Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

Prediction of transposable element derived enhancers using chromatin modification profiles

Experimentally characterized enhancer regions have previously been shown to display specific patterns of enrichment for several different histone modifications. We modelled these enhancer chromatin profiles in the human genome and used them to guide the search for novel enhancers derived from transposable element (TE) sequences. To do this, a computational approach was taken to analyze the genome-wide histone modification landscape characterized by the ENCODE project in two human hematopoietic cell types, GM12878 and K562. We predicted the locations of 2,107 and 1,448 TE-derived enhancers in the GM12878 and K562 cell lines respectively. A vast majority of these putative enhancers are unique to each cell line; only 3.5% of the TE-derived enhancers are shared between the two. We evaluated the functional effect of TE-derived enhancers by associating them with the cell-type specific expression of nearby genes, and found that the number of TE-derived enhancers is strongly positively correlated with the expression of nearby genes in each cell line. Furthermore, genes that are differentially expressed between the two cell lines also possess a divergent number of TE-derived enhancers in their vicinity. As such, genes that are up-regulated in the GM12878 cell line and down-regulated in K562 have significantly more TE-derived enhancers in their vicinity in the GM12878 cell line and vice versa. These data indicate that human TE-derived sequences are likely to be involved in regulating cell-type specific gene expression on a broad scale and suggest that the enhancer activity of TE-derived sequences is mediated by epigenetic regulatory mechanisms.     

Mechanisms of homocysteine-induced oxidative stress

Hyperhomocysteinemia decreases vascular reactivity and is associated with cardiovascular morbidity and mortality. However, pathogenic mechanisms that increase oxidative stress by homocysteine (Hcy) are unsubstantiated. The aim of this study was to examine the molecular mechanism by which Hcy triggers oxidative stress and reduces bioavailability of nitric oxide (NO) in cardiac microvascular endothelial cells (MVEC). MVEC were cultured for 0-24 h with 0-100 microM Hcy. Differential expression of protease-activated receptors (PARs), thioredoxin, NADPH oxidase, endothelial NO synthase, inducible NO synthase, neuronal NO synthase, and dimethylarginine-dimethylaminohydrolase (DDAH) were measured by real-time quantitative RT-PCR. Reactive oxygen species were measured by using a fluorescent probe, 2',7'-dichlorofluorescein diacetate. Levels of asymmetric dimethylarginine (ADMA) were measured by ELISA and NO levels by the Griess method in the cultured MVEC. There were no alterations in the basal NO levels with 0-100 microM Hcy and 0-24 h of treatment. However, Hcy significantly induced inducible NO synthase and decreased endothelial NO synthase without altering neuronal NO synthase levels. There was significant accumulation of ADMA, in part because of reduced DDAH expression by Hcy in MVEC. Nitrotyrosine expression was increased significantly by Hcy. The results suggest that Hcy activates PAR-4, which induces production of reactive oxygen species by increasing NADPH oxidase and decreasing thioredoxin expression and reduces NO bioavailability in cultured MVEC by 1) increasing NO2-tyrosine formation and 2) accumulating ADMA by decreasing DDAH expression.     

Mechanisms of endothelial dysfunction with development of type 1 diabetes mellitus: role of insulin and C-peptide

Complications associated with insulin-dependent diabetes mellitus (type-1diabetes) primarily represent vascular dysfunction that has its origin in the endothelium. While many of the vascular changes are more accountable in the late stages of type-1diabetes, changes that occur in the early or initial functional stages of this disease may precipitate these later complications. The early stages of type-1diabetes are characterized by a diminished production of both insulin and C-peptide with a significant hyperglycemia. During the last decade numerous speculations and theories have been developed to try to explain the mechanisms responsible for the selective changes in vascular reactivity and/or tone and the vascular permeability changes that characterize the development of type-1diabetes. Much of this research has suggested that hyperglycemia and/or the lack of insulin may mediate the observed functional changes in both endothelial cells and vascular smooth muscle. Recent studies suggest several possible mechanisms that might be involved in the observed decreases in vascular nitric oxide (NO) availability with the development of type-1 diabetes. In addition more recent studies have indicated a direct role for both endogenous insulin and C-peptide in the amelioration of the observed endothelial dysfunction. These results suggest a synergistic action between insulin and C-peptide that facilitates increase NO availability and may suggest new clinical treatment modalities for type-1 diabetes mellitus.     

High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.