Expression and Characterization of Yeast Derived Chikungunya Virus Like Particles (CHIK-VLPs) and Its Evaluation as a Potential Vaccine Candidate

<div><p>Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (<i>Pichia pastoris</i>) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The <i>in vitro</i> and <i>in vivo</i> evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both <i>in vitro</i> and <i>in vivo</i> neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by <i>Pichia pastoris</i> and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.</p></div

Gross pathology, viral replication and profile of inflammatory cytokines.

<p>A. Surgically removed hind limb muscles from mock- and CHIKV-infected mice showing gross pathology of the muscles (in terms of swelling) along with the 4 μm sections showing the localization of CHIKV antigen in the hind limb skeletal muscle on day 9 post infection. B. Virus titre in the hind limb muscles. C. Real time analysis showing relative fold change in inflammatory genes (MCP-1, MCP-3, IL-6, TNF-α, Rantes) in muscle tissue on day 9 post infection. ** Genes were considered significantly up-regulated if the change in their relative expression level was ≥2 fold at p<0.05 by student's <i>t</i> test.</p

Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.

<p>Summary of proteins differentially expressed in mouse muscle tissue infected by Chikungunya virus.</p

<i>In vivo</i> virus neutralization activity of mice sera immunized with CHIK-VLPs.

<p>(A) Percentage survival of the all the mice groups. CHIKV infected mice showed 100% mortality whereas treated mice (infected with DRDE 07) that received CHIK-VLPs IgG and then infected with CHIKV showed 100% survival rate same as mock infected mice that neither infected with CHIKV nor received specific IgG. However, treated mice (infected with DRDE 06), showed 90% survival. (B) Body weight gain measured on 1–7 day of post infection. Treated mice group (infected with DRDE 07 or DRDE 06) showed significantly higher (***P < 0.0001) body weight gain than CHIKV infected mice group; (C) Serum viremia at 3 dpi and 6 dpi. Serum viremia was found to be 10 fold lower at day 3 dpi in IgG treated groups (infected with DRDE 07 or DRDE 06) compared to CHIKV infected group (*P < 0.01). At day 6 dpi, 2500 fold lower CHIKV RNA copies were detected in treated groups (infected with DRDE 07 or DRDE 06) compared to CHIKV infected group (**P < 0.001).</p

Chikungunya virus (CHIKV) induced disease signs.

<p>A. Pictures of the hind limbs of mock- and CHIKV-infected animals. Chikungunya virus infection induced severe hind limb disease in new born mice. 2–3 day old mice inoculated with 10⁶ PFU (50 ul) of CHIKV by subcutaneous injection in the loose fold of skin on the back of the animal developed peak clinical signs on 8 day post inoculation whereas mock-infected group injected with uninfected tissue culture supernatant remained healthy. B. Mice were scored for the development of hind limb dysfunction and disease based on the following scale: 0, no disease signs; 1, ruffled fur; 2, mild hind limb weakness; 3, moderate hind limb weakness; 4, severe hind limb weakness and dragging and 5, moribund. Each data point represents the arithmetic mean ± SD for eight animals. Data is representative of three independent experiments.</p

Measurement of serum IgG isotypes titers in immunized BALB/c mice.

<p>Profile of IgG isotypes in sera from immunized animal groups (40 μg, 20 μg and 10 μg CHIK-VLPs). Data represented as mean antibody titers with S.D. of ten Balb/c mice in each group Analysis was done by one way ANOVA, (Fisher LSD) ^<b>#</b>P < 0.0001(significance with respect to control); ****P < 0.0001(significance with respect to 20 μg CHIK-VLPs); °P < 0.0001(significance with respect to 10 μg CHIK-VLPs); ^<b>$</b>P < 0.0001(significance with respect to IgG2b); ^§P < 0.001(significance with respect to IgG2b); ^<b>¤</b>P < 0.0001(significance with respect to IgG3).</p

Validation of proteomic results using Q-PCR and immunoblotting.

<p>A. Transcript alteration of the differentially expressed proteins in muscle tissue upon CHIKV infection. Total RNA of muscle tissue (infected/uninfected) was analysed by real time RT-PCR. House-keeping GAPDH gene was used for normalization purpose. RNA expression changes of vimentin, hemopexin, haptoglobin, Rho GDP, PKM2 and kininogen were in concordance with protein expression changes and were determined to be statistically significant (p≤0.05). *Genes were considered to be significantly up-regulated if the change in their relative expression levels was ≥2 fold. No significant difference in the RNA expression of ApoA1 and peroxiredoxin 6 was found. B. Immunoblot of representative proteins showing increased expressions in muscle tissue upon CHIKV infection.</p

Characterization of VLPs.

<p><b>(A) Immuno-blotting using different CHIKV specific Ab.</b> (a) Western blot using anti-E1 pAb; (b) WB using anti-E2 mAb; (c) WB using anti-CHIKV pAb; <b>(B) Transmission Electron Microscopy analysis of purified CHIK-VLPs and native CHIKV:</b> (a) Electron micrograph of purified CHIK-VLP at 2, 00,000 X; (b) Electron micrograph of purified native CHIKV at 2, 00,000 X.</p

<i>In vitro</i> virus neutralization activity of mice sera immunized with CHIK-VLPs.

<p><i>In vitro</i> neutralization activity of mice sera immunized against CHIK-VLP was evaluated against two different CHIKV strains (DRDE 07 and DRDE 06). Serial two fold dilution of mice sera starting from 1:8 to 1:4196 were used to neutralize 10² pfu virus (DRDE 07 and DRDE 06). The PRNT₅₀ titer of mice sera were 1:2048, 1:512 and 1:128 for 40 μg CHIK-VLPs, 20 μg CHIK-VLPs and 10 μg CHIK-VLPs respectively.</p

Functional classification of differentially affected proteins and their possible role in disease pathogenesis.

<p>A. Functional classification of the differentially affected proteins of muscle tissue in CHIKV infection. B. Schematic representation of the possible roles of identified proteins of different classes showing metabolic and rheumatic implications in CHIKV induced myopathy.</p