Evaluation of recombinant E2 protein-based and whole-virus inactivated candidate vaccines against chikungunya virus

Abstract

Objectives

With the re-emergence of chikungunya virus (CHIKV) in an explosive form and in the absence of a commercially available vaccine, we aimed to develop candidate vaccines employing recombinant E2 protein or chemically inactivated whole virus.

Design and methods

E2 gene of CHIKV isolate of ECSA genotype was cloned in pET15b vector, expressed and purified (rE2p). The virus was propagated in Vero cell line, purified and inactivated with formalin and BPL individually. Six to eight weeks old female BALB/c mice were immunized intramuscularly with two doses of 10 μg, 20 μg and 50 μg of vaccine formulations with or without adjuvants, 2 weeks apart. The adjuvants evaluated were alum, Mw, CadB (rE2p), alum/Mw (formalin inactivated CHIKV) and alum (BPL-inactivated CHIKV). Humoral immunity was assessed by ELISA and in vitro neutralization test using homologous and heterologous (Asian genotype) strains of CHIKV. Two cohorts of vaccinated mice were challenged separately via intranasal route with homologous virus two and 20 weeks after the 2nd dose. Viral load (CHIKV RNA by real time PCR) was determined in the serum and tissues (muscle, brain, spleen) of the mice challenged with the homologous virus.

Results

Anti-CHIK-antibody titres were dose dependent for all the immunogen formulations. BPL-inactivated vaccines led to the highest ELISA/neutralizing antibody (nAb) titres while alum was the most effective adjuvant. Asian genotype strain could be neutralized by the nAbs. In an adult mouse model, complete protection was offered by the alum-adjuvanted rE2p and both the inactivated vaccines as no virus was detected in the tissues and blood after challenge 2 weeks or 20 weeks-post-2nd dose. However, with rE2p-CadB, very low viremia was recorded on the 2nd day-post-challenge.

Conclusion

Both rE2p and BPL/formalin-inactivated virus are promising candidate vaccines deserving further evaluation.

Introduction

Chikungunya, a mosquito borne viral disease caused by chikungunya virus (CHIKV) has drawn substantial attention after its re-emergence causing massive outbreaks in tropical regions of Asia and Africa [1]. CHIKV, an alphavirus of family Togaviridae was first isolated from the serum of a febrile patient during the 1952–53 epidemics in Tanzania, Africa [2]. The virus is transmitted by mosquitoes (genus Aedes) and is maintained in nature through human–mosquito–human or sylvatic cycles [3].

Chikungunya fever is characterized by high fever, arthralgia, myalgia, nausea and rash. Though the disease is usually self limiting, persistence of arthralgia for several months to years have been recorded [1], [4], [5]. The other clinical complications include encephalopathy, haemorrhagic fever, neurological complications, renal failure, ophthalmic involvements, fulminant hepatitis, acute flaccid paralysis, etc. [6], [7], [8], [9], [10]. Patients with underlying medical conditions were associated with clinical complications and death [10], [11].

CHIKV is a positive sense, single stranded RNA virus of approximately 12 kb genome. It encodes four non-structural proteins (nsP1-4) and five structural proteins (Capsid and four envelope proteins E1, E2, E2 and 6K). Phylogenetic analysis based on the E1 gene sequences has classified CHIKV into three genotypes; Asian, East/Central/South African (ECSA) and West African. In India, all the previous outbreaks were attributed to the Asian genotype while a shift to ECSA genotype was noted during the current episode [12], [13].

Currently, no licensed vaccine or specific therapeutics are available for CHIKV infection. The earlier efforts include a formalin-inactivated vaccine employing Asian genotype [14] and attenuation of the same strain to formulate an attenuated vaccine [15]. However, during phase II clinical trials, the attenuated vaccine was reactogenic and withdrawn [16]. Following the recent resurgence of the disease and with the availability of newer platforms for vaccine development, different approaches have been tried [17], [18], [19], [20], [21], [22]. So far, none of the vaccines have been licensed.

Several Indian states experienced massive outbreaks of chikungunya since October 2005 and are continued till date (NIV unpublished data). Mortality among patients with underlying medical condition and a substantial proportion exhibiting continued arthralgia for several years are noteworthy [10]. Considering the widespread infections and associated sequalae/mortality, the need for an effective vaccine was obvious. We attempted to develop recombinant E2 protein-based and whole virus-inactivated vaccines.