An Essential Role of Antibodies in the Control of Chikungunya Virus Infection Fok-Moon Lum, Teck-Hui Teo, Wendy W. L. Lee, Yiu-Wing Kam, Laurent Rénia and Lisa F. P. Ng This information is current as of September 27, 2021. J Immunol 2013; 190:6295-6302; Prepublished online 13 May 2013; doi: 10.4049/jimmunol.1300304 http://www.jimmunol.org/content/190/12/6295 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/05/13/jimmunol.130030 Material 4.DC1 References This article cites 44 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/190/12/6295.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology An Essential Role of Antibodies in the Control of Chikungunya Virus Infection Fok-Moon Lum,*,†,1 Teck-Hui Teo,*,‡,1 Wendy W. L. Lee,*,‡ Yiu-Wing Kam,* Laurent Re´nia,*,2 and Lisa F. P. Ng*,†,2 In recent years, Chikungunya virus (CHIKV) was responsible for epidemic outbreaks in intertropical regions. Although acquired immunity has been shown to be crucial during CHIKV infection in both humans and mice, their exact role in the control of CHIKV infection remains unclear. In this study, wild-type (WT), CD42/2, and B cell (mMT) knockout mice were infected with CHIKV. Sera were taken at different days postinfection and measured for anti-CHIKV Ab levels. Isotype and neutralizing capacity of these Abs were assessed in vitro, and specific linear epitopes were mapped. Viremia in CHIKV-infected mMT mice persisted for more than a year, indicating a direct role for B cells in mediating CHIKV clearance. These animals exhibited a more severe disease than WT mice during the acute phase. Characterization of CHIKV-specific Abs revealed that anti-CHIKV Abs were elicited early and Downloaded from targeted epitopes mainly at the C terminus of the virus E2 glycoprotein. Furthermore, CD42/2 mice could still control CHIKV infection despite having lower anti-CHIKV Ab levels with reduced neutralizing capacity. Lastly, pre-existing natural Abs in the sera of normal WT mice recognized CHIKV and were able to partially inhibit CHIKV. Taken together, natural and CHIKV infection–induced specific Abs are essential for controlling CHIKV infections. The Journal of Immunology, 2013, 190: 6295–6302. hikungunya virus (CHIKV) is an arthropod-borne alpha- imminent risks for the spread of large-scale outbreaks. Hence, http://www.jimmunol.org/ virus transmitted by Aedes mosquitoes, namely, A. aegypti CHIKV remains a public threat that should not be ignored. C and A. albopictus (1). Infected patients experience develop- Anti-CHIKV immunity is poorly understood, and most studies ment of Chikungunya fever, characterized mainly by polyarthralgia have focused on innate immunity, particularly in elucidating the (2), febrile illness, maculopapular rashes, myalgia, headache, edema roles of type I IFN and their related antiviral pathways (10–18). of the extremities, and gastrointestinal complaints (3, 4). Patients Although these studies have demonstrated the importance of type I may also experience development of neurologic complications IFNs in restricting virus replication during the acute phase of in- and in some extreme cases, death has been reported (5, 6). Cur- fection, their effects are insufficient for the complete elimination rently, CHIKV is endemic in Africa, India, and many parts of Asia of virus in infected hosts. Moreover, CHIKV has been reported to by guest on September 27, 2021 (7), with occasional sporadic outbreaks (8, 9). The lack of herd persist in the tissues and organs in animal model studies even after immunity in countries surrounding these endemic areas presents viremia has subsided, and when levels of IFN-a/b have returned to normal (10, 14, 19). Nevertheless, CHIKV in the organs was *Singapore Immunology Network, Agency for Science, Technology and Research, shown to be eliminated progressively over time (20). Furthermore, Biopolis, Singapore 138648; †Department of Biochemistry, Yong Loo Lin School of persistently high levels of viremia with no signs of joint inflam- Medicine, National University of Singapore, Singapore 117597; and ‡National Uni- mation were observed in infected RAG22/2 mice that have no T versity of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456 and B cells (20). These observations strongly suggest that adaptive 1F.-M.L. and T.-H.T. contributed equally to this work. immunity has a crucial role in controlling and eliminating CHIKV after the initial IFN-a/b and other innate immune responses have 2L.R. and L.F.P.N. directed this work equally. subsided. It was further revealed that CD4+, but not CD8+, T cells Received for publication January 31, 2013. Accepted for publication April 9, 2013. play a major role in mediating the severity of joint inflammation, This work was supported by the Singapore Immunology Network, Agency for Sci- ence, Technology and Research. F.-M.L. is supported by a Yong Loo Lin School of although both subsets have no role in the control of CHIKV Medicine, National University of Singapore postgraduate scholarship. T.-H.T. is sup- replication and dissemination (20). In addition, passive transfer ported by an Agency for Science, Technology and Research postgraduate scholarship. of anti-CHIKV Abs was able to clear CHIKV infection in mice W.W.L.L. is supported by a National University of Singapore Graduate School for Integrative Sciences and Engineering postgraduate scholarship. (21, 22). These observations indicated that Abs could be the main The funders had no role in study design, data collection and analysis, decision to effectors in anti-CHIKV immunity. publish, or preparation of the manuscript. In this article, CHIKV infections in B cell–deficient (mMT) mice Address correspondence and reprint requests to Prof. Lisa F.P. Ng or Prof. Laurent Renia, (23) were investigated to clearly define the role of B cells in anti- Singapore Immunology Network, A*STAR, Immunos, #04-06, 8A Biomedical Grove, CHIKV immunity. We characterized the Ab response elicited dur- Biopolis, 138648 Singapore (L.F.P.N.) or Singapore Immunology Network, A*STAR, Immunos, #03-15, 8A Biomedical Grove, Biopolis, 138648 Singapore (L.R.). E-mail ing the infection and demonstrated that the breadth of protective + addresses: [email protected] (L.F.P.N.) or [email protected]. Ab response is dependent on CD4 T cells. Remarkably, we also edu.sg (L.R.) uncovered a role for natural Abs that are present in the sera of The online version of this article contains supplemental material. uninfected mice to control early CHIKV infection. Abbreviations used in this article: CHIKV, Chikungunya virus; dpi, days postinfec- tion; PBST, PBS containing 0.05% Tween 20; PDB, Protein Data Bank; WT, wild- type. Materials and Methods This article is distributed under The American Association of Immunologists, Inc., Mice Reuse Terms and Conditions for Author Choice articles. Six-week-old female wild-type (WT), mMT, and CD42/2 C57BL/6J mice Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 were used. All mice were bred and kept under specific pathogen-free www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300304 6296 Abs IN CHIKUNGUNYA VIRUS INFECTION conditions in the Biological Resource Centre, Agency for Science, Tech- Viral RNA extraction and quantification nology and Research, Singapore. In all experiments, age- and sex-matched WT and deficient mice were used. All experiments and procedures were Ten microliters blood was collected from the tail vein and diluted in 120 ml approved by the Institutional Animal Care and Use Committee (IACUC: PBS containing 10 ml citrate-phosphate-dextrose solution (Sigma-Aldrich). 120714) of the Agency for Science, Technology and Research, Singapore, Viral RNA was extracted with the QIAamp Viral RNA Kit (Qiagen) fol- in accordance with the guidelines of the Agri-Food and Veterinary Au- lowing manufacturer’s instructions. Viral RNA copies were quantified by thority and the National Advisory Committee for Laboratory Animal quantitative RT-PCR (18). Viral RNA copies were estimated from a stan- Research of Singapore. dard curve generated using serial dilutions of CHIKV negative-sense nsP1 RNA transcripts as reported previously (22). Virus Ab quantification and isotyping CHIKV SGP11 isolate, previously described (13), was used for all ex- Ab titers were assessed by a virion-based ELISA (24–26). CHIKV-coated periments. Viruses were further propagated in C6/36 cells and purified by 6 ultracentrifugation (24) before in vivo infections. Virus titer was deter- (10 virions/well in 50 ml PBS) polystyrene 96-well MaxiSorp plates minedbystandardplaqueassaysusingVero-E6cells(13). (Nunc) were blocked with PBS containing 0.05% Tween 20 (PBST) and 5% w/v nonfat milk for 1.5 h at 37˚C. Sera from normal or infected groups Virus infection and evaluation of disease were heat inactivated and serially diluted in Ab diluent (0.05% PBST + 2.5% w/v nonfat milk). One hundred microliters of diluted sera was added Mice were infected as previously described (18, 20, 24).