Chimeric Chikungunya Viruses are Nonpathogenic in Highly Sensitive. Mouse Models, but Efficiently Induce a Protective Immune Response.

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1 JVI Accepts, published online ahead of print on 22 June 2011 J. Virol. doi: /jvi Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Chimeric Chikungunya Viruses are Nonpathogenic in Highly Sensitive Mouse Models, but Efficiently Induce a Protective Immune Response. Eryu Wang 1, Dal Young Kim 2, Scott C. Weaver 1, and Ilya Frolov 2 *. 1 Institute for Human Infections and Immunity, Sealy Center for Vaccine Development, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America. 2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America. * Corresponding author. Mailing address: Department of Microbiology, BBRB 373/Box 3, University of Alabama, 1530 Third Avenue South, Birmingham, AL Phone (205) Fax: (205) ivfrolov@uab.edu. 1

2 ABSTRACT Chikungunya virus (CHIKV) is an important pathogen causing outbreaks of highly debilitating and often chronic, arthralgic human disease. We have designed chimeric alphaviruses encoding CHIKV-specific structural genes but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response. These chimeras demonstrate a highly attenuated phenotype in both immunocompetent and immunocompromised (A129) mice. However, after a single vaccination, they induced protective immune response against subsequent CHIKV challenge, characterized by high titers of neutralizing antibodies. The rational design of alphavirus genomes provides a strong basis for the development of new recombinant alphaviruses with irreversible, highly attenuated, cell type-restricted phenotypes. Downloaded from on June 29, 2018 by guest 2

3 Alphaviruses are a group of widely distributed human and animal pathogens (17). Most of them are transmitted by mosquito vectors among vertebrate hosts, in which they cause diseases of varying severities (21). Some of the alphaviruses, such as Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis viruses are known to induce encephalitis in humans, frequently with lethal outcomes (16, 20, 22, 23). Others, such as Ross River and chikungunya (CHIKV) viruses, induce rash and severe arthralgia that can persist for years (14, 15). The public health threat of alphaviruses has been strongly underestimated, and recent widespread outbreaks of CHIK on the islands of the Indian Ocean, India, Southeast Asia, and further autochthonous transmission in temperate regions of Italy and France demonstrate a possibility for alphavirus re-emergence and spread to new geographical areas (2, 7, 11). In spite of being a continuous health threat, no efficient licensed vaccines or therapeutic means have been developed against alphavirus infections. The existing experimental vaccines suffer from numerous drawbacks, such as low immunogenicity or residual reactogenicity (1, 13). In our previous studies, we started to develop a new strategy for designing CHIKV vaccine candidates, which we expected to meet a number of the following critical requirements. i) Replication of a successful vaccine candidate must not interfere with development of the innate immune response induced in infected cells (3-5, 8-10, 19). ii) The vaccine should be highly and stably attenuated, even in immunocompromised animals. iii) To avoid possible natural circulation from a vaccinee, the ideal candidate must be incapable of replication in mosquito vectors (18). iv) Finally, it should also replicate to high titers in Vero cells, which are widely used in vaccine production, and should demonstrate no evolution during this production procedure. 3

4 To achieve these goals, in this study, we applied the results of basic research, which demonstrated that alphaviruses use different means to interfere with the development of the innate immune response. The Old World alphavirus-specific nsp2, but not other structural and nonstructural viral proteins, inhibits cellular transcription during virus replication. In the New World alphaviruses, capsid protein, but not nsp2 exhibits the same function. Therefore, we took an advantage of these fundamental differences between the New and the Old World alphaviruses and made an attempt to develop chimeric viruses encoding neither capsid nor nsp2 proteins capable of interfering with nuclear functions and inhibition of the antiviral response (10). The designed alphaviruses with chimeric genomes encoding VEEV- or EEEV-derived nonstructural and CHIKV-specific structural genes (VEE/CHIKV and EEE/CHIKV) met this requirement and were found to be dramatically less cytopathic than the parental viruses (12, 19). In contrast to the nonchimeric viruses, they induced high levels of type I interferon, whose release prevented spread of the infection and led to virus clearance from cells within a few days post infection. As expected, recombinant viruses were also highly attenuated in immunocompetent mice (19). However, we intended to introduce additional modifications in order to make these chimeras safer and unable to replicate in mosquito cells. Therefore, we made their replication dependent on the function of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and tested three different strategies of IRES-mediated CHIKV structural protein(s) expression (12). The final design of these chimeric, IRES-dependent VEE/CHIKV variants included VEEVspecific nonstructural proteins adapted to the presence of a new genetic element, the EMCV IRES, in their genomes. They also contained mutations in CHIKV glycoprotein 4

5 genes (W 64 R and N 72 Y), which allowed for replication to higher titers in Vero cells (12). Their replication efficiently activated the antiviral response, which was undetectable in the same cells infected with parental viruses that express either wild-type nsp2 or capsid protein capable of inducing transcriptional shutoff in virus-infected cells (12). However, these IRES-dependent VEE/CHIKV variants were not characterized in terms of safety and immunogenicity, particularly in immunocompromised mice. In this new study, we present data from experiments aimed at comparative evaluation of these chimeras in vivo. VEE/IRES-CHIKV contained an inactivated subgenomic promoter, and thus, expression of all of the CHIKV-specific structural proteins was mediated by the EMCV IRES (Fig. 1). It contained a previously described Y 370 C mutation in nsp2, required for its more efficient replication (12, 18). VEE/IRES- C/CHIKV encoded CHIKV glycoprotein genes in the subgenomic RNA, and only the capsid protein was translated from genomic RNA via the IRES. It also contained previously identified, nsp2-specific adaptive mutations K 139 N, K 628 E, L 461 S and D 548 G (12). The third chimeric virus genome, VEE/CHIKV/IRES-C, encoded all of the structural genes in a subgenomic RNA and adaptive mutations K 139 N, K 628 E in nsp2 (12). Its glycoproteins were translated by the cap-dependent mechanism and translation of the CHIKV capsid was directed by the IRES. The IRES-independent construct VEE/CHIKV was applied in most of the experiments as a control virus. In initial experiments, all of the variants were tested for replication in different cell types, which led to a number of findings. First, replication of the viruses was cell type-dependent and strongly depended on the IRES position (Fig. 1A). For example, VEE/IRES-C/CHIKV replicated better than VEE/IRES-CHIKV in Vero cells, but 5

6 replicated less efficiently in HeLa cells. Thus, it is unlikely that the replication efficiency of chimeric viruses in a particular cell type is indicative of their in vivo phenotype, because the latter involves replication in numerous types of cells and tissues. Secondly, dependence of the capsid or the complete set of structural proteins translation on the IRES function noticeably increased cytopathogenicity of the chimeras. The IREScontaining constructs caused complete CPE in the NIH 3T3 cells if they were infected with high doses of viruses (Fig. 1B, infections with 10 5 PFU). However, in contrast to the nonchimeric viruses, low doses of the chimeras did not induce complete CPE (Fig. 1B, infections with 10 2 PFU), indicating that the inability of these viruses to inhibit antiviral response and IFN-β release (12) prevented spread of the infection. All of these viruses were able to generate plaques on Vero cells, which are incapable of type I IFN production. Therefore, titers of the infectious viruses were determined on Vero cells. Next, the designed variants were tested for the ability to induce neutralizing antibodies. 6-week old CD1 mice were subcutaneously (s.c.) immunized using viral stocks generated in Vero cells. All of the recombinant chimeric viruses were immunogenic, and after a single immunization with 10 6 PFU, all of the mice reacted with detectable titers of CHIKV-specific, neutralizing antibodies by day 28 post infection (Table 1). Regardless of the genome design, recombinant viruses did not induce detectable signs of disease in mice, such as fever, behavioral changes, or weight loss (data not shown). One of the most important characteristics of live vaccine candidates is their safety. Therefore, constructs were evaluated in terms of safety in one of the most sensitive models available, IFN-α/βR -/- (A129) mice. 6-9 week-old A129 mice were s.c. 6

7 infected with high doses (10 4 or 10 5 PFU) of the chimeras. The VEE/CHIKV and VEE/CHIKV/IRES-C variants induced high levels of viremia within the first 24 h post infection (Table 2), and all of the mice died by day 3 post infection. The other two constructs, VEE/IRES-C/CHIKV and VEE/IRES-CHIKV, induced no detectable viremia, and no weight loss was detected (data not shown). None of the VEE/IRES-C/CHIKVinfected mice died (Table 2), and the death of a single mouse infected with VEE/IRES- CHIKV at day 6 post infection was likely to be unrelated to infection, as the animal did not exhibit signs of disease (however, necropsy was not performed). Even though the latter two viruses, VEE/IRES-C/CHIKV and VEE/IRES-CHIKV, were incapable of inducing viremia they remained immunogenic in A129 mice. At 3 weeks post infection, PRNT 80 titers of CHIKV-specific antibodies were consistently above 1:640 (Table 2). A129 mice immunized with VEE/IRES-C/CHIKV and VEE/IRES-CHIKV were also challenged with 100 PFU of the highly pathogenic LaReunion (LR) strain of CHIKV. None of the immunized animals died, or developed detectable viremia on days 1 or 3 post infection (Table 3). In contrast, the sham-vaccinated group developed significant viremia and the animals died following the CHIKV challenge with an average survival time of 4 days (Table 3). In additional safety tests, groups of 6-day-old CD1 mice (10 mice per group) were s.c. infected with 5x10 4 PFU of VEE/IRES-C/CHIKV, and CHIKV 181/clone25 experimental vaccine strain (6). No viremia [<0.9 log 10 (PFU/ml)] was detected either on day 1 or 2 post infection in VEE/IRES-C/CHIKV-infected mice. Animals infected with 181/clone25 experimental vaccine developed viremia with mean titers of 3.2 log 10 and 4.3 log 10 (PFU/ml) at day 1 and 2 post infection, respectively. Importantly, none of ten 6-7

8 day-old CD1 mice died after i.c. infection with 2x10 4 PFU of VEE/IRES-C/CHIKV, further demonstrating its safety. The results of this study demonstrate that alphavirus genomes can be rationally modified to develop variants with unique characteristics, such as an inability to interfere with the development of a cellular antiviral response and an inability to replicate in arthropod cells (12). Based on the above-described, and our previously published data (12), we consider VEE/IRES-C/CHIKV to be the variant that is most appropriate for further testing as a vaccine candidate as it i) is reproducibly immunogenic in both immunocompromised and immunocompetent mice, ii) is not lethal for A129 mice, and iii) replicates to higher titers than VEE/IRES-CHIKV in Vero cells. VEE/IRES- C/CHIKV contains fragments of genetic material derived from three different viruses (VEEV, CHIKV and EMCV), which determine translation of viral proteins, RNA replication and formation of viral particles. Therefore, none of them can be deleted during virus passaging. The combined genes were also adapted towards the most efficient virus replication, and therefore, their further evolution is unlikely. This chimeric virus encodes nsp2 and capsid proteins naturally lacking transcription inhibition functions and virus evolution towards gaining this function is also a very unlikely event. One very useful characteristic of the VEE/IRES-C/CHIKV phenotype is the inability to develop viremia not only in adult and 6-day-old CD1, but also in A129 mice. Despite this remarkable lack of pathogenicity, it retained the ability to induce CHIKV-specific neutralizing antibodies, which protect even A129 mice against challenge with a lethal dose of CHIKV LR. This indicates that replication VEE/IRES-C/CHIKV takes place, but at a level insufficient for development of viremia and detectable disease. Taken together, 8

9 the data suggest that the accumulated knowledge about alphavirus pathogenesis at the molecular level provides a strong basis for the development of viruses that demonstrate irreversible, stable and highly attenuated phenotypes even in immunocompromised animals, but remain capable of inducing efficient, protective immune response. ACKNOWLEDGMETS We thank Maryna Akhrymuk for excellent technical assistance. This work was supported by a grant from the National Institute of Allergy and Infectious Disease (NIAID) through the Western Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research U01AI and the NIAID grant R01AI REFERENCES 1. Alevizatos, A. C., R. W. McKinney, and R. D. Feigin Live, attenuated Venezuelan equine encephalomyelitis virus vaccine. I. Clinical effects in man. Am J Trop Med Hyg 16: Arankalle, V. A., S. Shrivastava, S. Cherian, R. S. Gunjikar, A. M. Walimbe, S. M. Jadhav, A. B. Sudeep, and A. C. Mishra Genetic divergence of Chikungunya viruses in India ( ) with special reference to the explosive epidemic. J Gen Virol 88: Atasheva, S., A. Fish, M. Fornerod, and E. I. Frolova Venezuelan equine Encephalitis virus capsid protein forms a tetrameric complex with CRM1 9

10 and importin alpha/beta that obstructs nuclear pore complex function. J Virol 84: Atasheva, S., N. Garmashova, I. Frolov, and E. Frolova Venezuelan equine encephalitis virus capsid protein inhibits nuclear import in Mammalian but not in mosquito cells. J Virol 82: Atasheva, S., V. Krendelchtchikova, A. Liopo, E. Frolova, and I. Frolov Interplay of acute and persistent infections caused by Venezuelan equine encephalitis virus encoding mutated capsid protein. J Virol 84: Edelman, R., C. O. Tacket, S. S. Wasserman, S. A. Bodison, J. G. Perry, and J. A. Mangiafico Phase II safety and immunogenicity study of live chikungunya virus vaccine TSI-GSD-218. Am J Trop Med Hyg 62: Enserink, M Infectious diseases. Massive outbreak draws fresh attention to little-known virus. Science 311: Garmashova, N., S. Atasheva, W. Kang, S. C. Weaver, E. Frolova, and I. Frolov Analysis of Venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription. J Virol 81: Garmashova, N., R. Gorchakov, E. Frolova, and I. Frolov Sindbis virus nonstructural protein nsp2 is cytotoxic and inhibits cellular transcription. J Virol 80: Garmashova, N., R. Gorchakov, E. Volkova, S. Paessler, E. Frolova, and I. Frolov The Old World and New World alphaviruses use different virusspecific proteins for induction of transcriptional shutoff. J Virol 81:

11 11. Hapuarachchi, H. C., K. B. Bandara, S. D. Sumanadasa, M. D. Hapugoda, Y. L. Lai, K. S. Lee, L. K. Tan, R. T. Lin, L. F. Ng, G. Bucht, W. Abeyewickreme, and L. C. Ng Re-emergence of Chikungunya virus in South-east Asia: virological evidence from Sri Lanka and Singapore. J Gen Virol 91: Kim, D. Y., S. Atasheva, N. J. Foy, E. Wang, E. I. Frolova, S. Weaver, and I. Frolov Design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype. J Virol 85: Pittman, P. R., R. S. Makuch, J. A. Mangiafico, T. L. Cannon, P. H. Gibbs, and C. J. Peters Long-term duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and following booster vaccination with inactivated VEE vaccine. Vaccine 14: Rao, T. R Recent epidemics caused by chikungunya virus in India, Scientific Culture 32: Rao, T. R., D. E. Carey, and K. M. Pavri Preliminary isolation and identification of chikungunya virus from cases of dengue-like illness in Madras city. Indian Journal of Medical Research 53: Rico-Hesse, R., S. C. Weaver, J. de Siger, G. Medina, and R. A. Salas Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America. Proc. Natl. Acad. Sci. USA 92: Strauss, J. H., and E. G. Strauss The alphaviruses: gene expression, replication, evolution. Microbiol. Rev. 58:

12 18. Volkova, E., E. Frolova, J. R. Darwin, N. L. Forrester, S. C. Weaver, and I. Frolov IRES-dependent replication of Venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells. Virology 377: Wang, E., E. Volkova, A. P. Adams, N. Forrester, S. Y. Xiao, I. Frolov, and S. C. Weaver Chimeric alphavirus vaccine candidates for chikungunya. Vaccine 26: Weaver, S. C., and A. D. Barrett Transmission cycles, host range, evolution and emergence of arboviral disease. Nat Rev Microbiol 2: Weaver, S. C., and I. Frolov Togaviruses, p In B. W. J. Mahy and V. t. Meulen (ed.), Virology, vol. 2. ASM Press, Salisbury, UK. 22. Weaver, S. C., A. Hagenbaugh, L. A. Bellew, L. Gousset, V. Mallampalli, J. J. Holland, and T. W. Scott Evolution of alphaviruses in the eastern equine encephalomyelitis complex. J Virol 68: Weaver, S. C., R. Salas, R. Rico-Hesse, G. V. Ludwig, M. S. Oberste, J. Boshell, and R. B. Tesh Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348: FIGURE LEGENDS FIG. 1. Replication of chimeric viruses in different cell types. (A) The schematic representation of viral genomes and titers of the viruses obtained from different cell types. Indicated cell lines were infected at an MOI of 10 PFU/cell, and titers were measured at 24 h post infection by plaque assay on Vero cells. This panel presents the 12

13 results of one of two reproducible experiments. (B) Comparative analysis of virus spread and CPE development in NIH 3T3 cells. 5x10 5 NIH 3T3 cells in the 6-well Costar plates were infected with the indicated doses of viruses, presented in PFU, and staining with crystal violet was performed at 72 h post infection. 13

14 Table 1. Titers of CHIKV-specific, neutralizing antibodies in CD1 mice immunized with 10 6 PFU of chimeric viruses. 1 PRNT 80 VEE/CHIKV VEE/CHIKV/IRES-C VEE/IRES-C/CHIKV VEE/IRES-CHIKV >640 > >640 > >640 > > >640 > >640 > >640 >640 >640 1 Groups of 6-week old CD1 mice were immunized s.c. with 10 6 PFU of indicated viruses in 100 µl of PBS. Titers of neutralizing antibodies were determined on day 28 post infection using PRNT 80. Values represent titers in individual mice. Downloaded from on June 29, 2018 by guest 14

15 Table 2. Infection of A129 mice with chimeric viruses. Virus Dose (PFU) 1 Number of mice Mean viremia 2 Survival (%) PRNT 80 3 ± SD VEE/CHIKV ± N.A. VEE/CHIKV/IRES-C ± N.A. VEE/IRES-CHIKV < >640 VEE/IRES-C/CHIKV < >640 VEE/IRES-C/CHIKV < >640 1 Mice were infected s.c. with indicated doses of viruses in 100 µl of PBS. 2 Viremia levels are presented in log 10 (PFU/ml) at day 1 post infection. 3 Titers of neutralizing antibodies were assessed at day 21 post infection. >640 indicates that all of the mice had titers above 640. Downloaded from on June 29, 2018 by guest 15

16 Table 3. Viremia levels and survival times of A129 mice immunized with chimeric viruses and challenged with CHIKV strain LR. Virus used for vaccination Mean viremia ± SD (day 1) Mean viremia ± SD (day 3) Average survival time ± SD (days) Sham 4.6± ± VEE/IRES-CHIKV <0.9 <0.9 >14 VEE/IRES-C/CHIKV <0.9 <0.9 >14 A129 were immunized with chimeric viruses as indicated in Table 2, and challenged by s.c. infection with 10 2 PFU indicated doses of CHIKV LR at 5 weeks post immunization. Downloaded from on June 29, 2018 by guest 16

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