Designing a Novel Multiepitope Vaccine from the Human Papilloma Virus E1 and E2 Proteins for Indonesia with Immunoinformatics and Molecular Dynamics Approaches.

One of the deadliest malignant cancer in women globally is cervical cancer. Specifically, cervical cancer is the second most common type of cancer in Indonesia. The main infectious agent of cervical cancer is the human papilloma virus (HPV). Although licensed prophylactic vaccines are available, cervical cancer cases are on the rise. Therapy using multiepitope-based vaccines is a very promising therapy for cervical cancer. This study aimed to develop a multiepitope vaccine based on the E1 and E2 proteins of HPV 16, 18, 45, and 52 using in silico. In this study, we develop a novel multiepitope vaccine candidate using an immunoinformatic approach. We predicted the epitopes of the cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) and evaluated their immunogenic properties. Population coverage analysis of qualified epitopes was conducted to determine the successful use of the vaccine worldwide. The epitopes were constructed into a multiepitope vaccine by using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled with AlphaFold and was evaluated by Prosa-web. The results of vaccine construction were analyzed for B-cell epitope prediction, molecular docking with Toll like receptor-4 (TLR4), and molecular dynamics simulation. The results of epitope prediction obtained 4 CTL epitopes and 7 HTL epitopes that are eligible for construction of multiepitope vaccines. Prediction of the physicochemical properties of multiepitope vaccines obtained good results for recombinant protein production. The interaction showed that the interaction of the multiepitope vaccine-TLR4 complex is stable based on the binding free energy value -106.5 kcal/mol. The results of the immune response simulation show that multiepitope vaccine candidates could activate the adaptive and humoral immune systems and generate long-term B-cell memory. According to these results, the development of a multiepitope vaccine with a reverse vaccinology approach is a breakthrough to develop potential cervical cancer therapeutic vaccines.

Comparative biochemical analysis of full-length and truncated BaqA α-amylases from Bacillus aquimaris MKSC 6.2.

BaqA is a raw starch degrading α-amylase produced by the marine bacterium Bacillus aquimaris MKSC 6.2, associated with soft corals. This α-amylase belongs to a new subfamily Glycoside Hydrolases (GH) 13_45 which has several unique characteristics, namely, a pair of tryptophan residues Trp201 and Trp202, a distinct LPDIx signature in the Conserved Sequence Region-V (CSR-V), and an elongated C-terminus containing five aromatic residues. The research aims to investigate physicochemical, kinetics, and biochemical properties of BaqA. In this study, the full-length enzyme (BaqA) and a truncated form of BaqA (designated as BaqAΔC), lacking the C-terminal 34 amino acids were constructed and expressed in Escherichia coli ArcticExpress (DE3). BaqA formed inclusion bodies, while BaqAΔC was produced as a soluble protein. Purified and refolded BaqA exhibited a catalytic efficiency (k cat/K m) of 53.1 ± 6.3 mL mg-1 s-1 at 40 °C and pH 7.5, whereas the purified BaqAΔC displayed k cat/K m of 11.4 ± 1.3 mL mg-1 s-1 under the optimum condition of 50 °C and pH 6.5. Moreover, BaqAΔC showed a slight reduction in the binding affinity towards sago granules. Interestingly, BaqAΔC displayed robust stability and halotolerant properties compared to BaqA. BaqAΔC maintained 50 % amylolytic activity for up to 6 h, whereas BaqA lost over 50 % of its activity within 90 min. Furthermore, BaqAΔC showed a remarkable increase in amylolytic activity upon the addition of NaCl, with an optimum concentration of 0.5 M. Even at a high salt concentration (1.5 M NaCl), BaqAΔC retained over 50 % of its residual activity. Taken together, its solubility, amylolytic activity, stability, ability to degrade raw starch, and moderate halotolerance make BaqAΔC a promising candidate for various starch processing industries.

The SARS-CoV-2 Mpro Dimer-Based Screening System: A Synthetic Biology Tool for Identifying Compounds with Dimerization Inhibitory Potential.

The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (Mpro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 Mpro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-Mpro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 µg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 µg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 Mpro and also minimize the use of a high biosafety level laboratory.

The molecular interaction of six single-stranded DNA aptamers to cardiac troponin I revealed by docking and molecular dynamics simulation.

Cardiac troponin I (cTnI) is a cardiac biomarker for diagnosing ischemic heart disease and acute myocardial infarction. Current biochemical assays use antibodies (Abs) due to their high specificity and sensitivity. However, there are some limitations, such as the high-cost production of Abs due to complex instruments, reagents, and steps; the variability of Abs quality from batch to batch; the low stability at high temperatures; and the difficulty of chemical modification. Aptamer overcomes the limitations of antibodies, such as relatively lower cost, high reproducibility, high stability, and ease of being chemically modified. Aptamers are three-dimensional architectures of single-stranded RNA or DNA that bind to targets such as proteins. Six aptamers (Tro1-Tro6) with higher binding affinity than an antibody have been identified, but the molecular interaction has not been studied. In this study, six DNA aptamers were modeled and docked to cTnI protein. Molecular docking revealed that the interaction between all aptamer and cTnI happened in the similar cTnI region. The interaction between aptamer and cTnI involved hydrophobic interaction, hydrogen bonds, π-cation interactions, π-stack interactions, and salt-bridge formation. The calculated binding energy of all complexes was negative, which means that the complex formation was thermodynamically favorable. The electrostatic energy term was the main driving force of the interaction between all aptamer and cTnI. This study could be used to predict the behavior of further modified aptamer to improve aptamer performance.