The ongoing antibiotic-resistance crisis is becoming a global problem affecting public health. Urgent efforts are required to design novel therapeutics against pathogenic bacterial species. Brucella melitensis is an etiological agent of brucellosis, which mostly affects sheep and goats but several cases have also been reported in cattle, water buffalo, yaks and dogs. Infected animals also represent the major source of infection for humans. Development of safer and effective vaccines for brucellosis remains a priority to support disease control and eradication in animals and to prevent infection to humans. In this research study, we designed an in-silico multi-epitopes vaccine for B. melitensis using computational approaches. The pathogen core proteome was screened for good vaccine candidates using subtractive proteomics, reverse vaccinology and immunoinformatic tools. In total, 10 proteins: catalase; siderophore ABC transporter substrate-binding protein; pyridoxamine 5′-phosphate oxidase; superoxide dismutase; peptidylprolyl isomerase; superoxide dismutase family protein; septation protein A; hypothetical protein; binding-protein-dependent transport systems inner membrane component; and 4-hydroxy-2-oxoheptanedioate aldolase were selected for epitopes prediction. To induce cellular and antibody base immune responses, the vaccine must comprise both B and T-cells epitopes. The epitopes were next screened for antigenicity, allergic nature and water solubility and the probable antigenic, non-allergic, water-soluble and non-toxic nine epitopes were shortlisted for multi-epitopes vaccine construction. The designed vaccine construct comprises 274 amino acid long sequences having a molecular weight of 28.14 kDa and instability index of 27.62. The vaccine construct was further assessed for binding efficacy with immune cell receptors. Docking results revealed that the designed vaccine had good binding potency with selected immune cell receptors. Furthermore, vaccine-MHC-I, vaccine-MHC-II and vaccine-TLR-4 complexes were opted based on a least-binding energy score of −5.48 kcal/mol, 0.64 kcal/mol and −2.69 kcal/mol. Those selected were then energy refined and subjected to simulation studies to understand dynamic movements of the docked complexes. The docking results were further validated through MMPBSA and MMGBSA analyses. The MMPBSA calculated −235.18 kcal/mol, −206.79 kcal/mol, and −215.73 kcal/mol net binding free energy, while MMGBSA estimated −259.48 kcal/mol, −206.79 kcal/mol and −215.73 kcal/mol for TLR-4, MHC-I and MHC-II complexes, respectively. These findings were validated by water-swap and entropy calculations. Overall, the designed vaccine construct can evoke proper immune responses and the construct could be helpful for experimental researchers in formulation of a protective vaccine against the targeted pathogen for both animal and human use.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a human coronaviruses that emerged in China at Wuhan city, Hubei province during December 2019. Subsequently, SARS-CoV-2 has spread worldwide and caused millions of deaths around the globe. Several compounds and vaccines have been proposed to tackle this crisis. Novel recommended in silico approaches have been commonly used to screen for specific SARS-CoV-2 inhibitors of different types. Herein, the phytochemicals of Pakistani medicinal plants (especially Artemisia annua) were virtually screened to identify potential inhibitors of the SARS-CoV-2 main protease enzyme. The X-ray crystal structure of the main protease of SARS-CoV-2 with an N3 inhibitor was obtained from the protein data bank while A. annua phytochemicals were retrieved from different drug databases. The docking technique was carried out to assess the binding efficacy of the retrieved phytochemicals; the docking results revealed that several phytochemicals have potential to inhibit the SARS-CoV-2 main protease enzyme. Among the total docked compounds, the top-10 docked complexes were considered for further study and evaluated for their physiochemical and pharmacokinetic properties. The top-3 docked complexes with the best binding energies were as follows: the top-1 docked complex with a −7 kcal/mol binding energy score, the top-2 docked complex with a −6.9 kcal/mol binding energy score, and the top-3 docked complex with a −6.8 kcal/mol binding energy score. These complexes were subjected to a molecular dynamic simulation analysis for further validation to check the dynamic behavior of the selected top-complexes. During the whole simulation time, no major changes were observed in the docked complexes, which indicated complex stability. Additionally, the free binding energies for the selected docked complexes were also estimated via the MM-GB/PBSA approach, and the results revealed that the total delta energies of MMGBSA were −24.23 kcal/mol, −26.38 kcal/mol, and −25 kcal/mol for top-1, top-2, and top-3, respectively. MMPBSA calculated the delta total energy as −17.23 kcal/mol (top-1 complex), −24.75 kcal/mol (top-2 complex), and −24.86 kcal/mol (top-3 complex). This study explored in silico screened phytochemicals against the main protease of the SARS-CoV-2 virus; however, the findings require an experimentally based study to further validate the obtained results.

The emergence of antibiotic resistance in bacterial species is a major threat to public health and has resulted in high mortality as well as high health care costs. Burkholderia mallei is one of the etiological agents of health care-associated infections. As no licensed vaccine is available against the pathogen herein, using reverse vaccinology, bioinformatics, and immunoinformatics approaches, a multi-epitope-based vaccine against B. mallei was designed. In completely sequenced proteomes of B. mallei, 18,405 core, 3671 non-redundant, and 14,734 redundant proteins were predicted. Among the 3671 non-redundant proteins, 3 proteins were predicted in the extracellular matrix, 11 were predicted as outer membrane proteins, and 11 proteins were predicted in the periplasmic membrane. Only two proteins, type VI secretion system tube protein (Hcp) and type IV pilus secretin proteins, were selected for epitope prediction. Six epitopes, EAMPERMPAA, RSSPPAAGA, DNRPISINL, RQRFDAHAR, AERERQRFDA, and HARAAQLEPL, were shortlisted for multi-epitopes vaccine design. The predicted epitopes were linked to each other via a specific GPGPG linker and the epitopes peptide was then linked to an adjuvant molecule through an EAAAK linker to make the designed vaccine more immunologically potent. The designed vaccine was also found to have favorable physicochemical properties with a low molecular weight and fewer transmembrane helices. Molecular docking studies revealed vaccine construct stable binding with MHC-I, MHC-II, and TLR-4 with energy scores of −944.1 kcal/mol, −975.5 kcal/mol, and −1067.3 kcal/mol, respectively. Molecular dynamic simulation assay noticed stable dynamics of the docked vaccine-receptors complexes and no drastic changes were observed. Binding free energies estimation revealed a net value of −283.74 kcal/mol for the vaccine-MHC-I complex, −296.88 kcal/mol for the vaccine-MHC-II complex, and −586.38 kcal/mol for the vaccine-TLR-4 complex. These findings validate that the designed vaccine construct showed promising ability in terms of binding to immune receptors and may be capable of eliciting strong immune responses once administered to the host. Further evidence from experimentations in mice models is required to validate real immune protection of the designed vaccine construct against B. mallei.

The swift emergence of antibiotic resistance (AR) in bacterial pathogens to make themselves adaptable to changing environments has become an alarming health issue. To prevent AR infection, many ways can be accomplished such as by decreasing the misuse of antibiotics in human and animal medicine. Among these AR bacterial species, Plesiomonas shigelloides is one of the etiological agents of intestinal infection in humans. It is a gram-negative rod-shaped bacterium that is highly resistant to several classes of antibiotics, and no licensed vaccine against the aforementioned pathogen is available. Hence, substantial efforts are required to screen protective antigens from the pathogen whole genome that can be subjected easily to experimental evaluations. Here, we employed a reverse vaccinology (RV) approach to design a multi-antigenic epitopes based vaccine against P. shigelloides. The complete genomes of P. shigelloides were retrieved from the National Center for Biotechnological Information (NCBI) that on average consist of 5226 proteins. The complete proteomes were subjected to different subtractive proteomics filters, and in the results of that analysis, out of total proteins, 2399 were revealed as non-redundant and 2827 as redundant proteins. The non-redundant proteins were further checked for subcellular localization analysis, in which three were localized in the extracellular matrix, eight were outer membrane, and 13 were found in the periplasmic membrane. All surface localized proteins were found to be virulent. Out of a total of 24 virulent proteins, three proteins (flagellar hook protein (FlgE), hypothetical protein, and TonB-dependent hemoglobin/transferrin/lactoferrin family receptor protein) were considered as potential vaccine targets and subjected to epitopes prediction. The predicted epitopes were further examined for antigenicity, toxicity, and solubility. A total of 10 epitopes were selected (GFKESRAEF, VQVPTEAGQ, KINENGVVV, ENKALSQET, QGYASANDE, RLNPTDSRW, TLDYRLNPT, RVTKKQSDK, GEREGKNRP, RDKKTNQPL). The selected epitopes were linked with each other via specific GPGPG linkers in order to design a multi-epitopes vaccine construct, and linked with cholera toxin B subunit adjuvant to make the designed vaccine construct more efficient in terms of antigenicity. The 3D structure of the vaccine construct was modeled ab initio as no appropriate template was available. Furthermore, molecular docking was carried out to check the interaction affinity of the designed vaccine with major histocompatibility complex (MHC-)I (PDB ID: 1L1Y), MHC-II (1KG0), and toll-like receptor 4 ((TLR-4) (PDB: 4G8A). Molecular dynamic simulation was applied to evaluate the dynamic behavior of vaccine-receptor complexes. Lastly, the binding free energies of the vaccine with receptors were estimated by using MMPB/GBSA methods. All of the aforementioned analyses concluded that the designed vaccine molecule as a good candidate to be used in experimental studies to disclose its immune protective efficacy in animal models.

Staphylococcus hominis is a Gram-positive bacterium from the staphylococcus genus; it is also a member of coagulase-negative staphylococci because of its opportunistic nature and ability to cause life-threatening bloodstream infections in immunocompromised patients. Gram-positive and opportunistic bacteria have become a major concern for the medical community. It has also drawn the attention of scientists due to the evaluation of immune evasion tactics and the development of multidrug-resistant strains. This prompted the need to explore novel therapeutic approaches as an alternative to antibiotics. The current study aimed to develop a broad-spectrum, multi-epitope vaccine to control bacterial infections and reduce the burden on healthcare systems. A computational framework was designed to filter the immunogenic potent vaccine candidate. This framework consists of pan-genomics, subtractive proteomics, and immunoinformatics approaches to prioritize vaccine candidates. A total of 12,285 core proteins were obtained using a pan-genome analysis of all strains. The screening of the core proteins resulted in the selection of only two proteins for the next epitope prediction phase. Eleven B-cell derived T-cell epitopes were selected that met the criteria of different immunoinformatics approaches such as allergenicity, antigenicity, immunogenicity, and toxicity. A vaccine construct was formulated using EAAAK and GPGPG linkers and a cholera toxin B subunit. This formulated vaccine construct was further used for downward analysis. The vaccine was loop refined and improved for structure stability through disulfide engineering. For an efficient expression, the codons were optimized as per the usage pattern of the E coli (K12) expression system. The top three refined docked complexes of the vaccine that docked with the MHC-I, MHC-II, and TLR-4 receptors were selected, which proved the best binding potential of the vaccine with immune receptors; this was followed by molecular dynamic simulations. The results indicate the best intermolecular bonding between immune receptors and vaccine epitopes and that they are exposed to the host’s immune system. Finally, the binding energies were calculated to confirm the binding stability of the docked complexes. This work aimed to provide a manageable list of immunogenic and antigenic epitopes that could be used as potent vaccine candidates for experimental in vivo and in vitro studies.