Prioritization of potential vaccine candidates and designing a multiepitope-based subunit vaccine against multidrug-resistant Salmonella Typhi str. CT18: A subtractive proteomics and immunoinformatics approach
Introduction
Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) is a Gram-negative human-specific pathogen that causes typhoid fever, a severe and often fatal systemic infection. The infection spreads through the fecal-oral route via contaminated food or water from acutely infected or chronic carriers. Symptoms include persistent fever, influenza-like symptoms, headache, malaise, anorexia, and abdominal pain [1]. Previously widespread throughout the world, improvements in the provision of safe drinking water and proper sewage disposal have resulted in a significant decrease in the incidence of typhoid fever, with the disease burden now residing mostly in low- and middle-income regions such as Africa, East Asia, South Asia (India, Pakistan, and Bangladesh), Southeast Asia, and South America, with a higher fatality rate among children and older adults [2]. In developed countries, typhoid fever has predominately become a travel-associated disease, affecting travelers such as tourists, military personnel, temporary workers, or travelers visiting friends or relatives in endemic areas [3]. According to the World Health Organisation (WHO), an estimated 12–27 million typhoid fever cases are reported worldwide, resulting in 129,000–223,000 deaths annually [4].
While antibiotic therapy slows the progression of enteric fever and lowers the risk of death, the rapid emergence of multidrug-resistant (MDR) S. Typhi strains has significantly reduced the efficacy of antibiotics therapies posing a severe public health concern [5]. The first complete genome sequence of an MDR S. Typhi strain known as CT18 from Vietnam was published in 2000. The strain CT18 carries an MDR incH1 plasmid (pHCM1) as well as a second large cryptic plasmid (pHCM2) that is closely related to the Yersinia pestis virulence-associated plasmid pMT1 [6]. Over the past two decades, several outbreaks of MDR S.Typhi strains have been reported from around the world [7,8]. These MDR strains are resistant to first-line antibiotics such as ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. In some parts of Asia and Africa, MDR strains also display sporadic resistance to second-line antibiotics such as fluoroquinolones. As a result, in endemic countries, third-generation cephalosporins, particularly ceftriaxone, have become the antimicrobials of choice for typhoid treatment [9,10]. In November 2016, a major outbreak of extensive drug-resistant (XDR) S. Typhi strain has emerged in the Karachi and Hyderabad districts of Pakistan. The S. Typhi strain is resistant to all recommended typhoid fever drugs including first- and second-line antibiotics, and third-generation cephalosporins [11,12].
To avoid the spread of MDR S. Typhi strains, alternative methods for controlling and preventing typhoid fever must be developed. The best way to reduce the burden of typhoid fever is to improve water quality, sanitation, and food hygiene. However, the high expenses and lengthy process make this strategy impractical in low- and middle-income nations [13,14]. Vaccination is the most effective way to prevent infectious diseases such as typhoid fever. The inactivated whole-cell vaccine is no longer used because of its high toxicity. Currently, two typhoid vaccines are widely available-an orally administered live-attenuated Ty21a (Vivotif®) and an injectable TYPHIM Vi® containing purified Vi capsular polysaccharide. Both of these vaccines provide moderate protection (50–70%) in adults but have poor immunogenicity in young children, and are not licensed for children under the age of two. They are also considered to be expensive for low-middle income areas [3,15]. As a result, there is an urgent need to develop an effective, safe, and affordable vaccine against typhoid fever.
Conventional whole organism vaccines consisting of live-attenuated or killed organisms result in an unnecessary antigenic load and an increased risk of eliciting an allergic response [16]. Similarly, subunit vaccines, which are typically made of a single protein, have a high antigenic load and can trigger allergic reactions [16]. Peptide-based vaccines containing only epitopes, on the other hand, are both safe and specific in terms of an immune response. They are also chemically stable and easy to produce [16]. Nonetheless, they are only moderately immunogenic and have poor population coverage of T-cell epitopes [16]. To overcome these limitations, multi-epitope vaccines are an ideal alternative [16]. A multi-epitope vaccine has the following characteristics: (a) it contains multiple MHC-restricted epitopes that can be recognized by T-cell receptors (TCRs); (b) it is composed of cytotoxic T lymphocyte (CTL) epitopes, helper T lymphocyte (HTL) epitopes, and B-cell epitopes, allowing it to activate both cellular and humoral immune responses; (c) it contains multiple epitopes from different target proteins/antigens of the target pathogen, thereby expanding the range of the targeted pathogen; (d) to enhance multi-epitope vaccine immunogenicity the epitopes can be linked with adjuvants and (e) the chances of adverse effects or pathological immune responses are reduced because it contains fewer unwanted components [17]. The development of a successful multi-epitope vaccine begins with the identification of suitable vaccine candidates and their immunodominant epitopes [17]. Rapid advancement in sequencing technologies and the availability of genomic and proteomic data in international databases have aided in the prediction of pathogenic proteins as potential vaccine candidates through computational approaches [18,19]. Subtractive proteomics is a widely used computational-based approach to prioritize potential drug and vaccine targets [20,21]. It employs In silico filters to screen the pathogen proteome sequentially based on redundancy, host non-homology, subcellular localization, virulence and essentiality factors, and physicochemical properties including the number of transmembrane helices, molecular weight, and antigenicity [21]. Several immunoinformatics tools for antigenic epitope prediction with potential translational implications have been developed [22]. The immunoinformatics approach to construct multiepitope vaccine reduces time and costs while increasing the likelihood of successful vaccine design [[23], [24], [25], [26], [27], [28]]. The subtractive proteomics approach in combination with immunoinformatics has been widely used to develop an effective, low-cost multi-epitope subunit vaccine against various pathogens [16,21,[29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]].
In the present study, the proteome of MDR S. Typhi str. CT18 was first subjected to a subtractive proteomics pipeline to prioritize potential subunit vaccine candidates by eliminating paralogous, host homologous, non-essential, and non-virulent outer membrane proteins (OMPs). Prior work discovered antigenic peptides in S. Typhi utilizing a single vaccine candidate protein, DnaK [40]. DnaK was identified as cytoplasmic based on subcellular localization and was thus excluded from the present study. Another work employed the antigens OmpA, OmpC, and OmpF to identify B- and T-cell epitopes to construct a multiepitope vaccine for S. Typhi using two rigid linkers [41]. OmpA met the criteria for becoming a vaccine candidate in our study, however, OmpC and OmpF were ruled out due to the lack of virulence factors. The resultant OMPs were further prioritized based on their physicochemical properties. The selected vaccine candidates were then screened for antigenic CTL, HTL, and B-cell epitopes using various immunoinformatics tools and servers for designing a multiepitope-based subunit vaccine (MESV). MESVs are poorly immunogenic and need adjuvant coupling to enhance their immunogenicity [42]. Therefore, TLR4 agonists were selected as adjuvants and paired with the MESV to boost its immunogenicity and long-term immune responses [42]. The antigenicity, allergenicity, toxicity, solubility, and physicochemical properties of the MESV were evaluated, followed by 3D structure prediction, refinement, and validation. The ability of the MESV to interact with various immune cells, such as major histocompatibility complex molecules (MHC I and MHC II) and the TLR4/MD2 complex, was investigated using molecular docking. The binding free energy calculations of the receptor-vaccine complexes were performed to determine favorable protein-protein interactions as well as their affinity for accurate modeling of biological systems [43]. Furthermore, molecular dynamics (MD) simulation of the MESV with TLR4/MD2 was performed to validate the stability of the interactions. The in-silico cloning method was used to investigate the MESV's ability to be cloned and expressed in a suitable vector for large-scale production. Finally, an immune simulation was performed to assess the immunogenic potency in real life.
Section snippets
Materials and methods
The present study is divided into Phase-I: Subtractive proteomics analysis, Phase-II: Immunoinformatics analysis, and Phase-III: Vaccine construct features. The sequential workflow of the study is shown in Fig. 1. The data used in this study was accessed between Jan 15, 2020, and Jan 19, 2020, from various databases and servers.
Non-paralogous proteome
The S. Typhi str. CT18 harbors a chromosome of 4,809,037 bp and two plasmids: a 218,150 bp multiple-drug-resistance incH1 plasmid (pHCM1) and a 106,516 bp cryptic plasmid (pHCM2) that share ancestry with a Yersinia pestis virulence plasmid [6]. The proteome of CT18, including a chromosome and two plasmids, encodes 4718 proteins. CD-HIT analysis identified 4556 non-redundant proteins.
Subcellular localization
Subcellular localization using PSORTb v3.0.2 characterized 1978 (43.4%) proteins as cytoplasmic, 1025 (22.5%)
Discussion
Typhoid fever is a severe and often fatal systemic infection caused by S. Typhi, with an estimated global prevalence of 12–27 million cases and 129,000–223,000 fatalities each year [4]. It is primarily a disease of low- and middle-income countries with poor food hygiene and inadequate sanitation but with the emergence of multidrug-resistant (MDR) S. Typhi strains and increase in international travel, the threat is now global [2,3]. Commercially available typhoid vaccines are moderately
Conclusion
In summary, we designed a multiepitope-based subunit vaccine (MESV) against S. Typhi str. CT18 utilizing subtractive proteomics and immunoinformatics approaches. The study started with the screening of S. Typhi str. CT18 proteome using subtractive proteomics. It shortlisted twelve outer membrane proteins (OMPs) as potential vaccine candidates based on their non-homology to host proteome, essentiality, virulence factor, molecular weight, number of transmembrane helices, and antigenicity. The
CRediT authorship contribution statement
Yamini Chand: Methodology, Formal analysis, Writing – original draft, Writing – review & editing. Sachidanand Singh: Conceptualization, Validation, Writing – review & editing, Visualization, Supervision.
Declaration of competing interest
The authors declare that there is no conflict of interest.
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