Topical PerspectivesDesigning a multi-epitope vaccine against blood-stage of Plasmodium falciparum by in silico approaches
Graphical abstract
Introduction
Malaria is the most destructive parasitic disease affecting humans. It results from infection with a protozoan parasite belonging to the Plasmodium genus that is transmitted by infectious female Anopheles mosquito [1]. P. falciparum is the most pathogenic species of five known human malaria parasites. This strain accounts for the most severe form of the disease and the majority of mortality from the infection [2,3]. Malaria eradication will be achieved by an effective vaccine. An effective vaccine which can prevent human infection and also transmission from human to mosquito would reduce malaria morbidity and mortality and eventually accelerate global malaria eradication [4,5]. Despite great efforts, there is still no licensed vaccine against malaria [6]. Genetic and antigenic diversity are major obstacles hindering the development of effective malaria vaccines [[7], [8], [9], [10]]. For these reasons, many studies have focused on conserved regions or epitopes which are common between a vast range of plasmodium strains [[11], [12], [13], [14]].
Subunit-based vaccines as one of the leading subgroups of malaria vaccines target every stage of the parasite lifecycle including pre-erythrocytic and erythrocytic stages in humans, or lifecycle stages in the mosquito host [1,15]. Nevertheless, there is a strong rationale for developing erythrocytic-stage vaccines, because the pathogenesis of malarial disease results from blood-stage infection and all of the symptoms and clinical illness of malaria occur during this stage [1]. In this stage, the merozoites invade erythrocytes and replicate inside them which eventually result in the cell burst and allow the merozoites to release and infect new cells [16,17]. This infection-making method reasonably turns the invasion as an attractive vaccine target, especially in light of the fact that in the erythrocytic-stage parasite is directly exposed to the host humoral immune response [18,19]. An effective antibody-inducing vaccine which targets invasion of merozoites, not only can be prophylactic but also can reduce the disease severity and disease transmission form. Studies in animal models and humans have obviously established that humoral immune responses targeting blood-stage antigens can facilitate control of parasitemia or even protect against disease [3,20,21]. In this regard, immunization with blood-stage antigens, mostly merozoite antigens, has been indicated to be protective in animal models [[22], [23], [24], [25]] as well as to some extent in humans by inducing antibody immune response [26]. Additionally, studies have shown that simultaneous targeting more than one invasive antigen via a combination of antibodies acted synergistically against P. falciparum merozoites and induced more potent parasite growth inhibition [27].
Presently, the leading blood-stage vaccine candidates are merozoite surface proteins [1] which have critical roles in attachment and invasion of the erythrocytes [28]; Among these proteins, P. falciparum cysteine-rich protective antigen (PfCyRPA), P. falciparum reticulocyte binding homolog 5 (PfRh5), P. falciparum erythrocyte-binding antigen 140 (PfEBA-140) and merozoite surface protein-1 (MSP-1) [19] are crucial for merozoite invasion of the erythrocytes and have features which make them as attractive candidates for designing antibody-inducing vaccines [[29], [30], [31]]. PfCyRPA is a 42.8 kDa protein of 362 residues localized at the apex of merozoites [3,32]. Sequence analysis of the CyRPA gene from 227 P. falciparum clinical isolates showed that the entire protein is highly conserved [8,32]. It is worth noting that P. falciparum merozoites in which the CyRPA gene has been conditionally disrupted cannot invade human erythrocytes [29]. Consistently, a CyRPA-specific monoclonal antibody (mAb) has been shown to significantly inhibited the parasite growth in vitro, as well as in an P. falciparum animal model [32]. CyRPA cannot bind directly to the erythrocytes and instead it provides the structural support for PfRh5 as an essential P. falciparum erythrocyte invasion ligand. Indeed, PfRh5 forms a complex with the CyRPA on the merozoite surface and by the other side binds to the basigin/CD147 receptor on the surface of host erythrocytes [29]. Constituting this complex is crucial for merozoite invasion of erythrocytes [33]. PfRh5 protein with only 5 common non-synonymous single nucleotide polymorphisms (SNPs) is highly conserved [34]. Studies have shown that anti-PfRh5 mAbs, by blocking the PfRh5-basigin interaction can directly inhibit parasite growth in vitro [34]. In addition, human studies have shown that naturally anti-PfRh5 antibodies acquired during infection inhibit parasite growth in vitro and are correlate with better clinical outcome [35]. PfEBA-140 belong to erythrocyte binding-like (EBL) family is a vital invasion ligand on P. falciparum that binds to glycophorin C (GPC) on the erythrocytes during the malaria infection [30]. PfEBA-140 contains an extracellular region that is comprised of two conserved Duffy binding-like (DBL) domains named F1 (residues 143–422) and F2 (residues 447–740). These two homologous domains contain the minimal binding region of the P. falciparum EBL ligands named region II (RII) [36]. Neither DBL domain is sufficient to independently engage erythrocytes and both domains create essential contacts with GPC during invasion [30]. Only four SNPs have been found in the RII of PfEBA-140, all of which are located in the F1 domain [37]. A recent study has shown that the recombinant EBA-140 region II is immunogenic [38]. Besides, naturally acquired antibody to EBA-140 was found in the serum of malaria patients and people who live in malaria-endemic areas. High levels of IgG against EBA-140 were strongly associated with protection from parasitemia and symptomatic malaria [39]. MSP-1 is a ∼200 kDa GPI anchored merozoite surface protein. This protein is the most abundant GPI-anchored protein on the surface of P. falciparum merozoites and efforts for knocking out MSP-1 gene have been failed, suggested that this protein is crucial for parasite growth and/or invasion. MSP-1 is proteolytically cleaved into 83-, 38-, 30-kDa, and C-terminal 42-kDa fragments just prior to egress from the schizont [40]. At the time of merozoite invasion, the 42 kDa C-terminal fragment is further proteolytically cleaved into a soluble fragment of 33 kDa and a 19 kDa fragment (MSP-119) which remains on the merozoite surface [41]. During the P. falciparum invasion, MSP-119 binds to band 3 protein receptor on the surface of erythrocytes [31]. MSP-119 fragment includes 98 amino acids and by only four non-synonymous changes is highly conserved [42]. Additionally, the MSP-119 is a known target of naturally acquired humoral immune response that can inhibit erythrocyte invasion and is associated with protection from malaria infection [40,43].
According to the abovementioned evidence, the purpose of the present study was to design a multi-epitope vaccine candidate based on PfCyRPA-, PfRh5-, PfEBA-140- and MSP-119-derived epitopes to elicit humoral immune responses against blood-stage of P. falciparum by employing bioinformatics methods. These proteins are expressed and localized on the surface of merozoites and act as invasion ligand for host erythrocyte membrane receptors [[29], [30], [31]].
Section snippets
Sequence and structure retrieval
The crystal structure of PfCyRPA (resolution: 2.44 Å, PDB Id: 5TIH), PfRh5 (resolution: 2.18 Å, PDB Id: 4WAT), PfEBA-140 (RII) (resolution: 2.4 Å, PDB Id: 4GF2), MSP-119 (resolution: 2.9 Å, PDB Id: 1OB1), CyRPA in complex with Rh5 (resolution: 7.17 Å, PDB Id: 6MPV) and Rh5 in complex with CD147 (resolution: 3.1 Å, PDB Id: 4U0Q) were retrieved from RCSB at http://www.rcsb.org/. The protein sequences of PfCyRPA (Q8IFM8), PfRh5 (Q8IFM5), PfEBA-140 (Q76NM5) and MSP-1 (Q8I0U8) were extracted from
B cell epitope prediction
The common linear B cell epitopes between sequence-based and structure-based predictions which three software agreed upon were selected from each antigenic protein and considered as final epitopes for vaccine construction (Table 1). Among predicted B cell epitopes, those which were dimorphic or trimorphic, in order to induce immune response against all variants, the sequences of all variants were used for vaccine construction. A total of 14 linear B cell epitopes were selected from antigens
Discussion
P. falciparum causes the most serious form of malaria disease and is the major cause of infection-related mortalities [2,3]. Despite the availability of effective drugs, due to increasing in P. falciparum resistance to the first-line anti-malaria drugs, mortality caused by malaria remains a global health and economic concern [70,71]. Thus, an effective vaccine would be an effective tool for the control, elimination or even possible eradication of malaria.
Since all of the malaria symptoms occur
Conclusion
In the present work, with the purpose of designing a protective antibody-inducing multi-epitope vaccine against blood-stage of P. falciparum, the immunodominant B cell epitopes from highly conserved and protective antigens of P. falciparum (CyRPA, pfRh5, EBA-140 and MSP-1) were defined. HP91 and RS09, which act as adjuvants, as well as Th epitopes were also incorporated into the vaccine construct to enhance the immunogenicity of vaccine and to induce, enhance and deviate/direct the best form of
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
The authors would like to thank Shiraz University of Medical Sciences.
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