Immunoinformatics and molecular docking studies reveal a novel Multi-Epitope peptide vaccine against pneumonia infection

Highlights

• Type 3 fimbrial protein (mrkA) protein plays a major determinant in the virulence of K. pneumoniae.

• Immunoinformatic approaches was employed to design a novel multi-peptide vaccine to induce humoral and cellular immune responses.

• Immunological potential including the allergenicity, antigenicity, non-toxicity, and population coverage analysis of the construct were also evaluated.

• Molecular docking and MD simulation exhibited a strong and stable binding affinity between vaccine-TLR2.

Abstract

Pneumonia is a major endemic disease around the world, and an effective vaccine is the need of the hour to fight against the disease. When there are no appropriate antiviral and associated therapies available, vaccine development becomes even more essential. Therefore, in the present study, a variety of immunoinformatics techniques was utilized to develop a novel multi-epitope vaccine that targets the highly immunodominant type 3 fimbrial protein of Klebsiella pneumoniae, the causal organism for pneumonia. The putative B and T cell epitopes were predicted from the protein and screened for antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. Subsequently, the selected epitopes were joined with the help of linkers to form a robust vaccine construct. In addition, an adjuvant was applied to the N-terminal of the construct to improve the immunogenicity of the vaccine. The physicochemical properties, solubility, the secondary and tertiary structure of the final vaccine were also established. MD simulations for 100 ns were employed to assess the stability of the vaccine-TLR-2 docked complex. The final vaccine was optimized and cloned in pET28a (+) vector with His-tag to achieve maximum vaccine protein expression for ease of purification. Immune simulation results indicated the potency of this vaccine candidate as a probable therapeutic agent. In conclusion, the overall results of various immunoinformatics tools and methods employed revealed that the constructed multi-epitope vaccine exhibits a high potential for stimulating both B and T-cells immune responses against pneumonia infection. However, experimental immunological studies are required to corroborate the viability of the novel multi-epitope construct as a commercial vaccine.

Introduction

ESKAPE organisms are the leading cause of healthcare-associated infections all over the world, especially in critically ill and immune-compromised individuals [1]. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., collectively known as ESKAPE, have been identified as being increasingly involved in infectious diseases in humans. These organisms consistently “escape” the effects of commonly used antibiotics and therefore pose a critical threat to public health [2]. Klebsiella pneumoniae is one of the clinically significant ESKAPE organisms and the major cause of healthcare-associated infections [3]. K. pneumoniae is considered the most common bacterial human pathogen and is reported to cause up to 10% of all nosocomial bacterial infections [4]. The bacteria is a significant Enterobacteriaceae and is considered as one of the human pathogens that are widely found in the mouth, skin, and intestines causing broad spectra of diseases and infections such as urinary tract infections, cystitis, pneumonia, surgical wound infections, endocarditis and septicemia [5], [6]. It has also been linked to outbreaks in closed hospital units in the past, particularly in pediatric (neonatal) units [3]. The recent emergence of antibiotic-resistant strains of K. pneumoniae worldwide has caused global concern [7].

K. pneumoniae is intrinsically virulent and capable of invasive infection due to fimbrial adhesins and a thick capsule, which acts as a putative antiphagocytic factor [2]. The most frequently encoded fimbriae types of K. pneumoniae are type 1 fimbriae and type 3 fimbriae [4]. The majority of immune interventions against K. pneumoniae fimbriae have been directed against type 3 fimbriae, also known as MrkA, which is considered as a promising target antibody therapeutics and vaccines [3]. The mrkA encodes the fimbrial subunit and a major protein of the type III fimbriae complex on the surface of K. pneumoniae that is implicated in the attachment to host cell and biofilm formation [7]. Type 3 fimbriae mediate in vitro adhesion to epithelial cells and also kidney and lung tissues, most likely in a mannose-resistant manner [8]. Type 3 fimbriae protein is also the major contributor to K. pneumoniae biofilm-associated urinary infections [6]. In today’s scenario, the growing numbers of antimicrobial-resistant pathogens, which are increasingly related to nosocomial infection, place a significant burden on healthcare systems and could be a major economic problem as these pathogens are prevalent natural residents of the human and animal microbiome [1]. Specifically, Multidrug-resistant Klebsiella infections have spread worldwide and rendered most current antibiotic classes largely ineffective [9], thereby renewing interest in vaccine development. Recently experimental studies have recommended a number of vaccine candidates including inactivated whole cell vaccines, capsular polysaccharides, outer membrane vesicles, and highly conserved immunogenic proteins for prevention of K. pneumoniae infections [10]. Moreover, a vaccine called ‘Immunovac’ containing antigenic complexes derived from K. pneumoniae, P. vulgaris, S. aureus, and E. coli has been clinically trialed against K. pneumoniae prevention and was observed to be immunogenic in elderly patients and children [10]. However, in spite of studies in animal models and clinical trials, no vaccine candidate has been fully protective, safe, and commercially available yet for K. pneumoniae prevention, necessitating exploring other potential antigens to develop an effective vaccine which can be effective against multiple strains [3].

Fimbriae proteins of K. pneumoniae have been proven to be effective protein carriers and immunogens and also found highly conserved among bacteria of the Enterobacteriaceae family [11]. However, vaccines candidates based on these proteins are at the initial stages of development and require more studies [11]. Moreover, In vivo and in vitro vaccine studies requires a lot of time and are expensive processes. Also, these traditional methods are not appropriate to work against K. pneumoniae, which includes a variety of strains. Immunoinformatics approaches for epitope prediction can make the vaccine discovery process easier, safer, and faster by introducing vaccine candidates which provoke B, helper T, and cytotoxic T cells [12]. It has been observed that humoral (antibody) responses play a critical role in controlling K. pneumoniae infections [13], [14]. However, some supporting evidence also signifies the protecting role of CD4 + T cells against K. pneumoniae [15]. Therefore, it is advocated that an effective immunotherapeutic strategy against this pathogen should include both humoral and cell-dependent immune responses which are mediated by B and T cell-specific epitopes, respectively [14].

Due to the importance of humoral immune responses against K. pneumoniae, here we performed an in silico study on type 3 fimbriae protein of K. pneumoniae as vaccine candidates and tried to screen and characterize B-cell epitopes along with T cell epitopes. Furthermore, the antigenicity, allergenicity, immunogenicity, toxicity, and other physicochemical properties were also estimated to be used as potential vaccine candidates. Consequently, the multiepitope vaccine construct was designed using a homology modelling approach with appropriate adjuvant (Cholera Toxin Subunit B) and linkers. Also, docking and molecular dynamic simulation were carried out to estimate the binding stability between the vaccine and TLR2 receptor. Additionally, Immune simulation was performed to estimate the real-life immunogenic potency. At last, codon optimization and in silico cloning were performed to determine the efficiency of expression of the vaccine construct. The results conclusively demonstrate the efficacy and reliability of the vaccine construct which holds strong rationale for further experimental validation for the treatment and prevention of pneumonia infection.