Characterization of exclusively non-commensal Neisseria gonorrhoeae pangenome to prioritize globally conserved and thermodynamically stable vaccine candidates using immune-molecular dynamic simulations.

Neisseria gonorrhoeae (Ngo) has emerged as a global threat leading to one of the most common sexually transmitted diseases in the world. It has also become one of the leading antimicrobial resistant organisms, resulting in fewer treatment options and an increased morbidity. Therefore, in recent years, there has been an increased focus on the development of new treatments and preventive strategies to combat its infection. In this study, we have combined the most conserved epitopes from the completely assembled strains of Ngo to develop a universal and a thermodynamically stable vaccine candidate. For our vaccine design, the epitopes were selected for their high immunogenicity, non-allergenicity and non-cytotoxicity, making them the ideal candidates for vaccine development. For the screening process, several reverse vaccinology tools were employed to rigorously extract non-homologous and immunogenic epitopes from the selected proteins. Consequently, a total number of 3 B-cell epitopes and 6 T-cell epitopes were selected and joined by multiple immune-modulating adjuvants and linkers to generate a promiscuous immune response. Additionally, the stability and flexible nature of the vaccine construct was confirmed using various molecular dynamic simulation tools. Overall, the vaccine candidate showed promising binding affinity to various HLA alleles and TLR receptors; however, further studies are needed to assess its efficacy in-vivo. In this way, we have designed a multi-subunit vaccine candidate to potentially combat and control the spread of N. gonorrhoeae.

All Roads Lead to Rome: Enhancing the Probability of Target Attainment with Different Pharmacokinetic/Pharmacodynamic Modelling Approaches.

In light of rising antimicrobial resistance and a decreasing number of antibiotics with novel modes of action, it is of utmost importance to accelerate development of novel treatment options. One aspect of acceleration is to understand pharmacokinetics (PK) and pharmacodynamics (PD) of drugs and to assess the probability of target attainment (PTA). Several in vitro and in vivo methods are deployed to determine these parameters, such as time-kill-curves, hollow-fiber infection models or animal models. However, to date the use of in silico methods to predict PK/PD and PTA is increasing. Since there is not just one way to perform the in silico analysis, we embarked on reviewing for which indications and how PK and PK/PD models as well as PTA analysis has been used to contribute to the understanding of the PK and PD of a drug. Therefore, we examined four recent examples in more detail, namely ceftazidime-avibactam, omadacycline, gepotidacin and zoliflodacin as well as cefiderocol. Whereas the first two compound classes mainly relied on the 'classical' development path and PK/PD was only deployed after approval, cefiderocol highly profited from in silico techniques that led to its approval. Finally, this review shall highlight current developments and possibilities to accelerate drug development, especially for anti-infectives.

Vaccinomics-Aided Development of a Next-Generation Chimeric Vaccine against an Emerging Threat: Mycoplasma genitalium.

Mycoplasma genitalium, besides urethritis, causes a number of other sexually transmitted diseases, posing a significant health threat to both men and women, particularly in developing countries. In light of the rapid appearance of multidrug-resistant strains, M. genitalium is regarded as an emerging threat and has been placed on the CDC's "watch list". Hence, a protective vaccine is essential for combating this pathogen. In this study, we utilized reverse vaccinology to develop a chimeric vaccine against M. genitalium by identifying vaccine targets from the reference proteome (Strain G-37) of this pathogen. A multiepitope vaccine was developed using proteins that are non-toxic, non-allergic, and non-homologous to human proteins. Several bioinformatic tools identified linear and non-linear B-cell epitopes, as well as MHC epitopes belonging to classes I and II, from the putative vaccine target proteins. The epitopes that showed promiscuity among the various servers were shortlisted and subsequently selected for further investigation based on an immunoinformatic analysis. Using GPGPG, AAY, and KK linkers, the shortlisted epitope sequences were assembled to create a chimeric construct. A GPI anchor protein immunomodulating adjuvant was adjoined to the vaccine construct's N-terminus through the EAAK linker so as to improve the overall immunogenicity. For further investigations of the designed construct, various bioinformatic tools were employed to study the physicochemical properties, immune profile, solubility, and allergenicity profile. A tertiary chimeric design was computationally modeled using I-TASSER and Robetta and was subsequently refined through GalaxyRefine. ProSA-Web was exploited to corroborate the quality of the construct by detecting errors and the Ramachandran plot was used to identify possible quality issues. Simulation studies of the molecular dynamics demonstrated the robustness and flexibility of the designed construct. Following the successful docking of the designed model to the immune receptors, the construct was computationally cloned into Escherichia coli plasmids to affirm the efficient expression of the designed construct in a biological system.

Immunoinformatics-Based Proteome Mining to Develop a Next-Generation Vaccine Design against Borrelia burgdorferi: The Cause of Lyme Borreliosis.

The tick-borne bacterium, Borrelia burgdorferi has been implicated in Lyme disease-a deadly infection, formerly confined to North America, but currently widespread across Europe and Asia. Despite the severity of this disease, there is still no human Lyme disease vaccine available. A reliable immunoinformatic approach is urgently needed for designing a therapeutic vaccine against this Gram-negative pathogen. Through this research, we explored the immunodominant proteins of B. burgdorferi and developed a novel and reliable vaccine design with great immunological predictability as well as low contamination and autoimmunity risks. Our initial analysis involved proteome-wide analysis to filter out proteins on the basis of their redundancy, homology to humans, virulence, immunogenicity, and size. Following the selection of proteins, immunoinformatic tools were employed to identify MHC class I & II epitopes and B-cell epitopes, which were subsequently subjected to a rigorous screening procedure. In the final formulation, ten common MHC-I and II epitopes were used together with a suitable adjuvant. We predicted that the final chimeric multi-epitope vaccine could invoke B-cell responses and IFN-gamma-mediated immunity as well as being stable and non-allergenic. The dynamics simulations predicted the stable folding of the designed molecule, after which the molecular docking predicted the stability of the interaction between the potential antigenic epitopes and human immune receptors. Our studies have shown that the designed next-generation vaccine stimulates desirable immune responses, thus potentially providing a viable way to prevent Lyme disease. Nevertheless, further experimental studies in a wet lab are needed in order to validate the results.

Immunoinformatic Approach to Contrive a Next Generation Multi-Epitope Vaccine Against Achromobacter xylosoxidans Infections.

Achromobacter xylosoxidans, previously identified as Alcaligenes xylosoxidans, is a rod-shaped, flagellated, non-fermenting Gram-negative bacterium that has the ability to cause diverse infections in humans. As a part of its intrinsic resistance to different antibiotics, Achromobacter spp. is also increasingly becoming resistant to Carbapenems. Lack of knowledge regarding the pathogen's clinical features has led to limited efforts to develop countermeasures against infection. The current study utilized an immunoinformatic method to map antigenic epitopes (Helper T cells, B-cell and Cytotoxic-T cells) to design a vaccine construct. We found that 20 different epitopes contribute significantly to immune response instigation that was further supported by physicochemical analysis and experimental viability. The safety profile of our vaccine was tested for antigenicity, allergenicity, and toxicity against all the identified epitopes before they were used as vaccine candidates. The disulfide engineering was carried out in an area of high mobility to increase the stability of vaccine proteins. In order to determine if the constructed vaccine is compatible with toll-like receptor, the binding affinity of vaccine was investigated via molecular docking approach. With the in silico expression in host cells and subsequent immune simulations, we were able to detect the induction of both arms of the immune response, i.e., humoral response and cytokine induced response. To demonstrate its safety and efficacy, further experimental research is necessary.

Risk of Malignancy in Breast FNAB Categories, Classified According to the Newly Proposed International Academy of Cytology (IAC) Yokohama System.

Background: A new category system comprising five classes (C1-insufficient material, C2-benign, C3-atypical, C4-suspicious, and C5-malignant) has been proposed by the International Academy of Cytology (IAC) for fine needle aspiration biopsy cytology (FNAB) for proper diagnosis of breast cancer. Aims and Objectives: This study is designed to categorize institutional FNAB data according to the new system and calculation of the absolute risk of malignancy (ROM), sensitivity, specificity, positive predictive values, false negative and false-positive rate. Study Design: We conducted a retrospective cross-sectional study involving 2133 cases collected between June, 2008 and August, 2019, at Foundation University Medical College's Department of Histopathology and the Surgery and Oncology Department at the Fauji Foundation Hospital. All cases fulfilling the inclusion and exclusion criteria were retrieved from the archives and reviewed by two expert pathologists. Matching histopathology was compared with the cytology reports for concordance or discordance of results. Findings: We found 6.9% (n = 147) insufficient, 65.8% (n = 1403) benign, 7.2% (n = 153) atypical, 7.5% (n = 160) suspicious and 12.6% (n = 270) malignant cases. Cyto-histological correlation was found in 421 cases from the year 2014 to 2019 with 370 concordant and 51 discordant cases. The maximum number of concordant cases was 151 in the C5 category and discordant cases had a diagnosis of C3 and C4 on cytology with 16 cases in each category. The calculated values of ROM were 45.45%, 10.3%, 30.6%, 82.79% and 99.34% from C1 to C5, respectively. We calculated 83.42% absolute sensitivity and 85.24% specificity. The positive predictive value for category 3, 4 and 5 was 67.34%, 82.7% and 99.34%, respectively, while false-negative rate was 7.9% and false-positive rate was 0.66%. Conclusion: The ROM for C1 category calculated from this study is quite high (45.45%) compared to previous studies; therefore, it is recommended to perform core needle biopsy in all these cases. The higher sensitivity and specificity of this method of diagnosing malignant lesions supports its use.

Role of Therapeutic Plasmapheresis in SARS-CoV-2 Induced Cytokine Release Syndrome: A Retrospective Cohort Study on COVID-19 Patients.

Background: Cytokine release syndrome (CRS) significantly contributes to the pathophysiology and progression of COVID-19. It is speculated that therapeutic plasma exchange (TPE) can dampen CRS via elimination of pathogenic cytokines. Objectives: The study is intended to compare the outcomes of COVID-19 patients with CRS treated with TPE and standard care (SC) to their counterparts receiving SC alone. Methodology: A retrospective cohort study of severe COVID-19 confirmed patients presenting with CRS and admitted to the medical ICU was conducted between March and August 2021. Using case-control (CC) matching 1:1, 162 patients were selected and divided into two equal groups. The primary outcome was 28-day in-hospital survival analysis in severe COVID-19 patients with CRS. However, secondary outcomes included the effect of plasmapheresis on inflammatory markers, the need for mechanical ventilation, the rate of extubation, and the duration of survival. Results: After CC matching, the study cohort had a mean age of 55.41 (range 56.41±11.56 in TP+SC and 54.42±8.94 in SC alone; p=0.22). There were 25.95% males and 74.05% females in both groups. The mean time from first day of illness to hospitalization was 6.53±2.18 days. The majority of patients with CRS had comorbid conditions (75.9%). Diabetes mellitus was the most common comorbidity (40.1%), followed by hypertension (25.3%), and chronic kidney disease (21%). Notable reduction in some inflammatory markers (D-dimers, LDH, CRP and serum ferritin) (p<0.0001) was observed in the group that received TPE+SC. Moreover, the patients in the plasmapheresis plus standard care group required relatively less mechanical ventilation as compared to the group receiving SC alone (46.9% vs 58.1%, respectively; p>0.05). The rate of extubation in the TP+SC group vs SC alone was 60.5% vs 44.7%, respectively (p>0.05). Similarly, the mortality percentages in both groups were 19.8% and 24.7%, respectively. Conclusion: For this particular group of matched patients with COVID-19-induced CRS, TPE+SC was linked with relatively better overall survival, early extubation, and earlier discharge compared to SC alone. As these results were not statistically significant, multi-centered randomized control trials are needed to further elaborate the role of therapeutic plasmapheresis in COVID-19 induced CRS.

Efficacy and Safety of Remdesivir in COVID-19 Positive Dialysis Patients.

(1) Background: Immune compromised hemodialysis patients are more likely to develop COVID-19 infections, which increase the risk of mortality. The benefits of Remdesivir, despite less literature support on its effectiveness in dialysis patients due to renal toxicity, can outweigh the risks if prescribed early. The aim of this study was to evaluate the efficacy of Remdesivir on the 30-day in-hospital clinical outcome of hemodialysis population with COVID-19 infection and safety endpoints of adverse events. (2) Study design: A prospective quasi-experimental study design was used in the study. (3) Methods: The sample population consisted of 83 dialysis patients with COVID-19 who were administered Remdesivir at a dose of 100 mg before hemodialysis, as per hospital protocol. After the treatment with Remdesivir, we assessed the outcomes across two endpoints, namely primary (surviving vs. dying) as well as clinical and biochemical changes (ferritin, liver function test, C-reactive protein, oxygen requirements, and lactate dehydrogenase levels) and secondary (adverse effects, such as diarrhea, rise in ALT). In Kaplan-Meier analysis, the survival probabilities were compared between patients who received Remdesivir within 48 h of diagnosis and those who received it after 48 h. Cox regression analysis was employed to determine the predictors of outcome. (4) Results: Of the 83 patients, 91.5% survived and 8.4% died. Remdesivir administration did not reduce the death rate overall. Hospital stays were shorter (p = 0.03) and a nasopharyngeal swab for COVID-19 was negative earlier (p = 0.001) in survivors who had received Remdesivir within 48 h of diagnosis compared to those who had received Remdesivir after 48 h. The only variables linked to the 30-day mortality were serum CRP (p = 0.028) and TLC (p = 0.013). No major adverse consequences were observed with Remdesivir. (5) Conclusions: Remdesivir has the potential to shorten the recovery time for dialysis patients if taken within 48 h of onset of symptoms, without any adverse effects.

In-Silico Vaccine Design Based on a Novel Vaccine Candidate Against Infections Caused by Acinetobacter baumannii.

Acinetobacter baumannii is notorious for causing serious infections of the skin, lungs, soft tissues, bloodstream, and urinary tract. Despite the overwhelming information available so far, there has still been no approved vaccine in the market to prevent these infections. Therefore, this study focuses on developing a rational vaccine design using the technique of epitope mapping to curb the infections caused by A. baumannii. An outer membrane protein with immunogenic potential as well as all the properties of a good vaccine candidate was selected and used to calculate epitopes for selection on the basis of a low percentile rank, high binding scores, good immunological properties, and non-allergenicity. Thus, a 240 amino-acid vaccine sequence was obtained by manually joining all the epitopes in sequence-wise manner with the appropriate linkers, namely AAY, GPGPG, and EAAAK. Additionally, a 50S ribosomal protein L7/L12, agonist to the human innate immune receptors was attached to the N-terminus to increase the overall immune response towards the vaccine. As a result, enhanced overall protein stability, expression, immunostimulatory capabilities, and solubility of the designed construct were observed. Molecular dynamic simulations revealed the compactness and stability of the polypeptide construct. Moreover, molecular docking exhibited strong binding of the designed vaccine with TLR-4 and TLR-9. In-silico immune simulations indicated an immense increment in T-cell and B-cell populations. Bioinformatic tools also significantly assisted with optimizing codons which allowed for successful cloning of constructs into desired host vectors. Using in-silico tools to design a vaccine against A. baumannii demonstrated that this construct could pave the way for successfully combating infections caused by multidrug-resistant bacteria. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10316-7.

Antimicrobial Resistance and Genomic Characterization of Six New Sequence Types in Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Pakistan.

Pseudomonas aeruginosa (P. aeruginosa) is a major bacterial pathogen associated with a variety of infections with high mortality rates. Most of the clinical P. aeruginosa isolates belong to a limited number of genetic subgroups characterized by multiple housekeeping genes' sequences (usually 5-7) through the Multi-Locus Sequence Typing (MLST) scheme. The emergence and dissemination of novel multidrug-resistant (MDR) sequence types (ST) in P. aeruginosa pose serious clinical concerns. We performed whole-genome sequencing on a cohort (n = 160) of MDR P. aeruginosa isolates collected from a tertiary care hospital lab in Pakistan and found six isolates belonging to six unique MLST allelic profiles. The genomes were submitted to the PubMLST database and new ST numbers (ST3493, ST3494, ST3472, ST3489, ST3491, and ST3492) were assigned to the respective allele combinations. MLST and core-genome-based phylogenetic analysis confirmed the divergence of these isolates and positioned them in separate branches. Analysis of the resistome of the new STs isolates revealed the presence of genes blaOXA-50, blaPAO, blaPDC, blaVIM-2, aph(3')-IIb, aac(6')-II, aac(3)-Id, fosA, catB7, dfrB2, crpP, merP and a number of missense and frame-shift mutations in chromosomal genes conferring resistance to various antipseudomonal antibiotics. The exoS, exoT, pvdE, rhlI, rhlR, lasA, lasB, lasI, and lasR genes were the most prevalent virulence-related genes among the new ST isolates. The different genotypic features revealed the adaptation of these new clones to a variety of infections by various mutations in genes affecting antimicrobial resistance, quorum sensing and biofilm formation. Close monitoring of these antibiotic-resistant pathogens and surveillance mechanisms needs to be adopted to reduce their spread to the healthcare facilities of Pakistan. We believe that these strains can be used as reference strains for future comparative analysis of isolates belonging to the same STs.