Defeating a superbug: A breakthrough in vaccine design against multidrug-resistant Pseudomonas aeruginosa using reverse vaccinology.

BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa has become a major cause of severe infections. Due to the lack of approved vaccines, this study has presented putative vaccine candidates against it. METHODS: P. aeruginosa 24Pae112 as a reference strain was retrieved from GenBank database. The surface-exposed, antigenic, non-allergenic, and non-homologous human proteins were selected. The conserved domains of selected proteins were evaluated, and the prevalence of proteins was assessed among 395 genomes. Next, linear and conformational B-cell epitopes, and human MHC II binding sites were determined. Finally, five conserved and highly antigenic B-cell epitopes from OMPs were implanted on the three platforms as multi-epitope vaccines, including FliC, the bacteriophage T7 tail, and the cell wall-associated transporter proteins. The immunoreactivity was investigated using molecular docking and immune simulation. Furthermore, molecular dynamics simulation was done to refine the chimeric cell-wall-associated transporter-TLR4 complex as the best interaction. RESULTS: Among 6494 total proteins of P. aeruginosa 24Pae112, 16 proteins (seven OMPs and nine secreted) were ideal according to the defined criteria. These proteins had a molecular weight of 110 kDa and were prevalent in ≥ 75% of P. aeruginosa genomes. Among the presented multi-epitope vaccines, the chimeric cell-wall-associated transporter had the strongest interaction with TLR4. Moreover, the immune simulation response revealed that the bacteriophage T7 tail chimeric protein had the strongest ability to stimulate the immune system. In addition, molecular docking and molecular dynamic simulation indicated the proper and stable interactions between the chimeric cell-wall-associated transporter and TLR4. CONCLUSION: This study proposed 16 shortlisted proteins as promising immunogenic targets. Two novel platforms (e.g. cell-wall-associated transporter and bacteriophage T7 tail proteins) for designing of multi-epitope vaccines (MEVs), showed the better performance compared to FliC. In our future studies, these two MEVs will receive more scrutiny to evaluate their immunoreactivity.

Immunization with recombinant DcaP-like protein and AbOmpA revealed protections against sepsis infection of multi-drug resistant Acinetobacter baumannii ST2Pas in a C57BL/6 mouse model.

BACKGROUNDS: The prevalence of infections associated with multi-drug resistant (MDR) Acinetobacter baumannii is increasing worldwide. Therefore, the introduction of effective vaccines against this bacterium seems necessary. METHODS: AbOmpA and DcaP-like protein were selected as promising and putative immunogenic candidates based on previous in silico studies. Three formulations including AbOmpA, DcaP-like protein, and AbOmpA + DcaP-like protein were injected into C57BL/6 mice three times with Alum adjuvant. The specific production of IgG antibodies (e.g. total IgG, IgG1 and IgG2c) and cytokines (e.g. IL-4, IL-6, and IL-17A), were evaluated. LD50% of MDR A. baumannii ST2Pas was measured using Probit's method. After the challenge with bacteria, a decrease in bacterial loads (DLs) in the lung and spleen of mice was measured. Then serum bactericidal assay was performed to determine the function of antibodies on day 42. In addition, histopathological examinations of the spleen and lung, the number of macrophage and neutrophil, as well as the rate of lymphocyte infiltration were assessed. RESULTS: The highest level of total IgG was reported in the group immunized with DcaP-like protein on day 42. The survival rate of mice was 80% in the AbOmpA immunized group and 100% for the rest of two groups. DLs in the spleen of mice immunized with AbOmpA, DcaP-like protein, and combination form were 3.5, 3, and 3.4 Log10 (CFU/g), respectively. While in the lung, the DLs were 7.5 Log10 (CFU/g) for the AbOmpA group and 5 for the rest of two groups. The levels of IL-6, IL-4, and IL-17A were significantly decreased in all immunized groups after the bacterial challenge (except for IL-17A in the group of AbOmpA). The bactericidal effect of antibodies against DcaP-like protein was more effective. No histopathological damage was observed in the combination immunized group. The DcaP-like protein was more effective in neutrophil and macrophage deployment and decreased lymphocyte infiltration. CONCLUSION: The results of immunization with AbOmpA + DcaP-like protein induced a protective reaction against the sepsis infection of MDR A. baumannii. It seems that in the future, these proteins can be considered as promising components in the development of the A. baumannii vaccine.

Genome-Wide Subtraction Analysis and Reverse Vaccinology to Detect Novel Drug Targets and Potential Vaccine Candidates Against Ehrlichia chaffeensis.

Human monocytotropic ehrlichiosis is an emerging tick-borne infection caused by the obligate intracellular pathogen, Ehrlichia chaffeensis. The non-specific symptoms can range from a self-limiting fever to a fatal septic-like syndrome and may be misdiagnosed. The limited treatment choices including doxycycline are effective only in the initiation phase of the infection. It seems that novel therapeutic targets and new vaccine strategies could be effective to control this pathogen. This study is comprised of two major phases. First, the common proteins retrieved through subtractive analysis and potential drug targets were evaluated by subcellular localization, homology prediction, metabolic pathways, druggability, essentiality, protein-protein interaction networks, and protein data bank availability. In the second phase, surface-exposed proteins were assessed based on antigenicity, allergenicity, physiochemical properties, B cell and T cell epitopes, conserved domains, and protein-protein interaction networks. A multi-epitope vaccine was designed and characterized using molecular dockings and immune simulation analysis. Six proteins including WP_011452818.1, WP_011452723.1, WP_006010413.1, WP_006010278.1, WP_011452938.1, and WP_006010644.1 were detected. They belong to unique metabolic pathways of E. chaffeensis that are considered as new essential drug targets. Based on the reverse vaccinology, WP_011452702.1, WP_044193405.1, WP_044170604.1, and WP_006010191.1 proteins were potential vaccine candidates. Finally, four B cell epitopes, including SINNQDRNC, FESVSSYNI, SGKKEISVQSN, and QSSAKRKST, were used to generate the multi-epitope vaccine based on LCL platform. The vaccine showed strong interactions with toll-like receptors and acceptable immune-reactivity by immune simulation analysis. The findings of this study may represent a turning point in developing an effective drug and vaccine against E. chaffeensis. However, further experimental analyses have remained.

Comprehensive pan-genomic, resistome and virulome analysis of clinical OXA-48 producing carbapenem-resistant Serratia marcescens strains.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) have been thoroughly studied as the pathogens associated with hospital acquired infections. However, data on Serratia marcescens are not enough. S. marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. METHODS: Four carbapenem-resistant S. marcescens (CR-SM) isolates were obtained from hospitalized patients through routine microbiological experiments. We assembled the isolates genomes using whole genome sequencing (WGS) and compared their resistome and virulome patterns. RESULTS: The average length and CG content of chromosomes was 5.33 Mbp and 59.8%, respectively. The number of coding sequences (CDSs) ranged from 4,959 to 4,989. All strains had one single putative conjugative plasmid with IncL incompatibility (Inc) group. The strains harbored blaCTX-M-15, blaTEM-1 and blaSHV-134. All plamsids were positive for blaOXA-48. No blaNDM-1, blaKPC, blaVIM and blaIMP were identified. The blaSRT-2 and aac(6')-Ic genes were chromosomally-encoded. Class 1 integron was detected in strains P8, P11 and P14. The Escher_RCS47 and Salmon_SJ46 prophages played major role in plasmid-mediated carraige of extended spectrum ß-lactamases (ESBLs). The CR-SM strains were equipt with typical virulence factors of oppotunistic pathogens including biofilm formation, adhesins, secretory systems and siderophores. The strains did not have ability to produce prodigiosin but were positive for chitinase and EstA. CONCLUSION: The presence of conjugative plasmids harboring major ß-lactamases within prophage and class 1 integron structures highlights the role of different mobile genetic elements (MGEs) in distribution of AMR factors and more specifically carbapenemases. More molecular studies are required to determine the status of carbapenem resistance in clinical starins. However, appropriate strategies to control the global dissemination of CR-SM are urgent.