Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Design, synthesis, molecular docking and DFT studies on novel melatonin and isatin based azole derivatives.

In order to address the pressing demand for newer broad-spectrum antifungal medicines with enhanced activity, computer modelling was utilised to rationally develop newer antifungal azole-based drugs. Based on the drug and active sites of the Lanosterol 14 alpha-Demethylases (LAD) of the prominent fungal pathogen Candida albicans interaction, Novel triazole-linked melatonin and isatin derivatives 7a-d and 8a-d were synthesised using bioisosterism. Besides the experimental synthesis and subsequent characterization, the present study focused on obtaining optimised geometries, frequency calculations, and TD-DFT studies of the synthesised molecules. We also performed molecular docking studies to explore the inhibitory ability of the synthesised compounds against the active sites of the Lanosterol 14 alpha-Demethylases (LAD) of the prominent fungal pathogen Candida albicans. The binding interactions resulted in positive findings, demonstrating the involvement of the synthesised compounds in the suppression of fungal growth. Comparative analysis of the binding potential of the synthesised molecules and commercially available drug fluconazole revealed a remarkable note: the docking scores for the designed drugs 7b, 7c, and 8c are much greater than those of the fluconazole molecule. The in silico study of the designed series of drug molecules serves as an important guideline for further exploration in the quest for potent antifungal agents.

Synthesis of benzothiazole-appended bis-triazole-based structural isomers with promising antifungal activity against Rhizoctonia solani.

In order to explore new antifungal agrochemicals, we reported the synthesis of two series 5a-f, 6 and 7a-f, 8 of benzothiazole-appended bis-triazole derivative-based structural isomers using a molecular hybridization approach. The synthesized compounds were tested for fungal growth inhibition against the plant pathogen Rhizoctonia solani. All the synthesized compounds showed excellent antifungal activity in their minimum concentrations (10-0.62 µM). Among all the synthetics, compounds 5b (ED50: 2.33 µM), 5f (ED50: 0.96 µM), and 7f (ED50: 1.48 µM) exerted a superior inhibitory effect in comparison to the commercially available fungicide, hexaconazole (ED50: 2.44 µM). The binding interactions of the active compounds 5f, 7f, 6, and 8 within the active site of the sterol 14α-demethylase enzyme were studied with the help of molecular docking studies. The studies revealed that these hybrid pharmacophores could be used as an important intermediate to demonstrate new structural isomer-based fungicides.

Contrasting behavior between the three human monocyte subsets in dengue pathophysiology.

Monocytes are known to play a critical role in dengue pathophysiology. However, which monocyte subset expresses what inflammatory mediator(s) and what transcriptional features distinguish each of the monocyte subset in vivo remain poorly understood. In this study we provide a detailed transcriptional analysis of the three human monocyte subsets in healthy children and in children with dengue febrile illness. Notably, we found that the CD14+ CD16high intermediate monocyte subset from dengue patients highly upregulated key genes involved in mediating inflammation, endothelial dysfunction, vascular permeability, tissue extravasation, and clot prevention compared to healthy children. The CD14+CD16low classical monocytes shared some of these features. These two subsets increased massively in patients with severe dengue. By contrast, the CD14-CD16high nonclassical monocyte subset upregulated key genes involved in vasoconstriction, endothelial barrier stability, and are involved in endothelial patrolling while showing a significant decline from circulation. These findings improve our understanding of monocyte responses in dengue.

Immunophenotyping and Transcriptional Profiling of Human Plasmablasts in Dengue.

Plasmablasts represent a specialized class of antibody-secreting effector B cells that transiently appear in blood circulation following infection or vaccination. The expansion of these cells generally tends to be massive in patients with systemic infections such as dengue or Ebola that cause hemorrhagic fever. To gain a detailed understanding of human plasmablast responses beyond antibody expression, here, we performed immunophenotyping and RNA sequencing (RNA-seq) analysis of the plasmablasts from dengue febrile children in India. We found that plasmablasts expressed several adhesion molecules and chemokines or chemokine receptors that are involved in endothelial interactions or homing to inflamed tissues, including skin, mucosa, and intestine, and upregulated the expression of several cytokine genes that are involved in leukocyte extravasation and angiogenesis. These plasmablasts also upregulated the expression of receptors for several B-cell prosurvival cytokines that are known to be induced robustly in systemic viral infections such as dengue, some of which generally tend to be relatively higher in patients manifesting hemorrhage and/or shock than in patients with mild febrile infection. These findings improve our understanding of human plasmablast responses during the acute febrile phase of systemic dengue infection. IMPORTANCE Dengue is globally spreading, with over 100 million clinical cases annually, with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody-dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the early cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the transcriptional profiles of human plasmablasts from dengue patients.

Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.