Risk Stratification for Prevention of Sudden Cardiac Death

Sudden cardiac death (SCD) is a very prevalent cause of death in the United States. The majority of individuals who experience SCD do not survive the episode. Although there are ongoing efforts to improve resuscitation (ie, training in cardiopulmonary resuscitation, easy access to automatic external defibrillators), the primary modality addressing this public health problem is prevention by identification and treatment of high-risk cohorts. Current screening techniques have focused on identifying patients for primary prevention of ventricular tachyarrhythmias. Primary prevention therapies include medications, such as beta-blockers, statins, and angiotensin-converting enzyme inhibitors, and the implantable cardioverter defibrillator (ICD), whose use is currently focused on only the highest-risk subpopulations. The high-risk groups that are currently screened for consideration of an ICD for either primary or secondary prevention of SCD include those with a variety of cardiomyopathies, those with a history of previous aborted SCD, and those with genetic predispositions. In patients with ischemic cardiomyopathy and nonischemic dilated cardiomyopathy, the primary screening parameter to identify the highest-risk group (which is then subsequently screened for consideration of an ICD) is left ventricular ejection fraction (LVEF). Various other clinical factors and noninvasive test results are often combined with this information, but the optimal way in which this should be done has not been established. The array of noninvasive tests available includes those focusing on depolarization abnormalities, repolarization abnormalities, disturbed autonomic responses, and imaging. Unfortunately, current risk-stratification paradigms do not identify the majority of patients who will experience SCD. The fundamental reason for this is that the risk of SCD is truly lower in those without high-risk features such as depressed LVEF; however, the much larger number of patients with these lower-risk features translates into a larger absolute number of SCDs in this lower-risk group. In order to widen the scope of risk stratification, careful clinical study will be needed to develop appropriate testing strategies that can reliably identify patients at significant risk for ventricular tachyarrhythmias in the broader population

Identification of 2,4-Diaminoquinazoline Derivative as a Potential Small-Molecule Inhibitor against Chikungunya and Ross River Viruses

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor

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

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

Expression level of cytokines in control (PBS immunized) and vaccinated (CHIK-VLPs immunized) animal groups, stimulated with inactivated CHIKV.

<p>Expression level of cytokines in control (PBS immunized) and vaccinated (CHIK-VLPs immunized) animal groups, stimulated with inactivated CHIKV.</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

<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 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

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