Data_Sheet_2.DOC

<p>The pathogenesis of dengue hemorrhagic fever (DHF), following dengue virus (DENV) infection, is a complex and poorly understood phenomenon. In view of the clinical need of identifying patients with higher likelihood of developing this severe outcome, we undertook a comparative genome-wide association analysis of epitope variants from sequences available in the ViPR database that have been reported to be differentially related to dengue fever and DHF. Having enumerated the incriminated epitope variants, we determined the corresponding HLA alleles in the context of which DENV infection could potentially precipitate DHF. Our analysis considered the development of DHF in three different perspectives: (a) as a consequence of primary DENV infection, (b) following secondary DENV infection with a heterologous serotype, (c) as a result of DENV infection following infection with related flaviviruses like Zika virus, Japanese Encephalitis virus, West Nile virus, etc. Subject to experimental validation, these viral and host markers would be valuable in triaging DENV-infected patients for closer supervision owing to the relatively higher risk of poor prognostic outcome and also for the judicious allocation of scarce institutional resources during large outbreaks.</p

Data_Sheet_1.XLS

<p>The pathogenesis of dengue hemorrhagic fever (DHF), following dengue virus (DENV) infection, is a complex and poorly understood phenomenon. In view of the clinical need of identifying patients with higher likelihood of developing this severe outcome, we undertook a comparative genome-wide association analysis of epitope variants from sequences available in the ViPR database that have been reported to be differentially related to dengue fever and DHF. Having enumerated the incriminated epitope variants, we determined the corresponding HLA alleles in the context of which DENV infection could potentially precipitate DHF. Our analysis considered the development of DHF in three different perspectives: (a) as a consequence of primary DENV infection, (b) following secondary DENV infection with a heterologous serotype, (c) as a result of DENV infection following infection with related flaviviruses like Zika virus, Japanese Encephalitis virus, West Nile virus, etc. Subject to experimental validation, these viral and host markers would be valuable in triaging DENV-infected patients for closer supervision owing to the relatively higher risk of poor prognostic outcome and also for the judicious allocation of scarce institutional resources during large outbreaks.</p

Bandgap Engineering of Conjugated Materials with Nonconjugated Side Chains

Controlling the optical properties of conjugated materials, especially their bandgaps, is critical to nearly all of applications of these materials. The most prevalent strategy involves changes to the structures of conjugated backbones, while side chains are generally reserved for imparting solubility. This paper, using a series of donor–acceptor conjugated oligo- and poly­(arylene–ethynylene)­s that have terephthalate units as the electron-deficient unit, demonstrates examples of how the structures of side chains that are not formally part of the conjugated backbone can have significant effects on bandgaps of these materials. In organic solution, changing alkoxy substituents on the terephthalate unit yields changes in absorbance onsets of, in some cases, greater than 20 nm; the position of absorbance spectra of these materials correlates with the Taft σ* values of the ester alkoxy groups, consistent with the side chains inductively altering the electron-accepting nature of the terephthalate ring. This structure–property relationship persists in the solid state. These results indicate that synthetically simple side-chain substitutions of formally nonconjugated groups may be useful in rational design of the optoelectronic properties of conjugated materials in both solution and the solid state

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

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

Measurement of cytokines expression in immunized mice splenocytes.

<p>Graphs showing concentrations of IL-2 <b>(A);</b> TNF-α <b>(B);</b> IFN-γ <b>(C)</b>; IL-4 <b>(D)</b> and IL-10 <b>(E)</b> in picograms per millilitre. Each bar represents the average of 10 mice/group ± S.D. and is representative of three independent experiments. Analysis was done by one way ANOVA, (Fisher LSD Method). ****P < 0.001 (significance between 72 and 48 hrs); ^<b>#</b>P < 0.001(significance with respect to control); ^<b>¤</b>P < 0.001(significance with respect to 10 μg inactivated CHIKV); *P < 0.05, **P < 0.01, ***P < 0.001(significance with respect to 20 μg inactivated CHIKV).</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

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

<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

Measurement of serum IgG antibody titers in Balb/C mice immunized with CHIK-VLPs.

<p>Sera collected after first booster 14, 28, 42, 56 and 140 days of post-vaccination from immunized groups with 40 μg, 20 μg and 10 μg CHIK-VLPs and antibody titer was measured by indirect ELISA. 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 Method). ****P <0.0001(significance with respect to 20 μg CHIK-VLPs); ^$P < 0.0001; (significance with respect to 10μg CHIK-VLPs); ^#P < 0.0001; ***P < 0.001; *P < 0.01 (significance with respect to control).</p