Multi-epitope vaccine against drug-resistant strains of Mycobacterium tuberculosis: a proteome-wide subtraction and immunoinformatics approach.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Toll-like receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

Feasibility of MinION Nanopore Rapid Sequencing in the Detection of Common Diarrhea Pathogens in Fecal Specimen.

The need for fast detection of etiological agents outside the narrow target range of pathogens that may cause an event of an infectious disease epidemic necessitates rapid sequencing technologies to be implemented in routine diagnostic procedures. We tested the performance of a PCR-free rapid nanopore barcoding assay to detect microbial species by analyzing genomic contents extracted from acute diarrheal case specimens. Sequenced reads were processed in an automated analysis module for species identification, whereas pathogenic subspecies detection was aided by a sequence similarity search against a gene-specific database. Evaluation of assay and analysis parameters (e.g., run-time, sequence length, and species hit abundance level) was carried out using a standard bacterial community for assessing detection accuracy. It was observed that longer sequence length (≥500 nucleotides) along with higher species abundance level (≥1%) can be critical for exclusion of false-negative outcomes, while increased sequencing run-time can affect the proportional abundance of true-positive species. Under optimal parameters, the sensitivity of the rapid assay remained 100% for the detection of a target species in a background of nontarget fecal (diarrheal) DNA that weighed up to 64 times the DNA of the target species. The method was applied to acute diarrheal samples. Among these, 62.5% (5/8) were in agreement with target-specific traditional diagnosis methods for the presence/absence of pathogenic agent(s), 12.5% (1/8) were in disagreement, and pathogenic agents that were not targeted by the traditional methods were revealed by sequencing for 25% (2/8) of samples. These observations suggest that further optimization and evaluation of the rapid nanopore sequencing method could potentiate the widening of the range of pathogens that can be detected in acute diarrheal samples in the context of regular diagnostic needs as well as epidemics.

Non-lactose fermenting Escherichia coli: Following in the footsteps of lactose fermenting E. coli high-risk clones.

Multi-resistant pathogenic strains of non-lactose fermenting Escherichia coli (NLF E. coli) are responsible for various intestinal and extraintestinal infections. Although several studies have characterised such strains using conventional methods, they have not been comprehensively studied at the genomic level. To address this gap, we used whole-genome sequencing (WGS) coupled with detailed microbiological and biochemical testing to investigate 17 NLF E. coli from a diagnostic centre (icddr,b) in Dhaka, Bangladesh. The prevalence of NLF E. coli was 10%, of which 47% (8/17) exhibited multi-drug resistant (MDR) phenotypes. All isolates (17/17) were confirmed as E. coli and could not ferment lactose sugar. WGS data analysis revealed international high-risk clonal lineages. The most prevalent sequence types (STs) were ST131 (23%), ST1193 (18%), ST12 (18%), ST501 (12%), ST167 (6%), ST73 (6%) and ST12 (6%). Phylogenetic analysis corroborated a striking clonal population amongst the studied NLF E. coli isolates. The predominant phylogroup detected was B2 (65%). The bla CTX-M-15 extended-spectrum beta-lactamase gene was present in 53% of isolates (9/17), whilst 64.7% (11/17) isolates were affiliated with pathogenic pathotypes. All extraintestinal pathogenic E. coli pathotypes demonstrated ß-hemolysis. Our study underscores the presence of critical pathogens and MDR clones amongst non-lactose fermenting E. coli. We suggest that non-lactose fermenting E. coli be considered equally capable as lactose fermenting forms in causing intestinal and extraintestinal infections. Further, there is a need to undertake systematic, unbiased monitoring of predominant lineages amongst non-lactose fermenting E. coli that would help in better treatment and prevention strategies.

Proteome Exploration of Legionella pneumophila To Identify Novel Therapeutics: a Hierarchical Subtractive Genomics and Reverse Vaccinology Approach.

Legionella pneumophila is the causative agent of a severe type of pneumonia (lung infection) called Legionnaires' disease. It is emerging as an antibiotic-resistant strain day by day. Hence, identifying novel drug targets and vaccine candidates is essential to fight against this pathogen. Here, attempts were taken through a subtractive genomics approach on the complete proteome of L. pneumophila to address the challenges of multidrug resistance. A total of 2,930 proteins from L. pneumophila proteome were investigated through diverse subtractive proteomics approaches, e.g., identification of human nonhomologous and pathogen-specific essential proteins, druggability and "anti-target" analysis, subcellular localization prediction, human microbiome nonhomology screening, and protein-protein interaction studies to find out effective drug and vaccine targets. Only three fulfilled these criteria and were proposed as novel drug targets against L. pneumophila. Furthermore, outer membrane protein TolB was identified as a potential vaccine target with a better antigenicity score. Antigenicity and transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis, and a molecular docking approach were adopted to generate the most potent epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes, along with suitable adjuvant and linkers. The designed vaccine construct showed higher binding interaction with different major histocompatibility complex (MHC) molecules and human immune TLR-2 receptors with minimum deformability at the molecular level. The present study aids the development of novel therapeutics and vaccine candidates for efficient treatment and prevention of L. pneumophila infections. However, further wet-lab-based phenotypic and genomic investigations and in vivo trials are highly recommended to validate our prediction experimentally. IMPORTANCE Legionella pneumophila is a human pathogen distributed worldwide, causing Legionnaires' disease (LD), a severe form of pneumonia and respiratory tract infection. L. pneumophila is emerging as an antibiotic-resistant strain, and controlling LD is now difficult. Hence, developing novel drugs and vaccines against L. pneumophila is a major research priority. Here, the complete proteome of L. pneumophila was considered for subtractive genomics approaches to address the challenge of antimicrobial resistance. Our subtractive proteomics approach identified three potential drug targets that are promising for future application. Furthermore, a possible vaccine candidate, "outer membrane protein TolB," was proposed using reverse vaccinology analysis. The constructed vaccine candidate showed higher binding interaction with MHC molecules and human immune TLR-2 receptors at the molecular level. Overall, the present study aids in developing novel therapeutics and vaccine candidates for efficient treatment of the infections caused by L. pneumophila.

Immunoinformatics and molecular dynamics approaches: Next generation vaccine design against West Nile virus.

West Nile Virus (WNV) is a life threatening flavivirus that causes significant morbidity and mortality worldwide. No preventive therapeutics including vaccines against WNV are available for human use. In this study, immunoinformatics approach was performed to design a multi epitope-based subunit vaccine against this deadly pathogen. Human (HLA) and Mice (H-2) allele specific potential T-cell and B-cell epitopes were shortlisted through a stringent procedure. Molecular docking showed selected epitopes that have stronger binding affinity with human TLR-4. Molecular dynamics simulation confirmed the stable nature of the docked complex. Furthermore, in silico cloning analysis ensures efficient expression of desired gene in the microbial system. Interestingly, previous studies showed that two of our selected epitopes have strong immune response against WNV. Therefore, selected epitopes could be strong vaccine candidates to prevent WNV infections in human. However, further in vitro and in vivo investigations could be strengthening the validation of the vaccine candidate against WNV.

Structural analogues of existing anti-viral drugs inhibit SARS-CoV-2 RNA dependent RNA polymerase: A computational hierarchical investigation.

The Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic, resulting in an exponentially increased mortality globally and scientists all over the world are struggling to find suitable solutions to combat it. Multiple repurposed drugs have already been in several clinical trials or recently completed. However, none of them shows any promising effect in combating COVID-19. Therefore, developing an effective drug is an unmet global need. RdRp (RNA dependent RNA polymerase) plays a pivotal role in viral replication. Therefore, it is considered as a prime target of drugs that may treat COVID-19. In this study, we have screened a library of compounds, containing approved RdRp inhibitor drugs that were or in use to treat other viruses (favipiravir, sofosbuvir, ribavirin, lopinavir, tenofovir, ritonavir, galidesivir and remdesivir) and their structural analogues, in order to identify potential inhibitors of SARS-CoV-2 RdRp. Extensive screening, molecular docking and molecular dynamics show that five structural analogues have notable inhibitory effects against RdRp of SARS-CoV-2. Importantly, comparative protein-antagonists interaction revealed that these compounds fit well in the pocket of RdRp. ADMET analysis of these compounds suggests their potency as drug candidates. Our identified compounds may serve as potential therapeutics for COVID-19.

Identification of novel drug targets for humans and potential vaccine targets for cattle by subtractive genomic analysis of Brucella abortus strain 2308.

Brucella abortus is the causative agent of brucellosis, a neglected endemic zoonotic disease. It causes devastating economic losses in low income and developing countries. Clinical symptoms of infected cows include abortion, poor weight, reduced fertility gain and reduction in milk production. Transmission of the zoonotic disease from cattle to human can occur through direct contact with infected cows, their tissues (e.g. placenta or aborted tissues), or their products (e.g. dairy) whereas human-to-human transmission can occur transplacentally or via breastfeeding. Malaise, fatigue, fever, arthritis are some clinical symptom of the disease in humans. Recent studies have revealed that Brucella abortus show resistance to several antibiotics. There are worldwide concerns about rising levels of antibiotic resistance resulting in the treatment failure as well as the reduced usefulness of older broad-spectrum antibiotics. Hence, a rather novel method has been in use to combat resistant pathogens since the last decade. To overcome this challenge, subtractive genomic analysis has been successfully carried out with the whole proteome of Brucella abortus strain 2308 using various bioinformatic tools and servers. Proteins nonhomologous to cattle and human were selected for metabolic analysis. Only three membrane proteins (ABC transporter permease, acriflavine resistance protein B, penicillin-binding protein 2) were found to be potential novel vaccine candidates with cattle as the host whereas one membrane protein (ABC transporter permease) was selected as novel drug target with human as the host. Development of novel vaccines and therapeutics through targeting inhibition of the functions of any of these essential proteins can lead to disruption of pathogen-specific metabolic pathways and thereby to the destruction and the eradication of this pathogen from respective hosts. The results of this study could facilitate the discovery and release of new and effective drugs and help in designing and producing potent vaccines against Brucella abortus strain 2308.