Endophytic bacteria have been utilized as an alternative source to chemical fertilizers and pesticides to enhance plant productivity and defense mechanisms against biotic and abiotic stress. Five endophytic bacterial strains were isolated from the seeds of three different Pakistani wheat varieties (Ghaneemat-e-IBGE, Atta-Habib, and Siren). The isolated strains AH-1, S-5, S-7, GI-1, and GI-6 showed phylogenetic similarity with Bacillus altitudinis, B. aryabhattai, B. wiedmannii, Pseudomonas aeruginosa, and Burkholderia gladioli, respectively. All strains showed catalase activity (except AH-1) and Indole-3-acetic acid production, with the highest concentration (16.77 μg·mL⁻¹) found for GI-6, followed by S-5 (11.5 μg·mL⁻¹), nitrogen assimilation (except S-7), phosphorus solubilization (except S-7 and AH-1), and ability to produce siderophores, with maximum productions for GI-6 (31 ± 3.5 psu) and GI-1 (30 ± 2.9 psu). All five analyzed strains possessed antimicrobial activity, which was particularly strong in GI-6 and S-5 against Klebsiella pneumonia, Escherichia coli, and Bacillus subtilis. Increasing salinity stress with NaCl negatively affected the bacterial growth of all isolates. However, strains GI-6 and S-5 showed salt tolerance after three days of incubation. A drought tolerance test resulted in a negative impact of poly ethylene glycol on bacterial growth, which was, however, less pronounced in GI-6 strain. The GI-6 strain revealed growth-promoting effects on inoculated wheat plants.

The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computer-aided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1–3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1–TLR3 (–99.09 kcal/mol), MPXV-2–TLR3 (–98.68 kcal/mol), and MPXV-3–TLR2 (–85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine.

Crimean–Congo haemorrhagic fever (CCHF), caused by Crimean–Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10–40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012–2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

Lymphocytic choriomeningitis virus (LCMV) infects many individuals worldwide and causes severe infection in the immunosuppressant recipient, spontaneous abortion, and congenital disabilities in infants. There is no specific vaccine or therapeutics available to protect against LCMV infection; thus, there is a need to design a potential vaccine to combat the virus by developing immunity in the population. Herein, we attempted to design a potent multi-epitope vaccine for LCMV using immunoinformatics methods. The whole proteome of the virus was screened and mapped to extract immunodominant B-cell and T-cell epitopes which were fused with appropriate linkers (EAAAK, GGGS, AAY, GPGPG, and AAY), PADRE sequence (13aa) and an adjuvant (50 S ribosomal protein L7/L12) to formulate a multi-epitope vaccine ensemble. Codon adaptation and in silico cloning of the constructed vaccine were carried out using bioinformatics tools. The secondary and tertiary structure of the vaccine construct was predicted and refined. The physicochemical profile of the designed vaccine was analyzed, and the multi-epitope vaccine's potential to bind Toll-like receptors (TLR2 and TLR4) was evaluated through molecular docking and molecular dynamics simulations. Computational immune simulation of the designed vaccine antigen was performed using the C-ImmSim server. The designed multi-epitope-based vaccine (613 aa) comprised 26 immunodominant (six B-cell, nine cytotoxic T lymphocytes, and 11 helper T lymphocytes) epitopes and is predicted antigenic, non-toxic, non-allergen, soluble, and topographically accessible with a suitable physicochemical profile. The designed vaccine is expected to cover a broad worldwide population (96.35 %) and stimulate a robust adaptive immune response against the virus upon administration. In silico cloning of the constructed vaccine in PET28a (+) vector ensured its optimal expression in the Escherichia coli system. Molecular docking, molecular dynamics simulation, and binding free energy estimation collectively support the stability and energetically favourable interaction of the modeled vaccine-TLR2/4 complexes. The designed multi-epitope vaccine in the present study could serve as a potential vaccine candidate to protect against LMCV infection; however, the experimental validation and safety testing of the vaccine is warranted to validate the study's outcomes.

Neo-Coronavirus (NeoCoV) is a novel Betacoronavirus (β-CoVs or Beta-CoVs) discovered in bat specimens in South Africa during 2011. The viral sequence is highly similar to Middle East Respiratory Syndrome, particularly that of structural proteins. Thus, scientists have emphasized the threat posed by NeoCoV associated with human angiotensin-converting enzyme 2 (ACE2) usage, which could lead to a high death rate and faster transmission rate in humans. The development of a NeoCoV vaccine could provide a promising option for the future control of the virus in case of human infection. In silico predictions can decrease the number of experiments required, making the immunoinformatics approaches cost-effective and convenient. Herein, with the aid of immunoinformatics and reverse vaccinology, we aimed to formulate a multi-epitope vaccine that may be used to prevent and treat NeoCoV infection. Based on the NeoCoV proteins, B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were shortlisted. Four vaccines (Neo-1–4) were devised by fusing shortlisted epitopes with appropriate adjuvants and linkers. The secondary and three-dimensional structures of final vaccines were then predicted. The binding interactions of these potential vaccines with toll-like immune receptors (TLR-2, TLR-3, and TLR-4) and major histocompatibility complex molecules (MHC-I and II) reveal that they properly fit into the receptors’ binding domains. Besides, Neo-1 and Neo-4 vaccines exhibited better docking energies of -101.08 kcal/mol and -114.47 kcal/mol, respectively, with TLR-3 as compared to other vaccine constructs. The constructed vaccines are highly antigenic, non-allergenic, soluble, non-toxic, and topologically assessable with good physiochemical characteristics. Codon optimization and in-silico cloning confirmed efficient expression of the designed vaccines in Escherichia coli strain K12. In-silico immune simulation indicated that Neo-1 and Neo-4 vaccines could induce a strong immune response against NeoCoV. Lastly, the binding stability and strong binding affinity of Neo-1 and Neo-4 with TLR-3 receptor were validated using molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). The final vaccines require experimental validation to establish their safety and effectiveness in preventing NeoCoV infections.

Respiratory syncytial virus (RSV) usually causes respiratory tract infections of upper airways in infants and young children. Despite recent medical advances, no approved vaccine is available to control RSV infections. Therefore, we conducted an immunoinformatics study to design and evaluate a potential multi-epitope vaccine against RSV. Sequence-based analyses of the glycoproteins F and G revealed a total of eight CD8 T-cell and three CD4 T-cell epitopes after considering antigenicity, binding affinity and other parameters. Molecular docking analysis confirmed that these T-cell epitopes developed strong structural associations with HLA allele(s). By integrating these prioritized epitopes with linkers and a cholera toxin-derived adjuvant, a multi-epitope vaccine was designed. The developed vaccine was found to be stable, non-allergenic, flexible and antigenic. Molecular docking analysis revealed a striking mean HADDOCK score (−143.3) of top-ranked vaccine-TLR cluster and a Gibbs free energy change (ΔG) value of −11.3 kcal mol⁻¹. As per computational immune simulation results, the vaccine generated a high titer of antibodies (especially IgM) and effector T-cells. Also, codon optimization and in silico cloning ensured the increased expression of vaccine in Escherichia coli. Altogether, we anticipate that the multi-epitope vaccine reported in this study will stimulate humoral and cellular responses against RSV infection, subject to follow-up experimental validation.