Contriving a novel multi-epitope subunit vaccine from Plasmodium falciparum vaccine candidates against malaria

INTRODUCTION

Malaria is a deadly tropical disease caused by an infection of the protozoan Plasmodium, and it continues to be the most significant human parasitic illness in the world. It is a vector-borne illness. It spreads through the bites of female Anopheles mosquitoes infected with Plasmodium parasites [1]. Over the last century, despite the increased research efforts and control measures to drive down the malaria burden globally, eradication strategies and interventions have only been fairly successful. The parasite has co-evolved with new interventions, and eradication remains ongoing [2]. Plasmodium species have developed resistance to all known classes of antimalarial compounds and drugs which is one of the major challenges in the fight against malaria [3], in addition to inadequate research funding, healthcare professionals, facilities, research efforts in malaria-endemic regions, and limited knowledge about naturally acquired immunity to malaria [4,5].

Vaccination is an economical and successful solution to stop infectious diseases [6–10]. Advances have been made in blood-stage vaccine development, as they have reached clinical trials; however, they were unsuccessful in controlling human malaria infection on the field [11–13]. Antigen polymorphism, redundancy, parasite immune evasion, and low effectiveness of vaccine candidates have greatly hindered the rapid development of a licensed vaccine to neutralize malaria [14–18]. Furthermore, increasing insecticide resistance and asymptomatic infections have also been major setbacks [19,20], and cases of resurgence and increased malaria deaths have been reported in South American nations [21]. Vaccination has been identified as a critical therapeutic option for eliminating malaria; hence, it is a priority for sustained, substantial, and cost-effective control [22–24]. The only available licensed vaccine against malaria, RTS, S/AS01 shows low to modest efficacy in thwarting malaria caused by Plasmodium falciparum, which is enough to prevent clinical malaria but not enough for the global eradication of malaria [1]. Several other vaccines are currently under development, including the R21/Matrix-M (with 77% efficacy in preliminary trials), P. falciparum sporozoite, and the self-amplifying RNA vaccine. However, no vaccine with 100% efficacy remains available [25–27].

Post-genomic era vaccine development often involves preliminary computational analysis using bioinformatics tools to predict protective antigens rather than experimental studies with the pathogen, as in conventional vaccinology. Antigen characterization using in silico strategies and bioinformatics tools is crucial to the protein-based vaccine design and development [28]. Reverse vaccinology is named so because the vaccine discovery process employs computational methods to analyze genomic data instead of wet lab experimental studies with a pathogen, as with conventional vaccinology [29]. This technique has gained increasing global popularity and usage by research groups in the last 20 years for vetting the whole genome for vaccine antigens against several pathogens including Leishmania [30], Lassa fever [31], Dengue [32], Schistosomiasis [33], and P. falciparum [34], among others. This strategy is effective as it saves cost and time, and decrease the risk of failure compared to conventional vaccinology [35].

The World Health Organization set the goal to eradicate malaria in 1948, and efforts have been intensified to this end. Correspondingly, malaria-related fatalities progressively decreased, but this progress stalled due to the COVID-19 pandemic and attributed to the interference with malaria services and diagnostic practices [36]. In 2021, the number of malaria cases worldwide was recorded to be 241 million, with 627,000 related deaths [37]. In light of the available data and the current state of global malaria burden, this research aimed to develop a potent multi-epitope blood stage subunit vaccine candidate against P. falciparum by integrating epitopes from the most immunogenic regions of three merozoite surface proteins namely, liver-stage antigen 3 (LSA3-C), merozoite surface antigen 180 (MSA 180), and merozoite surface antigen 10 (MSA 10).

METHODOLOGY

A systematic flow chart was followed stepwise to identify immunodominant B- and T-cell epitopes and the design of a subunit vaccine via immunoinformatics techniques. A subsequent biophysical study was performed using integrated docking, immune simulation, and molecular docking. Finally, codon optimization was executed to ensure optimal expression in the microbial host (Fig. 1).

Amino acid sequence retrieval

To retrieve the primary sequence of liver stage antigen 3, MSA 180, and merozoite surface protein 10, the PlasmoDB [38] server was employed. However, only the most immunogenic regions of these proteins–(LSA3-C; V750-K1433), merozoite surface antigen 180 truncate-4 (MSA 180-T4; A805-P1093), and merozoite surface protein 10 region 1 (MSP10 R1; D29-N188) were further exploited for the vaccine construct (VC), following results from prior studies [39–41].

Figure 1. Methodology flow chart showing the step-by-step procedures used. [Click here to view]

RESULTS AND DISCUSSION

Proteins that encrust the extracellular surface of merozoite and are released from its specialized secretory apical organelles are considered potential vaccine candidates for the erythrocytic stage malaria [68]. Robust experimental evidence supports the importance of these surface proteins to host cell invasion, parasite growth, and survival and as potential preventive measures against P. falciparum malaria in humans [11,40,69–75]. In this study, three experimentally validated vaccine candidates against falciparum malaria were selected for further downstream immunoinformatics analysis.

The LSA3-C, MSA 180-T4, and MSP10 R1 are regions of blood-stage P. falciparum proteins that possess characteristics which have been experimentally validated to be malaria vaccine candidates, and this makes them attractive targets for developing a sub-unit vaccine [39–41]. Liver-stage antigen 3 (PF3D7_0220000) is a ~175kDa protein expressed in liver and blood stages on sporozoites surface, infected hepatocytes, and blood-stage merozoites, and several research groups have reported it to be a viable pre-erythrocytic and blood stage vaccine target [39,76–78]. Furthermore, LSA3 was reported to be highly conserved following genomic sequence analysis of 20 P. falciparum clinical isolates from diverse geographical regions and localized in the parasitophorous vacuole during the ring-stage following merozoite ingress [78,79]. Previous studies on the full-length and discrete regions of LSA3-C have reported its viability as a pre-erythrocytic and blood-stage vaccine candidate in addition to being the most immunologic antigen segment [39,78,79].

Plasmodium falciparum merozoite surface antigen 180 (PfMSA180) is a 170kDa protein that is essential and conserved in all Plasmodium sp.; it is expressed on the periphery of merozoites and has been implicated in merozoite ingress and egress during the asexual blood stage of the parasite’s lifecycle [41,80]. PfMSA180 (PF3D7_1014100) has been reported to be critical for parasite invasion of the erythrocyte, and antibodies against the C-terminal region of PfMSA180 [MSA 180 Tr-4 (A805-P1093)] abrogated merozoite invasion in vitro and conferred protective immunity against malaria [41]. Furthermore, the PfMSA180 (PfMSA180-Tr4) C-terminal region was reported to be highly conserved across isolates, interacts with the red blood cell (RBC) surface protein-CD47, and stimulates antibodies that abrogate parasite invasion; hence, Nagaoka et al. [41] proposed MSA 180 Tr-4 as a potential vaccine candidate. PfMSP10 (PF3D7_0620400) is an 80 kDa protein that directly interacts with PfGAMA, which is key for erythrocyte invasion. The PfMSP10 R1 (D29-N188) region was hypothesized to be the interacting region [40]. Also, bioinformatics genome-wide screening has predicted PfMSP10 to be a putative vaccine candidate [81].

Polypeptide subunit vaccine design

The finalized B- and T-cell epitopes were integrated using suitable linkers alongside two ADVs. ADVs present a classical approach to targeted delivery of subunit vaccines and maximizing protective immunity, as protein vaccine candidates are poorly immunogenic [88]. They bridge the antigens with the APCs by projecting visibility of the otherwise non or weakly immunogenic antigen to the immune cells to trigger a better immune response [89]. RS09, a synthetic TLR4 agonist comprising seven amino acid residues [89], was incorporated as ADVs at the N-terminal of the VC [90,91]. The EAAAK linker increases the consistency of the overall structure and limits protein components from joining through effective detachment; hence, it was used to join the ADV with the polypeptide vaccine [92]. The AAY linker improves the vaccine’s ability to elicit an immunological response [93]. The need to impinge the development of junctional epitopes and improve epitope presentation necessitates the GPGPG linkers [94]. The KK linker was used to connect linear B-cell epitopes so that their self-regulating immunogenic responses may be preserved [95]. The VC follows the sequence “ADV-CD8+ epitope-CD4+ epitopes-B-cell epitopes- 6X His tag,” joined by appropriate linkers to form a 437 amino acid-long peptide as illustrated in Figure 2.

Table 1. Selected linear B-cell epitopes with their antigenicity, allergenicity, toxicity, and conservancy. [Click here to view]

Table 2. Details of selected CD4+ T-cell epitopes predicted by NetMHCIIpan 4.0. [Click here to view]

Table 3. Selected CD8+ T-cell epitopes with MHC supertypes and immunogenicity score. [Click here to view]

Secondary and tertiary structure prediction, evaluation, and validation

The VCs comprise 26% α-helix, 7% β-strand, and 66% coil, as predicted by Psipred, and RaptorX in plots representing the structural moieties (Fig. 3). Also, 76% was forecasted to be exterior, 8% mildly exterior, and 14% to be buried in relation to the availability of amino acid residues. The α-helix and β-turn have internal localization within the protein and are less likely to act as epitopes; instead, they maintain the structure of the protein due to the high chemical bond energy they contain. Random coils are exposed on the protein surface, have fewer rigid regions of the protein, and may be probable epitopes [96]. Over half the overall structure of the construct comprises random coils, hinting at the greater potential of the protein to trigger immune responses. The 3D structure was predicted by the Robetta server, of which five models were generated, and model 1 was selected as it had higher comparative model quality following subjection to the Saves v.6.0 server for the ERRAT and Ramachandran plot analysis. GalaxyRefine server was used to refine and improve the quality of the selected model (Fig. 4A). Structural validation involves the identification of probable faults in the forecast 3D structure [97]. The model had a Z-score of -7.37, predicted by ProSA-web (Fig. 4B). Also, to determine the nonbonded atom-atom interactions, the ERRAT server was utilized [57], which revealed an overall quality factor of 94.72, suggesting a high quality. The Ramachandran plot enables the visualization of energetically favorable and unfavorable dihedral angles psi (ψ) and phi (?) of amino acids, which delineates the stereochemical quality of the 3D structure [98]. The Ramachandran plot analysis revealed that 93.8% of the initial model was in the favored regions, 5.2% in the allowed regions, and 0.8% in the disallowed regions. However, the refinement process improved the protein quality to 95.3% favoured, 3.4% allowed, and 0.5% disallowed regions, representing a very good model (Fig. 4C and D).

Figure 2. (A) Schematic diagram of polypeptide vaccine. It comprises several segments: an ADV, RS09 (TLR4 agonists), 10 CD8+ epitopes, 7 CD4+ epitopes, 9 B-cell epitopes, and a His tag. These segments are integrated by EAAK, AAY, GPGPG, and kk linkers. (B) Sequence of polypeptide vaccine. [Click here to view]

Conformational B-cell epitopes

B-cell epitopes usually form discontinuous epitopes resulting from the spatial proximity of a bunch of amino acid residues on the tertiary structure of the antigen [99]. Ellipro forecasted 4 epitopes with a maximum score of 0.758 and a minimum score of 0.512, and the number of epitope residues ranged from 3 to 81 (Table S3). Figure 5 reflects the structure of the epitopes in relation to the VC.

Figure 3. Schematic diagram of the secondary structure of VC. (A) Predicted secondary structure (H: α-helix, E: β-strand, C: coil) by PSIPRED. The bar chart represents the percentage of confidence. (B) RaptorX generated the chimeric protein’s secondary structure, and the results show a structure made up of 26% α-helix, 7% β-strand and 66% coil. [Click here to view]

Structural and functional characterization of VC

The physicochemical properties prediction revealed that the protein has a molecular weight and theoretical pI of 49.2 kDa and 5.99, respectively. The half-life was predicted to be 4.4 hours (mammalian reticulocytes, in vitro), >20 hours (yeast, in vivo), and >10 hours (Escherichia coli, in vivo). The instability index and GRAVY were forecasted to be 39.36 and −1.294, respectively. The score from the instability index suggested that the protein is stable. The GRAVY score represented that the protein is hydrophilic, which is a desired quality for a vaccine as it indicates the ability to trigger an elevated humoral immune response [100]. The aliphatic index was predicted to be 61.40, reflecting thermostability (Table S2).

Figure 4. (A) 3D structure cartoon representation (Red, green, and yellow represent α-helix, coil, and β-sheet). (B) ProSA map of the sub-unit vaccine with Z-score of −7.37. (C) Ramachandran plot before protein refinement with 93.8% in the favored region, 5.2% allowed region, 0.8% in the disallowed region. (D) Ramachandran plot after protein refinement with 95.3% favored region, 3.4% allowed region, and 0.5% disallowed region. [Click here to view]

Figure 5. 3-D representation of the four conformational B-cell epitopes. (A–D) A yellow surface represents the epitopes. [Click here to view]

Molecular docking of vaccine construct with toll-like receptors

The contact between ligand and receptor molecules is embroiled in molecular docking to generate a stable ligand-receptor product [101]. The interaction between antibodies and their targeted antigens is critical to the humoral immune response to aid pathogen elimination. The VC-TLR2 complex has a total of 81 members in its cluster, while VC-TLR4 has 72 members in its cluster. The lowest energy score and binding affinity of −945.1 and −919.8 kcal/mol were obtained for the VC-TLR2 and VC-TLR4 complexes, respectively (Table 4). These complexes were stable as shown in Figure S1.

Table 4. Molecular docking score between VC and TLR-2 and -4. [Click here to view]

MD simulation of vaccine construct-TLR complexes

MD simulation is useful for analyzing the stability of protein-ligand complex by incorporating Newton’s laws of motion [102]. It was done by enumerating protein dynamics to their normal modes using the iMODS server [103]. NMA is commonly used to evaluate the collective functional motions of docked protein-protein complexes [66]. The 3D interaction between VC-TLR2 and VC-TLR4 complexes is presented, with the arrows representing the direction of amino acids (Fig. 7A and B). The peaks on the deformability graph delineate deformability, as higher peaks depict higher deformability. From the result, the deformability plots report the stability of the complexes with individual amino acid residues having a lesser likelihood of deforming (Fig. 7C and D). The B-factor graph comparatively evaluates the NMA and the protein data bank field of the docked complexes (Fig. 7E and F). The eigenvalue corresponds with motion stiffness; a lower value is congruent with higher deformability and vice versa. The predicted high values of 9.12572e^-07 and 1.117255e^-06 for VC-TLR2 and VC-TLR4, respectively, reflect the less deformability and high stability of both complexes (Fig. 7G and H). The variance corresponding to each normal mode is inversely proportional to the eigenvalue (Fig. 7I and J) [104]. The covariance matrix reflects if the pairs of residues’ motions were correlated (red), uncorrelated (white), or anti-correlated (blue) (Fig. 7K and L). The elastic network graph reflects atom pairs joined by springs, which are correspondingly colored by the degree of stiffness. The lighter grey area corresponds to less rigid springs and vice versa (Fig. 7M and N).

Figure 6. Characterisation of the immune profile of the construct. (A) The antibody production denotes a rise in immune response following the vaccine shot. Antibody subtypes (IgM, IgG1, and IgG2) are depicted as colored peaks. (B) The active B-cell population is observed with the vaccine shots. (C) B-lymphocyte population per entity-state (D) CD4+ T-helper lymphocytes count sub-divided per entity-state (E) The generation of cytotoxic-T cells. (F) CD8+ T-cytotoxic lymphocytes count per entity-state (G) Dendritic cells for MHC class I and II. Shows the total number of active, resting, internalized, and presenting antigen (H) Macrophages. Total count, internalized, presenting on MHC class-II, active and resting macrophages (I) Concentration of cytokines and interleukins. [Click here to view]

Figure 7. (A, B) NMA mobility, (C, D) deformability, (E, F) B factor, (G, H) Eigenvalue, (I, J) variance map, (K, L) Covariance map and (M, N) Elastic network graph of docked complexes VC-TLR2 and VC-TLR4, respectively. For the covariance map, red: correlated, white: uncorrelated and blue: anti-correlated motions, while for the elastic network graph of docked complexes, darker grey regions correlate with stiffer regions. VC: Vaccine construct. [Click here to view]

Figure 8. In silico cloning of the leading VCs. The red fragment designates the inserted vaccine and the pET-28a(+) vector is presented in a black circle. [Click here to view]

CONCLUSION

A multi-immune inducer that targets several antigens or various points of the parasite’s entire life cycle may effectively engender robust clinical protection against malaria. To the best of our knowledge, this is the first study to use short regions rather than full-length proteins of experimentally validated vaccine candidates to contrive an antibody-inducing multi-epitope subunit vaccine targeting the blood stage of falciparum malaria. This is important because regions of vaccine candidates that have been experimentally validated to be most immunogenic will be most attractive for mapping immunodominant epitopes capable of being integrated to form a potent subunit vaccine. The VC demonstrated the ability to evoke combined innate and adaptive immune responses via interaction with the TLR-2 and -4, triggering cellular and humoral immunity in addition to forming a stable VC-receptor complex. The limitation of this study is that it is completely performed in silico. Therefore, an experimental study is necessary to determine the full potential of the proposed vaccine as a possible intervention in neutralizing malaria.

ACKNOWLEDGMENT

Covenant Applied Informatics and Communication Africa Centre of Excellence (CApIC-ACE) scholarship to COM is acknowledged. The deference of APC by Covenant University Centre for Research, Innovation and Discovery is appreciated

AUTHOR CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took p art in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.

ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY

All the data is available with the authors and shall be provided upon request.

PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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SUPPLEMENTARY MATERIAL

The supplementary material can be accessed at the journal's website: Link Here [https://japsonline.com/admin/php/uploadss/4324_pdf.pdf].