VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1681

Therapeutics and Vaccines Against Chikungunya Virus Tero Ahola,1 Therese Couderc,2,3 Lisa F.P. Ng,4 David Hallenga¨rd,5 Ann Powers,6 Marc Lecuit,2,3,7 Mariano Esteban,8 Andres Merits,9 Pierre Roques,10 and Peter Liljestro¨m 5

Abstract

Currently, there are no licensed vaccines or therapies available against chikungunya virus (CHIKV), and these were subjects discussed during a CHIKV meeting recently organized in Langkawi, Malaysia. In this review, we chart the approaches taken in both areas. Because of a sharp increase in new data in these fields, the present paper is complementary to previous reviews by Weaver et al. in 2012 and Kaur and Chu in 2013. The most promising antivirals so far discovered are reviewed, with a special focus on the virus-encoded replication proteins as potential targets. Within the vaccines in development, our review emphasizes the various strategies in parallel development that are unique in the vaccine field against a single disease. Key Words: Chikungunya—Vaccine—Antiviral—Polymerase—Protease—Antibodies—Attenuated virus—Viruslike particles—DNA chimeric viruses.

Antivirals

A

ntiviral molecules can act by blocking any of the virus life cycle stages through interaction with either virusencoded or cellular components essential for virus replication. The prime example of a successful treatment and clinical trials among positive-strand RNA viruses is hepatitis C virus (Manns and von Hahn 2013). As in other viral diseases with existing treatment, most success has been achieved by targeting the viral proteins, especially proteins involved in genome replication. Like all members of the alphavirus genus, chikungunya virus (CHIKV) proteins include four structural proteins (capsid, 6K, E1, and p62) and four nonstructural proteins (nsP1–4) that are multifunctional, display different enzymatic activities, and represent indispensable virus-encoded components of replication complexes. For CHIKV or other alphaviruses, very few screens directed against the viral proteins have been reported. In general, although polymerase inhibitors have been a highly successful class of antivirals, the alphavirus polymerase has

been extremely difficult to target because the core polymerase subunit nsP4 is inactive on its own (Rubach et al. 2009). Thus, only some polymerase inhibitors discovered for other viruses have been tested with some success. Among these, favipiravir is promising because it can protect mice against CHIKV-caused disease (Delang et al. 2014). Favipiravir, also known as T-705 or 6-fluoro-3-hydroxy-2-pyrazinecarboxamide, is an RNA-base analog that becomes phosphoribosylated in cells, and inhibits, e.g., the influenza virus polymerase (Furuta et al. 2013). Other modified nucleosides active against CHIKV include 6-azauridine and 3-deazaadenosine (Briolant et al. 2004, Scholte et al. 2013), but they may not act as polymerase blockers (Albulescu et al. 2014). To date, the functions of the alphavirus replicase protein nsP3 are still uncertain, and no inhibitors have been reported. nsP2 is both an RNA helicase and a protease cleaving the nonstructural polyprotein. Whereas there are no viral helicase blockers, protease inhibitors have generally been a good second class of antivirals. Even for the hepatitis C virus protease, whose active site was considered a challenging

1

Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland. Institut Pasteur, Biology Infection Unit, Paris, France. Inserm U1117, Paris, France. 4 Singapore Immunology Network, A*STAR, Singapore. 5 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 6 Division of Vector Borne Diseases, US Centers for Disease Control and Prevention, Fort Collins, Colorado. 7 Paris Descartes University, Sorbonne Paris Cite´, Division of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades University Hospital, Institut Imagine, Paris, France. 8 Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia, CSIC, Madrid, Spain. 9 Institute of Technology, University of Tartu, Tartu, Estonia. 10 CEA, Division of Immuno-Virology, iMETI, Fontenay-aux-Roses, France and University Paris-Sud XI, Orsay, France. 2 3

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target due to its flatness, excellent inhibitors have been found (Delang et al. 2013). In contrast, the alphavirus nsP2 cysteine protease has a partially constricted substrate-binding site (Golubtsov et al. 2006). Accordingly, there have been initial efforts to exploit the known structure of the alphavirus protease as a basis of molecular modeling and inhibitor studies (Singh Kh et al. 2012, Bassetto et al. 2013). Notably, one of the identified inhibitors and derivatives were substantiated as effective antivirals against CHIKV (Bassetto et al. 2013). Finally, the unique enzymatic activities of nsP1 involved in RNA capping should be promising targets (Magden et al. 2005). However, in this case, the membrane association propensity of the protein has hampered studies, and so far there are no published reports of specific guanylyltransferase or cap methyltransferase inhibitors. Cell-impermeable cap analogs can inhibit the enzyme in vitro, but they would not be useful for in vivo applications (Lampio et al. 1999). Interestingly, the effect of ribavirin (see below) against alphaviruses may be mediated by the lowering of intracellular guanosine triphosphate (GTP) concentration, thus impairing the guanine 7-methyltransferase function of nsP1 (Scheidel and Stollar 1991). So far, the large majority of reported anti-CHIKV molecules have been derived from cell-based screens or from testing of previously known compounds with antiviral properties (Kaur and Chu 2013). The general antivirals ribavirin and interferon are active against CHIKV and act synergistically (Briolant et al. 2004). Mycophenolic acid seems to be even more active than ribavirin against CHIKV (Khan et al. 2011, Scholte et al. 2013). Thus, these compounds could potentially be used in treatment, but further evaluation is required. For many of the known antivirals, or for natural products or other bioactive molecules derived from screening, the exact targets are not known, but in some cases it is possible to infer the part of the viral life cycle affected. Thus, chloroquine and arbidol, as well as compounds containing the 10H-phenothiazine core, are likely to inhibit CHIKV entry (Khan et al. 2010, Delogu et al. 2011, Pohjala et al. 2011). Remarkably, a small drug trial failed to find support for the use of chloroquine, a well-established antimalarial drug, in CHIKV treatment in patients (De Lamballerie et al. 2008). Separately, harringtonine, a complex plant alkaloid was able to inhibit translation of CHIKV, but it could also have other effects against CHIKV (Kaur and Chu 2013). For other compounds, such as plant terpenoid derivatives betulins, trigocherrins, and trigocherrolids, as well as the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate, the mechanisms of anti-CHIKV activity remain to be determined (Pohjala et al. 2009, Allard et al. 2012, Bourjot et al. 2012). Examples of compounds interfering with CHIKV replication through targeting of host processes have also been reported, including inhibition of the heat shock protein hsp90 as well as the inhibition of kinases and possibly other cellular signaling pathways (Cruz et al. 2013, Rathore et al. 2014). Currently, the best-identified inhibitors, such as mycophenolic acid, harringtonine, and 6-azauridine, are able to reduce CHIKV replication by 50% in the low or even submicromolar concentration range. However, this may not be sufficient to yield a good antiviral effect in vivo. Therefore, more work is required not only in the screening and synthetic chemistry of anti-CHIKV compounds, but also in the development of suitable testing platforms in relevant preclinical

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animal models. One could also potentially explore different treatment modalities relevant for human disease—treatment of acute CHIKV infection versus treatment of chronic symptoms in the joints. Thus, one must conclude that the antiviral studies in the case of CHIKV are still in very early stages. Biologics (Antibodies)

Human polyclonal antibody preparation and viroinactivated hyperimmune sera have been generally used for the treatment of human viral infections (Dessain et al. 2008). An alternative to antiviral polyclonal preparations has emerged through the development of antiviral monoclonal antibodies, either alone or in combination therapy (Both et al. 2013). The protective effect of passive immunization against alphaviruses, including Venezuelan equine encephalitis virus, Sindbis virus, and Semliki Forest virus, was demonstrated in mouse models a long time ago (Boere et al. 1983, Schmaljohn et al. 1983, Mathews et al. 1985). In recent years, passive immunization has been investigated for CHIKV in animal models susceptible to CHIKV infection (Weaver et al. 2012 and articles in this issue). These animal models are either mice deficient for type I interferon receptor (IFNAR - / - ) or neonatal C57BL/6 mice, which develop a lethal disease after CHIKV inoculation, or adult C57BL/6 mice, which develop viremia and foot swelling after footpad infection with CHIKV. The first study reporting the use of passive immunization against CHIKV has been performed with human polyvalent antibodies purified from human plasma donors in the convalescent phase of CHIKV infection (Couderc et al. 2009). These antibodies exhibited strong neutralizing activity in vitro and had a full protective efficacy in highly susceptible mouse models, i.e., adult IFNAR - / - mice and neonatal C57BL/6 mice. Moreover, they displayed a total therapeutic efficacy in these animal models when administrated early after infection (8 h) and a partial therapeutic efficacy when administrated 24 h after infection. Similarly, purified polyclonal antibodies from monkeys immunized with CHIKV virus-like particle (VLP) vaccine protected IFNAR - / - mice from CHIKV-induced disease and death (Akahata et al. 2010). As for neutralizing polyclonal antibodies, neutralizing monoclonal antibodies have been recently shown to protect mice against CHIKV, as reported at the CHIKV-2013 meeting by Andreas Suhrbier and Therese Couderc (Chikungunya 2013 conference, Malaysia, 2013). Two human neutralizing monoclonal antibodies directed against E2 and E1 have been isolated that significantly delay lethality in CHIKV-infected mice, both in prophylactic or therapeutic settings (Fric et al. 2013). Similarly, Selvarajah et al. (2013) isolated a human neutralizing monoclonal antibody directed against E2 protein able to prophylactically protect adult C57BL/6 mice from viremia and foot swelling, and, provided 8 or 18 h postinfection, to therapeutically protect neonatal C57BL/6 mice from death. In other studies, mouse neutralizing monoclonal antibodies have been isolated and tested against CHIKV disease in mice. Two murine neutralizing monoclonal antibodies directed against the E2 protein provided prophylactic protection from viremia and foot swelling of adult C57BL/6 mice (Goh et al. 2013). Pal and co-workers (2013) isolated

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four mouse neutralizing monoclonal antibodies directed against the E1 or E2 protein that provided complete prophylactic protection against CHIKV-induced lethality in adult IFNAR - / - mice or against CHIKV-induced foot swelling in adult C57BL/6 mice. Interestingly, they also show that combinations of two of them administrated after CHIKV infection completely prevent mortality of IFNAR - / - mice. Altogether, these studies suggest that passive immunization may constitute an effective medical intervention for humans with a known exposure to CHIKV who are at risk of severe disease. This prophylaxis approach could thus be recommended especially during birth for neonates born to viremic mothers, who are at high risk of developing severe infection (Gerardin et al. 2008), as well as for exposed at-risk patients with underlying conditions known to be associated with severe disease (Burt et al. 2012). Vaccines

There is currently no licensed vaccine available to combat CHIKV infection. Given the expanding incidence of CHIKV infections globally affecting millions of people mostly in Africa and Asia, but with recent outbreaks in the Caribbean islands and French Guiana, many teams have become engaged in the development of a CHIKV vaccine. This quest has recently been reviewed in greater depth than will be possible within the scope of this review (Weaver et al. 2012). As early as the 1960s, various attempts to develop a CHIKV vaccine included formalin-inactivated virus preparation and attenuated strains. However, none of these turned out to be very promising (Harrison et al. 1971, DeMeio et al. 1979). An early and conceptually important strategy was the

development of an attenuated vaccine candidate based on a clinical isolate originating from Thailand in 1962. The US Army Medical Research Institute of Infectious Disease (USAMRIID) passaged this strain in human MRC-5 cells, which resulted in an attenuated strain named TSI-GSD-218 or 181/clone 25 (Levitt et al. 1986, Hoke et al. 2012) (Table 1). Although this vaccine candidate showed promise in phase I (McClain et al. 1998) and phase II (Edelman et al. 2000) clinical trials, its further development was discontinued partly because of side effects in 8% of volunteers and also because of uncertainties about the production process (Hoke et al. 2012). Nevertheless, it is still considered for development as a product by Indian Immunological Ltd. Of note, a recent study showed that 181/clone 25 is only attenuated by two point mutations, suggesting that reversions may occur (Gorchakov et al. 2012) and thus putting safety into question for this vaccine. As indeed live attenuated viruses often are highly efficacious, a number of novel CHIKV candidate vaccines have been developed using this approach. Recently, two virus strains with large deletions in either the nsP3 gene or covering the 6K gene were shown to generate robust immune responses after a single immunization and to fully protect mice from a very high-dose challenge with wild-type CHIKV strain (Hallenga¨rd et al. 2014a). In another approach, attenuated mutants were generated via nine amino acid deletions of the transmembrane domain of the envelope protein E2 and by selecting host range (HR) mutants. This approach led to a reduction in titers. Finally, this vaccine displayed no reactogenicity in a mouse model and generated good humoral responses that were protective against CHIKV challenge (Piper et al. 2013). In a third approach, Gardner et al. passaged CHIKV strain 181/clone 25

Table 1. Chikungunya Vaccine Candidates Vaccine type

Strategy/antigen

Live attenuated

TSI-GSD-218 (181/clone25) LR-E2-E79K DnsP3 D6K HR mutant/D17-2TTMD CHIKV/IRES

Chimeric virus

VEE/EEE/CHIKV structural Adeno/CHIKV structural VSV/CHIKV E3-E2-6K-E1 MeaslesV/CHIKV structural MVA/CHIKV structural DNA CE2E1; nsP2 DREP D5nsP3 DREP D6K DREP-ENV iDNA; 181/clone 25 E1; E2 E2; E1-E2 Formalin-inactivated virus Baculovirus-infected insect cells DNA-transfected HEK293

DNA

Subunits Inactivated VLP

References Hoke et al. 2012 Gardner et al. 2014 Hallenga¨rd et al. 2014a Hallenga¨rd et al. 2014a Piper et al. 2013 Plante et al. 2011, Chu et al. 2013, Roy et al. 2014 Wang et al. 2008, Wang et al. 2011b Wang et al. 2011a Chattopadhyay et al. 2013 Brandler et al. 2013 Garcia-Arriaza et al. 2014 Mallilankaraman et al. 2011, Bao et al. 2013 Hallenga¨rd et al. 2014a,b Hallenga¨rd et al. 2014a,b Hallenga¨rd et al. 2014a Tretyakova et al. 2014 Khan et al. 2012 Kumar et al. 2012 Tiwari et al. 2009, Kumar et al. 2012 Metz et al. 2013, Wagner et al. 2014 Akahata et al. 2010a,b, Akahata and Nabel 2012

HR, host range; TMD, transmembrane domain; VLP, virus-like particles; VEE, Venezuelan equine encephalitis; EEE, eastern equine encephalitis virus; DREP, DNA replication; HEK289, human embryonic kidney cells; CHIKV, chikungunya virus; IRES, internal ribosome entry site; VSV, vesicular stomatitis virus; MVA, modified vaccinia virus Ankara; HEK, human embryonic kidney.

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on evolutionary divergent cell types and generated attenuated variants with increased electrostatic potential in their attachment proteins (Gardner et al. 2014). One particular mutant was identified as displaying new mutation in the E2 protein at position 79 (Table 1). One of the most advanced attenuated vaccine candidates is a strain derived from the La Reunion 2006 outbreak isolate. It was engineered to carry an internal response sequence (IRES) element between the nonstructural and the structural genes to attenuate the virus and to prevent it from replicating in the transmitting Aedes mosquito host. The vaccine was tested in several mouse models and induced good levels of neutralizing antibodies and protected them from challenge (Plante et al. 2011, Chu et al. 2013). It also resulted in cross-protective immunity against the O’Nyong-Nyong virus in a mouse model (Partidos et al. 2012). This vaccine and a novel variant were recently tested in nonhuman primates where they demonstrated strong immunogenicity without signs of disease. Both vaccine candidates prevented viremia upon challenge with wild-type CHIKV (Roy et al. 2014). The CHIK-IRES vaccine is now projected for phase I clinical trials by Takeda Inc. Another approach was taken by others who constructed chimeric viruses between CHIKV and Venezuelan encephalitis (TC-83 strain) or eastern equine encephalitis viruses. These strains were attenuated, replicated well in cell culture, and induced robust protective immune responses in mice (Wang et al. 2008, Wang et al. 2011b). However, it is unclear whether these vaccine candidates remain in the clinical development pipeline. A number of other strategies have focused on using nonalphavirus vectors for expression of CHIKV structural genes. Accordingly, an adenovirus vector was constructed carrying the structural polyprotein cassette of CHIKV. The vaccine completely protected mice from viremia and arthritis after challenge with the La Reunion and Asian isolates (Wang et al. 2011a). This vaccine candidate was in the research and development pipeline of GenPhar Inc.; however, because the company is no longer viable, it is unclear whether this vaccine will ever be evaluated clinically. Other recombinant viruses constructed include a vesicular stomatitis virus VSVDG-CHIK with the glycoprotein (G) gene replaced by the entire CHIKV structural polyprotein (Chattopadhyay et al. 2013) and a recombinant measles virus MV-CHIK (Brandler et al. 2013). Both vaccines generated robust immune responses and protected mice from lethal challenge. The MV-CHIK vaccine was constructed using the MV platform developed by Institut Pasteur and is projected to enter clinical trials performed by Themis Inc. (Vienna). Finally, a recombinant poxvirus–CHIK vaccine candidate was recently tested successfully in mice (Garcia-Arriaza et al. 2014). This vaccine is based on the modified vaccinia virus Ankara (MVA) strain expressing the CHIKV structural genes, which triggers robust B and T cell responses with high protection efficacy. The advantage of the MVA strain is its clinical safety record, which is being evaluated as a recombinant vaccine against a number of diseases (Go´mez et al. 2011, Volz and Sutter 2013). In contrast to traditional attenuated viruses or vaccines vectored by other viruses, the use of DNA vaccines to combat CHIKV has also been pursued. One of the first options was a DNA vaccine expressing the envelope proteins E3, E2, and E1 (Muthumani et al. 2008). An improved version of this

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vaccine employed a C–E2–E1 construct that was found to generate neutralizing antibodies that protected mice against virus challenge. The vaccine was also tested in nonhuman primates where neutralizing antibody responses were obtained after five immunizations employing electroporation as means for enhancing delivery. However, the vaccinated animals were never challenged with wild-type CHIKV (Mallilankaraman et al. 2011). In continuation, the same group further investigated the use of the CHIKV nsP2 gene as an adjuvant in an attempt to improve the protective capacity of a CHIK DNA–Env vaccine. It was claimed that immune responses were improved and led to better protection in virus challenge experiments (Bao et al. 2013). A different approach to DNA vaccination was employed by placing the complete CHIKV-encoding region of the DnsP3 and D6K replicative mutants under a cytomegalovirus (CMV) promoter. When these DNAs were delivered by intradermal electroporation, robust immune responses were obtained that included both binding and neutralizing antibodies. This strategy was to allow productive replication of the attenuated viruses by delivery of naked DNA to circumvent the need to grow large quantities of CHIKV in cell cultures (Hallenga¨rd et al. 2014a). A similar approach was recently taken (the i-DNA strategy) where the attenuated strain 181/clone 25 strain of CHIKV was expressed from the pCMV promoter and delivered as a DNA vaccine (Tretyakova et al. 2014). Finally, a replicon-based DNA vaccine expressing the CHIKV replicase and envelope proteins E3E2-6K-E1 was shown to be highly immunogenic, despite being unable to produce new virus particles due to the lack of the capsid gene (Hallenga¨rd et al. 2014b). New attempts have also been pursued with the goal of developing subunit vaccines or vaccines based on inactivated whole virions. One such study used bacterially produced rE2 and rE1 protein antigens delivered in combination with a number of different adjuvants. Balanced Th1/Th2 immune responses were obtained, including generation of neutralizing antibodies; however, the mice were not challenged in this study (Khan et al. 2012). In a separate report also using bacterially produced rE2 antigen, a number of adjuvants were tested. Although good immune responses were obtained with protection from challenge, not all adjuvants were effective (Kumar et al. 2012). Finally, two studies have been involved in testing formalin-inactivated whole virus preparations that were grown on monkey Vero cells. In both studies, Th1/Th2 balanced humoral responses were obtained. One study did not perform challenge studies (Tiwari et al. 2009), but the other could demonstrate good protection in a mouse model (Kumar et al. 2012). The use of VLPs has proven to be an interesting development. One approach has involved the transfection of human embryonic kidney cells (HEK) 293 cells with plasmid DNA encoding the CHIKV proteins C-E3-E2-6K-E1, resulting in the production of VLPs. These VLPs proved to be immunogenic in nonhuman primates where they induced good levels of neutralizing antibodies that were protective against a stringent challenge with wild-type virus (Akahata et al. 2010, Akahata and Nabel 2012). Protection was mainly antibody dependent because passive transfer of serum into naı¨ve mice rendered these animals immune against challenge. This VLP vaccine has completed a phase I clinical trial (NCT01489358) with a primary completion date of April of

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2013, the results of which are still to be disclosed. It will be important and interesting to assess how this technology will prevail in terms of production yields and stability (Kramer et al. 2013). Another similar approach engages the infection of insect cells in culture with recombinant baculovirus expressing the structural gene cassette of CHIKV (Metz et al. 2013a). Immunization of mice with nonadjuvanted VLPs resulted in the generation of high levels of neutralizing antibodies and provided complete protection against challenge. The same group further demonstrated that VLPs were more immunogenic than corresponding subunit antigens E1 or E2 that were produced in insect cells (Metz et al. 2011, Metz et al. 2013b). Due to limitations in scale-up production of VLPs in HEK293 cells or in common baculovirus-infected insect cell cultures, an improved method was recently developed. This method employs high pH–adapted Spodoptera frugiperda insect cells that resulted in a 10-fold increase in production yields. The resulting VLPs seemed to be equally immunogenic in guinea pigs when compared to the HEK293produced VLPs (Wagner et al. 2014). To date, all of the vaccine candidates developed have, with the exception of two, only been tested in mice. Because almost all of the candidates induced good protective immune responses, it is difficult to draw strong conclusions as to the merits of each candidate. Moreover, it is difficult, if not impossible, to determine correlates of protection that could drive design and testing of future vaccine candidates in the clinical pipeline. For example, the exact roles of antibodies and T cells in protection against CHIKV infection and/or chronic disease have yet to be clearly defined. Preclinical studies have certainly suggested that antibodies play an important protective role against acute infection. Thus, whereas adoptive transfer of T cells did not confer protection (Chu et al. 2013), passive transfer of immune sera did (Akahata et al. 2010, Plante et al. 2011, Chu et al. 2013). Furthermore, vaccines inducing mainly CD8-specific T cells did not protect, whereas vaccines inducing neutralizing antibodies did (Mallilankaraman et al. 2011). Most importantly, these preclinical results are strongly supported by findings from natural CHIKV infections in humans. Protective immune responses in CHIKV-infected human patients are mainly targeted against the E2, E3, and nsp3 protein antigens. Interestingly, although the production of early neutralizing IgG3 antibody levels was associated with high levels of viremia during the early disease phase, patients with such clinical manifestations recovered fully. On the other hand, patients with low levels of viremia showing slower development of IgG3 responses appeared to stand a higher risk of developing chronic arthralgia (Kam et al. 2012a, 2012b, 2012c). A number of linear and nonlinear epitopes have been associated with neutralization (or escape therefrom), with most of them being located in areas of the receptor-binding domain in E2, in E2 areas that interact with the E1 fusion loop or result in disturbance of the functions of the E1 fusion loop. Identification of such domains and epitopes will give guidance for monitoring and designing of vaccines candidates. For example, a potent human neutralizing monoclonal antibody C9 isolated from an individual who recovered from CHIKV infection defines an E2 epitope that includes residue A162 within the acid-sensitive region (ASR). The ASR has

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been shown to be involved in spike rearrangements during fusion and viral entry and is key in the structure–function of the spike complex. The C9 antibody binds to the outer and top edge of the trimeric spike complex, and another human monoclonal antibody designated E8 (involving residues Y69, F84, V113, G114, T116, D117) binds to the central part of the spike and appears not to be neutralizing (Selvarajah et al. 2013). Furthermore, the epitope V216 in the E2 domain B (distal ectodomain that may interact with the cellular receptor) and amino acid residue T101 in E1 (fusion groove) have been defined by the two broadly neutralizing antibodies, 5F10 and 8B10, respectively. Importantly, these sites were also shown to be involved in escape from neutralization, resulting in cell-to-cell transmission of CHIKV (Lee et al. 2011, Fric et al. 2013). The E2EP3 epitope (STKDNFNVYK) at the amino terminus of E2 and proximal to the E3E2 furin cleavage site was shown to be a dominant linear epitope that strongly associated with virus neutralization (Kam et al. 2012b). In corresponding murine studies, the residue K252 was demonstrated to be involved in the stabilization of the envelope complex (Akahata and Nabel 2012). Separately, monoclonal antibody CHK-152 was shown to protect the fusion loop of E1, whereas CHK-9, m10, and m242 antibodies define the receptor-binding site (Sun et al. 2013). In summary, it will be important for CHIKV vaccine candidates to address not only the strength and longevity of the specific immune responses but also the quality of the response. In this instance, one can expect greater differences between vaccine platform technologies. Further investigation for the establishment of well-defined correlates of protection and the key roles played by innate, adaptive, and memory immune cells triggered by the different vectors will be needed. In addition, safety, stability of the vaccines/vectors, and yields required for manufacture need to be assessed. The goal will be to have a longterm highly protective vaccine against chikungunya virus infection. Conclusion

The identification of specific drugs as inhibitors of CHIKV is in its infancy. Screening of inhibitors against the virusencoded enzymes has been hampered by the lack of suitable high-throughput assays and of high-resolution structural information. Thus, more potent candidates were obtained through cell-based assays. However, their exact targets remain unknown and specificity relatively low: Up to now, none of them has reached the level of preclinical study. The increasing interest in this area will probably provide more potent candidates in the near future. Thus, today the usage of biologics (antibodies) is not a second option but might be a very useful one, because most reports using either purified natural antibodies or artificial constructs claim high efficiency, up to the preclinical level for some of them. This strategy however, because of the risk and cost of production, will probably remain a provisional (temporary) therapy. Vaccine development is the most advanced topic, and some of the vaccine candidates have reached the preclinical stage (in nonhuman primates, [NHP]) and first-level evaluation in humans. Because most vaccine candidates generated protective immunity against wild-type CHIKV challenge in

CHIKUNGUNYA PREVENTION AND TREATMENT

mice, our review emphasizes the need to improve the usage of preclinical models closer to human infection and disease as well as a long-term follow-up of both quality and sustainability of the immune response. If not obtained in NHP, then such data must also be explored in historically infected people. The clinical implementation of therapeutics and vaccine candidates that will emerge from the current research will largely depend on the economical constraints, i.e., cost of production, stability of the products, and strategies of prevention. Aspects of general prophylactic vaccination must be considered against the strategy of limiting the use of treatment to only travelers and possibly prophylaxis during an outbreak. Acknowledgments

This work was supported by the Swedish Research Council and by the European Union FP7 project ‘‘Integrated Chikungunya Research’’ (ICRES), grant agreement no. 261202. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Pierre Roques CEA, Division of Immuno-Virology iMETI, Fontenay-aux-Roses France E-mail: [email protected] and Peter Liljestro¨m Department of Microbiology, Tumor and Cell Biology Karolinska Institutet Stockholm Sweden E-mail: [email protected]