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Blastomyces dermatitidis is a thermally dimorphic fungus that can cause serious and sometimes fatal infections, including blastomycosis. After spore inhalation, a pulmonary infection develops, which can be asymptomatic and have lethal effects, such as acute respiratory distress syndrome. Its most common extra-pulmonary sites are the central nervous system, bones, skin, and genito-urinary systems. Currently, no vaccine has been approved by the FDA to prevent this infection. In the study, a peptide-based vaccine was developed against blastomycosis by using subtractive proteomics and reverse vaccinology approaches. It focuses on mining the whole genome of B. dermatitidis, identifying potential therapeutic targets, and pinpointing potential epitopes for both B- and T-cells that are immunogenic, non-allergenic, non-toxic, and highly antigenic. Multi-epitope constructs were generated by incorporating appropriate linker sequences. A linker (EAAAK) was also added to incorporate an adjuvant sequence to increase immunological potential. The addition of adjuvants and linkers ultimately resulted in the formation of a vaccine construct in which the number of amino acids was 243 and the molecular weight was 26.18 kDa. The designed antigenic and non-allergenic vaccine constructs showed suitable physicochemical properties. The vaccine's structures were predicted, and further analysis verified their interactions with the human TLR-4 receptor through protein-protein docking. Additionally, MD simulation showed a potent interaction between prioritized vaccine-receptor complexes. Immune simulation predicted that the final vaccine injections resulted in significant immune responses for the T- and B-cell immune responses. Moreover, in silico cloning ensured a high expression possibility of the lead vaccine in the E. coli (K12) vector. This study offers an initiative for the development of effective vaccines against B. dermatitidis; however, it is necessary to validate the designed vaccine's immunogenicity experimentally.

Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.

Tuberculosis (TB), an infectious disease caused by multi-drug resistant Mycobacterium tuberculosis (Mtb), has been a global health concern. Mtb affects over a third of the world’s population, causing two million deaths annually due to its dormancy and propensity to spread infection during this period. Resuscitation-promoting factor B (RpfB) plays a pivotal role in the growth of Mtb during dormant periods, making it a critical target for eliminating Mtb and curing TB. Gymnema sylvestre is a famous medicinal plant with several medicinal properties, including antimicrobial activity; however, the therapeutic potential of the various reported metabolites of this plant against Mtb has not yet been explored. The aim of this study was to explore the reported natural products of G. sylvestre against the RpfB of the Mtb. A total of 131 reported secondary metabolites of this plant were collected and virtually screened against the RpfB. We particularly targeted the Glu292 residue of RpfB as it is crucial for the catalysis of this protein. From our in-house library, 114 compounds showed a binding affinity higher than the standard drug. The binding stability of the top three lead compounds was further confirmed through MD simulation analysis. Drug likeness analyses indicated that the ten hits had zero violations of the Lipinski rule of five. In addition, analyses of pharmacokinetics, toxicity, and target prediction revealed that the top compounds are devoid of toxicity and do not affect human proteins. Additionally, they reflect multifaceted approach as anti-TB agents. Our selected hits not only exhibit molecular properties favoring physiological compatibility but also exhibit properties enhancing their potential efficacy as therapeutic candidates. The compounds investigated here are worthy of experimental validation for the discovery of novel treatments against TB. Further, this study also provides a promising avenue for research on the pharmacological potential of G. sylvestre.

Primary Amoebic Meningoencephalitis (PAM), a severe lethal brain disease, is caused by a parasite, Naegleria fowleri, also known as the “brain-eating amoeba”. The chances of a patient’s recovery after being affected by this parasite are very low. Only 5% of people are known to survive this life-threatening infection. Despite the fact that N. fowleri causes a severe, fatal infection, there is no proper treatment available to prevent or cure it. In this context, it is necessary to formulate a potential vaccine that could be able to combat N. fowleri infection. The current study aimed at developing a multi-epitope subunit vaccine against N. fowleri by utilizing immunoinformatics techniques and reverse vaccinology approaches. The T- and B-cell epitopes were predicted by various tools. In order to choose epitopes with the ability to trigger both T- and B-cell-mediated immune responses, the epitopes were put through a screening pipeline including toxicity, antigenicity, cytokine-inductivity, and allergenicity analysis. Three vaccine constructs were designed from the generated epitopes linked with linkers and adjuvants. The modeled vaccines were docked with the immune receptors, where vaccine-1 showed the highest binding affinity. Binding affinity and stability of the docked complex were confirmed through normal mode analysis and molecular dynamic simulations. Immune simulations developed the immune profile, and in silico cloning affirmed the expression probability of the vaccine construct in Escherichia coli (E. coli) strain K12. This study demonstrates an innovative preventative strategy for the brain-eating amoeba by developing a potential vaccine through immunoinformatics and reverse vaccinology approaches. This study has great preventive potential for Primary Amoebic Meningoencephalitis, and further research is required to assess the efficacy of the designed vaccine.

The management of infectious diseases has become more critical due to the development of novel pathogenic strains with enhanced resistance. Prevotella melaninogenica, a gram-negative bacterium, was found to be involved in various infections of the respiratory tract, aerodigestive tract, and gastrointestinal tract. The need to explore novel drug and vaccine targets against this pathogen was triggered by the emergence of antimicrobial resistance against reported antibiotics to combat P. melaninogenica infections. The study involves core genes acquired from 14 complete P. melaninogenica strain genome sequences, where promiscuous drug and vaccine candidates were explored by state-of-the-art subtractive proteomics and reverse vaccinology approaches. A stringent bioinformatics analysis enlisted 18 targets as novel, essential, and non-homologous to humans and having druggability potential. Moreover, the extracellular and outer membrane proteins were subjected to antigenicity, allergenicity, and physicochemical analysis for the identification of the candidate proteins to design multi-epitope vaccines. Two candidate proteins (ADK95685.1 and ADK97014.1) were selected as the best target for the designing of a vaccine construct. Lead B- and T-cell overlapped epitopes were joined to generate potential chimeric vaccine constructs in combination with adjuvants and linkers. Finally, a prioritized vaccine construct was found to have stable interactions with the human immune cell receptors as confirmed by molecular docking and MD simulation studies. The vaccine construct was found to have cloning and expression ability in the bacterial cloning system. Immune simulation ensured the elicitation of significant immune responses against the designed vaccine. In conclusion, our study reported novel drug and vaccine targets and designed a multi-epitope vaccine against the P. melaninogenica infection. Further experimental validation will help open new avenues in the treatment of this multi-drug-resistant pathogen.

Chikungunya virus is an alphavirus transmitted by mosquitos that develops into chikungunya fever and joint pain in humans. This virus’ name originated from a Makonde term used to describe an illness that changes the joints and refers to the posture of afflicted patients who are affected by excruciating joint pain. There is currently no commercially available drug or vaccine for chikungunya virus infection and the treatment is performed by symptom reduction. Herein, we have developed a computationally constructed mRNA vaccine construct featuring envelope glycoprotein as the target molecule to aid in the treatment process. We have utilized the reverse vaccinology approach to determine epitopes that would generate adaptive immune reactions. The resulting T and B lymphocytes epitopes were screened by various immunoinformatic tools and a peptide vaccine construct was designed. It was validated by proceeding to docking and MD simulation studies. The following design was then back-translated in nucleotide sequence and codons were optimized according to the expression host system (H. sapiens). Various sequences, including 3′ and 5′ UTR regions, Kozak sequence, poly (A) tail, etc., were introduced into the sequence for the construction of the final mRNA vaccine construct. The secondary structure was generated for validation of the mRNA vaccine construct sequence. Additionally, in silico cloning was also performed to design a vector for proceeding towards in vitro experimentation. The proposed designed vaccine construct may proceed with experimental testing for further efficacy verification and the final development of a vaccine against chikungunya virus infection.