Designing a novel multi-epitope vaccine to evoke a robust immune response against pathogenic multidrug-resistant Enterococcus faecium bacterium

Enterococcus faecium is among the most frequent causes of healthcare-associated infections with underlying co-morbidities such as UTIs, intra-abdominal infection, prostatitis, organ transplantation, diabetes, endocarditis [1]. E. faecium is also reported as the second most prevalent organism involved in bloodstream infections and rank fourth in terms of surgical site infections in the US and European hospitals [2, 3]. Cassini et al. reported 16,146 instances of vancomycin-resistant enterococcal infections and 1081 related fatalities in Europe in 2015 [4]. According to Ferede et al. the prevalence rate of E. faecium in India was found to be 2.3% [5]. On average, 2.5 million people become infected with antibiotic-resistant E. faecium, and more than 25,000 people die each year across the world. The annual incidence of E. faecium is 1.6 per 100,000 population [6]. The World Health Organization issued a list of 12 antibiotic-resistant pathogens in 2017 posing the greatest threat to human health, with E. faecium designated as a high priority “ESKAPE pathogen” for the discovery of novel therapies [7].

Several putative virulence factors have been reported in E. faecium such as Enterococcal Surface Protein, aggregation substance, pili, MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), cell wall, capsular polysaccharides, glycolipids, gelatinase, and fsr two-component system. These proteins are mainly involved in adherence to extracellular structures and biofilm formation, important processes in initiating colonization of and infection in the host. In Gram-positive bacteria E. faecium, the cell wall is composed of a peptidoglycan macromolecule that protects bacteria against environmental conditions and serves as an anchor for the attachment of capsular polysaccharides, teichoic acids, and proteins that are covalently or non-covalently attached to peptidoglycan [8]. Bacterial peptidoglycan polymerization is performed by multi enzymatic complexes that include high-molecular-weight class A and B penicillin-binding proteins (PBPs) [9]. PBPs are transpeptidases, carboxypeptidases, and endopeptidases that synthesize new and remodel existing peptidoglycan, the stress-bearing component of the bacterial cell wall [10]. They help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Class A PBPs combine the two activities essential for peptidoglycan polymerization, glycosyltransferase, and d,d-transpeptidase, in a single polypeptide chain. Class B PBPs are monofunctional d,d-transpeptidases that must cooperate with a glycosyltransferase to synthesize peptidoglycan. In E. faecium, resistance to β-lactam antibiotics is conferred by a low-affinity class B PBP, PBP 5. PBP 5 is the major carboxypeptidases that play a key role in the control of cell diameter, proper spore cortex synthesis, and correct septum formation [9, 11]. Due to its capacity to evolve toward high-level resistance in E. faecium, PBP 5 is widely reported as an attractive target for vaccine development [11].

Glycopeptides, penicillin, aminoglycosides, vancomycin macrolides, oxazolidinones, clindamycin, cephalosporins, trimethoprim/sulfamethoxazole, and beta-lactams are the major antibiotics available for the treatment of infections caused due to E. faecium. However, due to increased resistance to β-lactam, glycopeptide vancomycin was the antibiotic employed to treat these infections. In the last decade, the number of vancomycin-resistant enterococci (VRE) has increased in health care facilities globally. Infection caused by VRE leads to poor outcomes, and it remains a challenge. Quinupristin/dalfopristin, daptomycin, tigecycline, and linezolid have entered the clinical practice as alternative antibiotic agents to fight VRE infections; however, E. faecium is resistant to these agents has also emerged and are reported recently.

Combating multidrug-resistant E. faecium infections with newer medicines necessitates long-term treatments during which resistance may emerge, leaving physicians with limited treatment alternatives [12]. Several world wide efforts have been undertaken to fight agsinst the infection. For instance, Romero-Saavedra et al. identified six enterococcal proteins that could serve as potential vaccine candidates against enterococcal infections by the implementation of transcriptomic and proteomic approaches [13, 14]. In both studies, rabbits were immunized with the recombinant proteins, and the resulting sera were evaluated. Both results indicate the potential use of these proteins as vaccine candidates with a broad cross-reactivity and serotype-independent coverage against enterococcal infections. A few studies [14, 15] have also reported the efficacy of glycoconjugate vaccines as they induced opsonic antibodies against Enterococcus species experimentally. Despite these advances, complete protection against this infection is yet to be achieved. As a consequence, there is an urgent need to develop alternate vaccine-based strategies to prevent infections. Reverse vaccinology is a method that has revolutionized vaccine development in the past [16]. This allows the development of the novel vaccine antigens from the genome sequence information without the requirement to isolate and culture the pathogen [17].

This research article was developed with the objective of designing a potent in silico multi-peptide vaccine for E. faecium infection. The initial step of the vaccine development process was to identify epitopes that can be used as immunogens. Predicted epitopes with high antigenicity, non-allergenicity, nontoxicity, and positive immunogenicity score were combined with the linkers. An adjuvant was added to the N-terminal of the antigenic epitope to make the multiepitope vaccine more immunogenic. Different bioinformatics tools were used to investigate the physicochemical, structural, and immunological aspects of the final vaccine construct. Furthermore, the chimeric vaccine sequence has been codon-optimized for expression in E. coli.

The pictorial representation of the workflow is depicted in Fig. 1.

The C-ImmSim results show that the main immune response following vaccine administration is considerably promoted by the steady increase in immunoglobulin levels (i.e., IgM, IgG1, IgG2, IgG1 + IgG2, and IgG + IgM) during the secondary and tertiary immunological responses (Fig. 11A). During the secondary and tertiary immune responses, the antigen level fell as well. Furthermore, the raised and maintained concentrations of the B-cell population (total, active, and plasma B-cell) and T-cell (helper T-cell, cytotoxic T-cell, regulatory T-cell) (Fig. 11B–F) clearly indicated the efficacy of the designed vaccine. After each dose, the concentration of Th1 was shown to rise. Furthermore, a higher density of dendritic and macrophage cells suggests that antigen-presenting cells efficiently process and deliver antigen to CD4+  and CD8 + cells (Fig. 11G-H). It is also obvious that the levels of several cytokines rose following exposure (Fig. 11I).

Vaccination is a widely employed technique for improving the host immune mechanism against a specific pathogen [40, 41]. Producing a range of vaccines, such as live or attenuated vaccines, is expensive, time-consuming, and takes a long time to reach the market [42, 43]. Moreover, the attenuated vaccine delivers poor passive immunization and causes allergic responses due to the large antigenic load [44]. With the advancement of multi-omics technology, it is now easier for identification of epitopes that elicit strong immune response. Multi-epitopes-driven vaccines against N. meningitidis and M. leprae have been developed utilizing immunoinformatics methodologies [45, 46].

Enterococcus faecium is a life-threatening bacterium that has recently arisen at an alarming pace. Limited advances have been made in the elucidation of the host immune response against invasive E. faecium infections. The innate immune defense system depends on the recognition of the pathogen-associated molecular patterns (PAMPs). PAMPs are recognized through pattern recognition receptors, e.g., the components of the complement system and the Toll-like receptors (TLRs). There is evidence that TLR 2 (Toll-like receptor 2) plays a significant role in the innate immune response against E. faecium by recognizing peptidoglycan and lipoteichoic acid. Apart from this direct interaction of the pathogen with the phagocyte, there is also an indirect pathway mediated through opsonins, comprised of immunoglobulins and complement components. Activation of the alternative complement pathway elicits deposition of the complement component on the bacterial surface, which is subsequently recognized by complement receptors on the phagocytes [47]. The incompetence of the immune system to kill the intracellular enterococci may lead to their systemic spread.

As a result, developing possible vaccinations might be a boon in the battle against this bacteria. A possible vaccine has been designed as an optimal option to prevent E. faecium infection in the current investigation. Penicillin-binding protein, a protein associated with the bacteria's pathogenicity, was chosen based on its antigenicity score, since the higher the antigenicity, the greater the immune response. Various databases were used to anticipate potential T and B-cell epitopes, and their viability for use as a vaccine candidate was investigated. CTL epitopes help in the recognition of foreign antigen fragments on MHC-I (major histocompatibility complex-I) molecules and destroy target cells whereas HTL epitopes are required for both humoral and cell-mediated immune responses. B-cell epitopes are also significant in the development of epitopic vaccines as well as disease detection. As a result, prediction of these epitopes are critical for the creation of an immunotherapeutic and preventive vaccination. Antigenicity prediction of the protein sequence is important because it indicates whether the bacterial protein sequence is likely to be recognized by immunogenic cells in the human body. ABCpred, NetMHCpan 4.1, and IEDB MHC II prediction tools was used to predict putative B-cell, CTL, and HTL epitopes. All of the epitopes were found to be ‘antigenic’ as analyzed by the VaxigenV2.0 server. Another prominent obstacle in vaccine development is the probability of allergenicity since many vaccines stimulate the immune system into an allergenic reaction. AllerTOP server was used to predict potential allergenicity and all of the epitopes were observed as ‘non-allergen". IEDB class I immunogenicity tool was utilized to find the immunogenicity score of the peptides. Toxinpred server identified all peptides as ‘non-toxin’ along with hydrophobicity, hydropathicity, hydrophilicity, charge, SVM score, and molecular weight of the predicted peptides. Finally, two LBL, five CTL, and two HTL epitopes were chosen based on their conservancy, interaction with the greatest number of HLA alleles, antigenicity, immunogenicity, non-allergenicity, non-toxicity, and capacity to induce cytokine production. Geographical differences impact the development of subunit vaccines because they influence the variety of HLA allele expression throughout the world [48, 49]. The global population coverage of the finalized T-cell epitopes (CTL and HTL) with their corresponding HLA alleles was observed to be 99.25 %. Finally, to ensure an immune response, KK, AAY, and GPGPG linkers were inserted between the LBL, CTL, and HTL epitopes to create a stable and effective vaccine design. β-defensins, a TLR4 agonist, was employed as an adjuvant and coupled to the N-terminal of the vaccine design using an EAAAK linker to increase the immunological response.

The final developed vaccine is 172 amino acids long and has a molecular weight of 18,294.07 Da, which falls within the optimal molecular weight range for chimeric vaccines. The suggested vaccine's pI value (9.38), instability index (18.85), GRAVY score (−0.472), and aliphatic index (73.90) reflect its basic, stable, hydrophilic, and thermostable characteristics, respectively. Furthermore, the vaccine's antigenicity (1.1569) and non-allergenicity further demonstrated its efficacy as a vaccination. Because better solubility of the recombinant protein within the E. coli host leads to faster separation and purification, solubility is one of the most fundamental and critical factors in vaccine manufacture. When the vaccine protein was overexpressed in the E. coli host, it was discovered to be soluble. Short half-lives of epitopes are often a worry while constructing vaccines and the proposed chimeric vaccine successfully demonstrated an acceptable stability index, indicating its potential as a vaccine candidate.

The secondary structure of the intended protein was expected to have 15.11 % helix, 27.32 % strands, and 57.55 % random coils after conformational analysis. Ramachandra plot showed that final vaccine protein have 81.4 % amino acid in the favorable section, 12.9 % amino acid in the additionally allowed zone, and 3.6 % amino acid in the prohibited region, indicating that the designed model is satisfactory. Furthermore, the ERRAT's Z-score (92.661) and Verify3D showed that the structural quality of the vaccine construct was promising. As a consequence of the validation results, the tertiary structure appears to be suitable for future investigation.

Increasing protein stability is conferred the utmost importance in various biomedical and therapeutic applications. Disulfide engineering and a pair of mutations on the CYS11-CYS18 residue pair were used to improve the protein's thermostability. The E.coli cell culture system is commonly utilized for bulk recombinant protein synthesis. As a result, codon optimization for E. coli strain K12 was carried out in order to achieve effective expression in the host. To ensure a higher degree of protein expression in E. coli, a GC content of 51.93 % was found, along with a codon adaptability index (CAI) of 1. Furthermore, the molecular docking experiment demonstrated that the proposed vaccine binds with low binding energy (856.6 kcal/mol) at the TLR4 receptor-binding site, demonstrating higher binding affinity. The stability and mobility of the TLR4-vaccine complex in the biological environment were assessed using a molecular dynamics simulation of the docked complex. The graph also showed that the protein–protein combination is extremely stable, with each residue exhibiting a decreased degree of deformation. Overall, molecular dynamics studies revealed that the suggested vaccine is sufficiently stable and has a low risk of deformability at the molecular level.