Subunit vaccine formulations based on recombinant envelope proteins of Chikungunya virus elicit balanced Th1/Th2 response and virus-neutralizing antibodies in mice
Highlights
► The immunogenic potential of recombinant CHIKV envelope proteins was evaluated in mice. ► Recombinant protein elicited a strong humoral response and a balanced Th1/Th2 response. ► In vivo and in vitro efficacy of various adjuvant based formulations was also evaluated. ► Recombinant CHIKV antigens can be proposed as potent subunit vaccine candidates.
Introduction
Chikungunya is a debilitating viral illness that is becoming a disease of global concern due to its escalating outbreaks in different parts of the world particularly in Africa and South East Asia. Chikungunya 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 Chikungunya 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 emergence 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 myalgia, headache, muscle aches and retro-orbital pains (Dash et al., 2007). In 2006 outbreak of La Reunion, severe forms of Chikungunya 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). Chikungunya disease is rarely fatal but is associated with significant 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 individual 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 development.
Many vaccines effective against CHIKV infection have been reported previously. Formalin based preparation of CHIKV vaccine 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 appropriate 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) comprises various safety issues as the Alphaviruses can recombine and have ability to generate replication competent virions (Strauss and Strauss, 1997).
However, protein subunit vaccines that employ only a portion of virus are safe, easy to produce, easy to scale up and economically useful than live vaccines. Moreover, production of such vaccines involves low cost when compared to inactivated or virus-like particle (VLPs) based vaccines. To date no report is available about the immunogenicity of characterized envelope proteins of CHIKV. Since the efficacy of the protein/peptide based vaccines is determined by the strength of the humoral response in association with Th1/Th2 response (as there is no CTL activation), the present study was undertaken to evaluate the immunogenic potential of recombinant envelope proteins of CHIKV with special emphasis on induction of neutralizing antibodies that are reported to play an effective role in CHIKV infection (Couderc et al., 2009). This study not only elucidated the suitability of above proteins as attractive vaccine candidates but also explained the immuno-modulatory potential of CHIKV surface proteins that has never been evaluated before. The study demonstrated that the subunit vaccine formulations based on envelope proteins of CHIKV are capable of eliciting strong humoral and cell mediated response that was confirmed through high neutralizing antibody titres, elevated level of IgG1, IgG2a, IgG2b and upregulation of Th1/Th2 cytokines (TNF-α, IL-10, IL-12, IL-6, etc.).
Section snippets
Cells and viruses
An Indian strain of CHIKV DRDE-06 (Genbank accession no: EF-210157) of ECSA genotype was used in the present study (Dash et al., 2007). Other CHIKV strains A226V (DRDE-07) mutant (Santhosh et al., 2008) and African prototype strain of CHIKV S-27 (AF369024) were used for cross neutralization study. The virus was isolated during 2006 epidemic from a confirmed human patient in BHK-21 cells and was further passaged in Vero cell line (up to 5 passages) to increase the adaptability and the viral
Expression and purification of rCHIKE1 and rCHIKE2 proteins
Both the recombinant envelope proteins (rCHIKE1 and rCHIKE2) were successfully cloned, expressed in bacterial system and obtained at expected size (27 kDa and 47 kDa respectively). Expressed proteins were successfully purified by immobilized metal affinity chromatography (IMAC) by using Ni-NTA (Fig. 1a). Translation of desired ORF of both the proteins was confirmed by using anti-histidine antibody that targeted 6x histidine tag situated at C-terminal of the recombinant proteins and anti-CHIKV
Discussion
CHIKV is an important arbovirus causing infection in humans. In every consecutive outbreak, new character associated with the disease severity, better vector competence and altered tissue tropism is reported (Powers and Logue, 2007, Santhosh et al., 2008, Tsetsarkin et al., 2007). Hence, an effective vaccine would be extremely useful in preventing the future outbreaks of Chikungunya infection.
As there is no universal adjuvant, the vaccine formulation must be adapted in such a way that safety
Acknowledgements
M.K. is a recipient of DRDO senior research fellowship. The work was supported by the fund of DRDO, Ministry of Defence, Government of India. We also thank Dr. R. Vijayaraghavan, Director, DRDE for his support and providing basic infrastructure to carry out this study.
References (34)
- et al.
Focus on Chikungunya pathophysiology in human and animal models
Microbes and Infection
(2009) - et al.
Interleukin-10-deficient mice develop chronic enterocolitis
Cell
(1993) - et al.
Development of attenuated strain of chikungunya virus for use in vaccine production
Vaccine
(1986) - et al.
Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔCt method
Methods
(2001) - et al.
Immunogenicity of novel consensus-based DNA vaccines against chikungunya virus
Vaccine
(2008) - et al.
Development and evaluation of SYBR Green I based one step real time RT-PCR assay for detection and quantification of Chikungunya virus
Journal of Clinical Virology
(2007) - et al.
Comparative full genome analysis revealed A226V shift in 2007 Indian Chikungunya virus isolates
Virus Research
(2008) - et al.
Cloning and expression of domain III of the envelope gene of Japanese encephalitis virus: evaluation for early diagnosis by IgM ELISA
Journal of Virological Methods
(2009) - et al.
Recombination in alphaviruses
Seminars in Virology
(1997) - et al.
Assessment of immunogenic potential of Vero adapted formalin inactivated vaccine derived from novel ECSA genotype of Chikungunya virus
Vaccine
(2009)
Chimeric alphavirus vaccine candidates for chikungunya
Vaccine
Immunogenicity of a recombinant domain III protein of dengue virus type 4 with various adjuvant in mice
Vaccine
Chikungunya outbreaks; the globalization of vector borne diseases
New England Journal of Medicine
Prophylaxis and therapy for chikungunya virus infection
Journal of Infectious Diseases
East Central South African genotype as causative agent in re-emergence of Chikungunya outbreak in India
Vector Borne and Zoonotic Diseases
Chikungunya virus vaccine prepared by Tween–ether extraction
Applied Microbiology
Evaluation in humans of a new, inactivated vaccine for Venezuelan equine encephalitis virus (C-84)
Journal of Infectious Diseases
Cited by (68)
An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses
2022, Virologica SinicaCitation Excerpt :Epitopes recognized by the monoclonal neutralizing antibodies isolated from infected people were reported to locate on both E1 and E2 proteins, indicating that both E1 and E2 can induce neutralizing antibodies, which may neutralize virus through various processes, such as receptor binding, viral entry, membrane fusion and virus release (Masrinoul et al., 2014; Fox et al., 2015). Therefore, both E1 and E2 are often selected as immunogens in various vaccine platforms (Mallilankaraman et al., 2011; Khan et al., 2012; Kumar et al., 2012; Brandler et al., 2013). Previous studies show that recombinant E1 and E2 proteins often induce only low levels of antibody response(Khan et al., 2012; Metza et al., 2013); but mixing E1 and E2 into a cocktail can induce higher level of neutralizing antibodies than using E1 or E2 alone (Khan et al., 2012).
Prime-boost with Chikungunya virus E2 envelope protein combined with Poly (I:C) induces specific humoral and cellular immune responses
2021, Current Research in ImmunologyCitation Excerpt :Furthermore, both homologous E2CHIKV + Poly (I:C) and heterologous pVAX-E2CHIKV/E2CHIKV + Poly (I:C) induced neutralizing antibodies against a CHIKV isolate, a desired feature for disease control that is associated with CHIKV clearance in humans (Kam et al., 2012a). Similarly, some studies have also demonstrated the induction of neutralizing antibodies in BALB/c mice immunized with bacteria-expressed E2CHIKV recombinant protein (Kumar et al., 2012; Khan et al., 2012; Weber et al., 2015). Regarding cellular immune responses, splenocytes from mice immunized with homologous E2CHIKV + Poly (I:C) or heterologous pVAX-E2CHIKV/E2CHIKV + Poly (I:C) presented similar number of specific IFN-γ producing cells.
An in vitro refolding method to produce oligomers of anti-CHIKV, E2-IgM Fc fusion subunit vaccine candidates expressed in E. coli
2020, Journal of Immunological MethodsCitation Excerpt :A persistent rise in global temperatures is facilitating vector growth and the lack of licensed vaccines or therapeutics further increases the risk of severe outbreaks of chikungunya globally. Up until now various strategies have been employed to develop a robust vaccine against CHIKV e.g. virus-like particles (VLPs) (Metz et al., 2013a; Metz et al., 2013b; Arevalo et al., 2019), live attenuated (Levitt et al., 1986; Kumar et al., 2012), chimeric virus based vaccines (Ramsauer et al., 2015), DNA (Mallilankaraman et al., 2011; Hallengärd et al., 2014; Tretyakova et al., 2014), replication defective virus (Erasmus et al., 2017) and subunit vaccines (Metz et al., 2011; Khan et al., 2012; Weber et al., 2015) have been tried, however none has yet been approved for human use. Vaccines carrying whole viruses, generate robust immune response however there are various safety concerns associated with them, for example reversion of the attenuated/killed virus to its virulent form resulting in infection or otherwise development of symptoms like joint and muscle pain during elicitation of vaccine directed immunity in host (Schwameis et al., 2016; Young et al., 2019).