Research paper
Rational design of multimeric based subunit vaccine against Mycoplasma pneumonia: Subtractive proteomics with immunoinformatics framework

https://doi.org/10.1016/j.meegid.2021.104795Get rights and content

Highlights

  • Mycoplasma pneumoniae is a common infectious source in adults and children.

  • No vaccine is available against Mycoplasma pneumoniae.

  • Need to develop a vaccine to battle against Mycoplasma pneumoniae infection.

  • M. pneumoniae proteome was analyzed to determine the highest antigenic proteins

  • Multi-epitope vaccine was developed using the most potent epitopes with appropriate adjuvants and linkers.

Abstract

Mycoplasma pneumoniae is the prevalent cause of acquired respiratory infections around the globe. A multi-epitope vaccine (MEV) must be developed to combat infections of M. pneumoniae because there is no specific disease-modifying treatment or vaccination is present. The objective of this research is to design a vaccine that targets M. pneumoniae top five highly antigenic proteins using a combination of immunological techniques and molecular docking. T-cell (HTL & CTL), B-cell, and IFN-γ of target proteins were forecasted and highly conservative epitopes were chosen for further study. For designing of final vaccine, 4LBL, 7CTL, and 5HTL epitopes were joined by linkers of KK, AAY, and GPGPG. The N-end of the vaccine was linked to an adjuvant (Cholera enterotoxin subunit B) with a linker named EAAAK to enhance immunogenicity. After the addition of adjuvants and linkers, the size of the construct was 395 amino acids. The epitopes of IFN-γ and B-cells illustrate that the model construct is optimized for cell-mediated immune or humoral responses. To ensure that the final design is safer and immunogenic, properties like non-allergens, antigenicity, and various physicochemical properties were evaluated. Molecular docking of the vaccine with the toll-like receptor 4 (TLR4) was conducted to check the compatibility of the vaccine with the receptor. Besides, in-silico cloning was utilized for validation of the credibility and proper expression of the vaccine. Furthermore, to confirm that the multi-epitope vaccine created is protective and immunogenic, this research requires experimental validation.

Introduction

The “Mollicutes” class contains a genus “Mycoplasma”, that are bacteria with the missing cell wall. It has been suggested through comparative genomics and phylogenetic analysis, that the ancestors of this category of bacteria are perhaps Gram-positive bacteria (Razin et al., 1998; Schnee et al., 2012). The genus Mycoplasma contains bacteria that are referred to be the smallest microorganisms regarding the size of the genome and cell, and also have the capability to self-replicate (Wilson and Collier, 1976). Due to the fact that there are less than 1000 genes in the Mycoplasma genome, its metabolic ability is limited, so it requires unique cellular compounds for its survival. These microorganisms need specific components such as sterols that protect them against their osmotic fragility for their growth in cultural media. The lack of a cell wall in the Mycoplasma genus makes it difficult to identify these microorganisms as bacilli or cocci; besides, this feature gives β-lactams a natural resistance and impairs Gram staining (Baron, 1996; Kumar, 2018). Of the 16 species of mycoplasma that causes human infection, six are pathogenic, of which M.pneumoniae is clinically most important (Waites et al., 2003; Waites and Talkington, 2004).

The major case of community pneumonia is M. pneumonia, comprised of a genome with 800,000 base pairs that encoded around 700 several proteins (Himmelreich et al., 1996). Studies have shown a prevalence of CAP for M. pneumoniae that can exceed 40% in reported cases, but in asymptomatic cases it is still extremely difficult to make a clear diagnosis (Bajantri et al., 2018); Community-acquired pneumonia (CAP) is an acute pulmonary disease that causes high mortality and morbidity rates and continues to contract outside of the health system (Musher and Thorner, 2014). The Pathogenic bacteria isn't usually recognized and hence the diagnostic study is often based on solely clinical symptoms, so the treatment is not accurate and the medication does not guarantee effective care, endangers patient's lives, and can even help to develop antibiotic-resistant bacteria (Wang et al., 2012).

M. pneumoniae is a common infectious source in both adults and children. The prevalence of Mycoplasma is commonly observed since it is considered to be the most common “atypical” pathogens in adults (Hammerschlag, 2001). Unlike bacteria, Mycoplasma replicates in cell-free media by primary fission but it lacks a cell wall and is unlikely to be susceptible to antibiotics such as beta-lactams, that affect the synthesis of cell walls (Hammerschlag, 2001). The incidence of M. pneumoniae infection is population-dependent and appears to be age-related (Foy et al., 1973). Studies have revealed that pneumonia was the highest among children aged 5–9, and the second-highest incidence was among children aged 10–14.

The prevalence of pneumonia may vary greatly depending on the population of the sample and the diagnostic methods used, 32.5% of which occur in M. Pneumoniae infection was based on the fixation complement serology found in an active population study of the Ohio CAP surveillance (Marston et al., 1997). In recent research at the Federal Service Training Academy which identified an outbreak in 1996, pneumonia was highlighted to trigger outbreaks of respiratory disease in the institutional context. In total, more than 300 students out of the 600, identified with respiratory diseases (Feikin et al., 1999).

M. pneumoniae can be divided into three different groups. One group should be considered direct as the cytokines that cause inflammations induced in the vicinity by lipoproteins in the membrane of the bacterial cells and the second one is an indirect system in which immune systems should be included in interactions among human cells and bacterial cell components, such as autoimmunity (Narita, 2010). Antibiotics including tetracyclines, macrolides, and quinolones that interfere with protein or DNA synthesis are susceptible to M. pneumoniae. Macrolides are the most toxic in-vitro and also the most stable macrolides at the inhibitory concentrations between 0.0003 and 0.031 μg/mL (Taylor-Robinson and Bébéar, 1997). But there's no effective vaccine for M. pneumoniae treatment to date.

The classes of antibiotic macrolides, tetracyclines, and quinolones are used to treat infections caused by M. pneumoniae because it lacks a cell wall. (Saraya et al., 2014; Tong et al., 2015). It is acknowledged that resistance to macrolides in different strains was presented by a mutate in the 23S rRNA gene, minimizing treatment choice for two antibiotics only. The highest rate of resistance is in the continent of Asia, where this mutation has already exhibited about 90% of the strains of these bacteria. Recurring and haphazard antibiotic usage in this area was the reason behind the high pace. There have also been many reported cases of resistance in Europe and America, and the proportion of resistant strains has increased more and more (Vilela Rodrigues et al., 2019). Not just restrictions on the use of macrolides need to be promoted, but also developments in science to develop new drugs and prophylactic methods (Cao et al., 2017; Okada et al., 2012; Zheng et al., 2015). Hemophilus influenza and Streptococcus pneumoniae are the most common pneumonia causative agents and vaccines for them are available. For atypical disease pathogens such as chlamydia pneumonia and M. pneumoniae, analyses and studies are crucial to achieving the same level of development (Nascimento-Carvalho, 2001). Such studies can be very relevant because the World Health Organization believes (Rudan et al., 2008), Vaccines produced against pneumonia prevent nearly 2 million children's deaths each year (Madhi et al., 2008).

Effective vaccine production would not only prevent major pneumonia infections but also protect us from disasters, particularly in enclosed societies (Linchevski et al., 2009). Inactivating the human and/or cellular arm of the immune system, epitope-dependent immunization is complex (Buriani et al., 2012). Immunoinformatics is now a crucial constituent of modern immunology research and is a correlation between experimental immunology and computer science. It utilizes computational resources and methods to understand, generate, process, and propagate the immunological information. Recently, the use of immunoinformatics to model peptide vaccines against bacteria (Hajighahramani et al., 2017; Mamede et al., 2020; Nezafat et al., 2017), viruses (Khalid and Ashfaq, 2020; Shahid et al., 2020; Tahir ul Qamar et al., 2020; Ul Qamar et al., 2020a, Ul Qamar et al., 2020b), and parasites (Kar and Srivastava, 2018; Yadav et al., 2020) has been intensified.

In many bacterial infections, B cells or T cells play an important role in protecting the response of the immune system and, therefore, the development of effective epitope vaccines requires the definition of peptides that produce T and B reactions (Gupta et al., 2010). The supremacies of epitope-based vaccines involve the ability to select immune types, economic development, and increased protection (Sbai et al., 2001). Different approaches are usually followed by the prevalent epitope vaccine defects such as an increase in the number and introduction by immunogenic adjuvants and carrier proteins of antigenic epitopes (Coban et al., 2011).

In this study, M. pneumoniae proteome was analyzed to determine the highest antigenic protein(s) followed by the prediction of the different T and B cell epitopes with their respective alleles of the major histocompatibility complex (MHC). For their immunological profile, these epitopes were evaluated. Finally, a multi-epitope vaccine was developed using the most potent epitopes with appropriate adjuvants and linkers. The primary sequence of the vaccine construction was used for immunogenic and physiochemical profiling and homology modeling. The predicted 3D structure was refined to improve stability and subjected to disulfide engineering. Binding interaction and stability of the vaccine-receptor complex were analyzed through molecular docking and dynamic simulation. Besides, the immune responses induced by the vaccine antigen were simulated to investigate real-life potency. Finally, the vaccine codon was optimized for the E. coli system and in-silico cloning was performed.

Section snippets

Proteome retrieval and analysis

The entire proteome of M. pneumoniae pathogenic strain (strain ATCC 29342 / M129) was retrieved from UniProt in FASTA format (Kalita et al., 2019). Essential Proteins were obtained through Geptop 0.5 server. Vaccine candidates should not be human-protein homologous so that autoimmune response can be prevented. Therefore, the non-homologous protein was found through BLASTp (Azhagesan et al., 2018). Antigenicity is also the capacity to promptly react, resistant to an antigen. Therefore, it is

Selection of protein

The whole proteome of M. pneumoniae (strain ATCC 29342 / M129) has 686 proteins. Of these 208 were referred to as essential predicted by Geptop 0.5 server. Human homologs have been removed with BLASTp analysis and 104 proteins were found to be non-homologous. These were assessed based on antigenicity values. The top 5 proteins with high antigenicity values were chosen and all of them were cytoplasmic proteins (antigenicity ranging from 0.738 to 1.019) (Table 1).

Appraisal and selection of B-cell and T-cell epitopes

A total of 18 CTL epitopes

Discussion

M. pneumoniae is a high-mortality bacterium capable of causing cataclysm in endemic regions worldwide. Therefore, a preventive mechanism should be developed to treat these bacterial infections. (Manjelievskaia et al., 2016). MEV eliminates the unnecessary components compared to conventional vaccines, which can induce abnormal responses of the immunity system or can have detrimental consequences (Depla et al., 2008). Improved safety, cost effectiveness, reasonably engineered epitopes for

Conclusion

M. pneumoniae infection has long been a mysterious problem and is now a global health issue. As such, it is still not possible to treat this infection with any medication or with effective vaccines. Many antibacterial drugs have been studied, but none of these show good infection outcomes. The development of a subunit MEV humoral and cellular immune response that is centered on subtractive genomics and Immunoinformatic techniques. In combination with computer analyses and immune-information

Declaration of Competing Interest

No financial and other conflict of interest related to this article.

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