccines [107], VLPs are the most efficacious approach to render immunogenic formulations even at low doses [108]. For example, influenza plantbased vaccines are in an advanced developmental stage, being evaluated in clinical trials [109]. In conclusion, the concept of plant-based vaccines constitutes a relevant path for the development of CHIV vaccines with attractive features.

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Acknowledgements

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Current investigations from the group are supported by 562 Q5 CONACYT/México (grant CB-2008-01, 102109to SRM and grant CB563 2010-01, 151818 to CA) and FAI/UASLP/2015 to SRM. 561

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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

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