Virus Research 167 (2012) 236–246 Contents lists available at SciVerse ScienceDirect Virus Research journa l h o me pag e: www.elsevier.com/locate/virusres Subunit vaccine formulations based on recombinant envelope proteins of Chikungunya virus elicit balanced Th1/Th2 response and virus-neutralizing antibodies in mice Mohsin Khan, Rekha Dhanwani, Putcha Venkata Lakshamana Rao, Manmohan Parida ∗ Division of Virology, Defense Research and Development Establishment, Gwalior, MP, India a r t i c l e i n f o Article history: Received 24 February 2012 Received in revised form 8 May 2012 Accepted 9 May 2012 Available online 17 May 2012 Keywords: Chikungunya vaccine Envelope protein Humoral response Cell mediated response Cytokine profiling a b s t r a c t The recent resurgence of Chikungunya virus in India and Indian Ocean Islands with unusual clinical severity is a matter of great public health concern. Despite the fact that CHIKV resurgence is associated with epidemic of unprecedented magnitude, none of the vaccine candidate has been approved so far. The envelope protein E1 and E2 being the major immunodominant structural proteins with crucial role in virus attachment and entry, can prove to be potential vaccine candidates. In the present study, the immunogenic potential of bacterially expressed CHIKE1 and CHIKE2 recombinant proteins along with various adjuvants is reported. Assessment of the protective efficacy of both the vaccine formulations was further confirmed by both in vitro and in vivo neutralisation tests. Splenocytes from immunized mice, cultured in vitro when stimulated with the vaccine antigens revealed induction of very high levels of both pro- and anti- inflammatory cytokines indicating a balance of Th1 and Th2 response. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Chikungunya is a debilitating viral illness that is becoming a dis- ease of global concern due to its escalating outbreaks in different parts of the world particularly in Africa and South East Asia. Chikun- gunya disease was first recognized in the form of an epidemic in East Africa during 1952–53 (Jupp and McIntosh, 1988). Since then, a large number of outbreaks have been reported (Dash et al., 2007; Schuffenecker et al., 2006). The disease is caused by Chikun- gunya Virus (CHIKV), which is classified in the family Togaviridae, genus Alphavirus. CHIKV is transmitted by the bite of infected Aedes aegypti and Aedes albopictus (Strauss and Strauss, 1994). The emer- gence and sustained circulation of Chikungunya has driven the interest of the scientific community to this long neglected tropical disease (Enserink, 2006; Charell et al., 2007). Chikungunya infection is characterized by a triad of fever, rash and arthritis and has an approximate incubation period of 1–2 weeks (Johnson and Peters, 1996). Other symptoms include myal- gia, headache, muscle aches and retro-orbital pains (Dash et al., 2007). In 2006 outbreak of La Reunion, severe forms of Chikun- gunya infection were observed in adults like encephalopathy and haemorrhagic fever as well as mother-to-child transmission of CHIKV (Couderc and Lecuit, 2009; Gerardin et al., 2008). Chikun- gunya disease is rarely fatal but is associated with significant ∗ Corresponding author. Tel.: +91 9893186204. E-mail address: paridamm@rediffmail.com (M. Parida). morbidity. While the acute febrile phase of the illness resolves within a few days, the joint pain may persists for months to years causing serious economic and social impact on both the indi- vidual and the affected communities. Unfortunately, there is no specific treatment, approved vaccine or drug available for CHIKV infection, despite the improvements of vaccine trials for other viruses that co-circulate with Chikungunya such as Dengue. This could be due to either poor understanding of pathogenesis of CHIKV or lack of proper animal model to study immune response and protection that has hindered the CHIKV vaccine develop- ment. Many vaccines effective against CHIKV infection have been reported previously. Formalin based preparation of CHIKV vac- cine was found to be immunogenic in human, monkey and mice (Edelman et al., 1979; Harrison et al., 1971; Nakao and Hotta, 1973; Tiwari et al., 2009). However the growth and production of large quantity of CHIKV antigen is a major constrain as it requires appro- priate BSL-3 containment facility. A live attenuated CHIKV vaccine (TSI-GSD-218) was also reported to be effective but caused side effects in clinical trials (Levitt et al., 1986; Edelman et al., 2000). DNA based CHIKV vaccine encoding viral structural protein was shown to be immunogenic in mice (Muthumani et al., 2008) but in general the DNA vaccines are not effective in generating the strong humoral response and neutralizing antibodies which is crucial for virus clearance. Chimeric CHIKV vaccines (Wang et al., 2008) com- prises various safety issues as the Alphaviruses can recombine and have ability to generate replication competent virions (Strauss and Strauss, 1997). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.05.004