Topical Perspectives
Designing a multi-epitope vaccine against blood-stage of Plasmodium falciparum by in silico approaches

https://doi.org/10.1016/j.jmgm.2020.107645Get rights and content

Highlights

  • Due to lack of licensed vaccine and increasing P. falciparum drug resistance, designing an effective vaccine is urgent.

  • All malaria symptoms and clinical illness occur during the blood-stage of the disease.

  • Four protective merozoite surface antigens critical for invasion of the erythrocyte were selected for vaccine designing.

  • Immunoinformatics tools were used to design a multi-epitope vaccine against blood-stage of P. falciparum.

  • Physicochemical and immunological characteristics of the vaccine showed acceptable results.

Abstract

Plasmodium falciparum causes the most severe form of malaria disease and is the major cause of infection-related mortalities in the world. Due to increasing in P. falciparum resistance to the first-line antimalarial drugs, an effective vaccine for the control and elimination of malaria infection is urgent. Because the pathogenesis of malaria disease results from blood-stage infection, and all of the symptoms and clinical illness of malaria occur during this stage, there is a strong rationale to develop vaccine against this stage. In the present study, different structural-vaccinology and immuno informatics tools were applied to design an effective antibody-inducing multi-epitope vaccine against the blood-stage of P. falciparum. The designed multi-epitope vaccine was composed of three main parts including B cell epitopes, T helper (Th) cell epitopes, and two adjuvant motives (HP91 and RS09), which were linked to each other via proper linkers. B cell and T cell epitopes were derived from four protective antigens expressed on the surface of merozoites, which are critical to invade the erythrocytes. HP91 and RS09 adjuvants and Th cell epitopes were used to induce, enhance and direct the best form of humoral immune-response against P. falciparum surface merozoite antigens. The vaccine construct was modeled, and after model quality evaluation and refinement by different software, the high-quality 3D-structure model of the vaccine was achieved. Analysis of immunological and physicochemical features of the vaccine showed acceptable results. We believe that this multi-epitope vaccine can be effective for preventing malaria disease caused by P. falciparum.

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.

References (92)

  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • A. Yano et al.

    An ingenious design for peptide vaccines

    Vaccine

    (2005)
  • P. Sarobe et al.

    Enhancement of peptide immunogenicity by insertion of a cathepsin B cleavage site between determinants recognized by B and T cells

    Res. Immunol.

    (1993)
  • R. Saenz et al.

    HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen

    Vaccine

    (2010)
  • Mark James Abraham et al.

    GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers

    Software

    (2015)
  • M.K. Khan et al.

    In silico predicted mycobacterial epitope elicits in vitro T-cell responses

    Mol. Immunol.

    (2014)
  • S. Talebi et al.

    Hp91 immunoadjuvant: an HMGB1-derived peptide for development of therapeutic HPV vaccines

    Biomed. Pharmacother.

    (2017)
  • A.E. Barry et al.

    Strategies for designing and monitoring malaria vaccines targeting diverse antigens

    Front. Immunol.

    (2014)
  • G.E. Weiss et al.

    Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during Plasmodium falciparum invasion of erythrocytes

    PLoS Pathog.

    (2015)
  • P. Favuzza et al.

    Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

    eLife

    (2017)
  • C.V. Plowe et al.

    The Potential Role of Vaccines in the Elimination of Falciparum Malaria and the Eventual Eradication of Malaria

    (2009)
  • m.C.G.o. Vaccines

    A research agenda for malaria eradication: vaccines

    PLoS Med.

    (2011)
  • A. Ouattara et al.

    Vaccines against malaria

  • M. Manske et al.

    Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

    Nature

    (2012)
  • A.V. Hill

    Vaccines against malaria

    Philos. Trans. R. Soc. Lond. B Biol. Sci.

    (2011)
  • A. Scherf et al.

    Antigenic variation in Plasmodium falciparum

    Annu. Rev. Microbiol.

    (2008)
  • J. Healer et al.

    Vaccination with conserved regions of erythrocyte-binding antigens induces neutralizing antibodies against multiple strains of Plasmodium falciparum

    PloS One

    (2013)
  • S. Dutta et al.

    Overcoming antigenic diversity by enhancing the immunogenicity of conserved epitopes on the malaria vaccine candidate apical membrane antigen-1

    PLoS Pathog.

    (2013)
  • P. Daubersies et al.

    Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3

    Nat. Med.

    (2000)
  • P.G. McQueen et al.

    Competition for red blood cells can enhance Plasmodium vivax parasitemia in mixed-species malaria infections

    Am. J. Trop. Med. Hyg.

    (2006)
  • L.M. Kats et al.

    Protein trafficking to apical organelles of malaria parasites–building an invasion machine

    Traffic

    (2008)
  • S. Cohen et al.

    Gamma-globulin and acquired immunity to human malaria

    Nature

    (1961)
  • J.S. Richards et al.

    Identification and prioritization of merozoite antigens as targets of protective human immunity to plasmodium falciparum malaria for vaccine and biomarker development

    The Journal of Immunology Author Choice

    (2013)
  • A.N. Kamali et al.

    Experimental immunization based on plasmodium antigens isolated by antibody affinity

    Journal of Immunology Research

    (2015)
  • P.M. Petritus et al.

    Suppression of lethal Plasmodium yoelii malaria following protective immunization requires antibody-, IL-4-, and IFN-gamma-dependent responses induced by vaccination and/or challenge infection

    J. Immunol.

    (2008)
  • D.L. Narum et al.

    Immunization with parasite-derived apical membrane antigen 1 or passive immunization with a specific monoclonal antibody protects BALB/c mice against lethal Plasmodium yoelii yoelii YM blood-stage infection

    Infect. Immun.

    (2000)
  • B. Genton et al.

    A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea

    J. Infect. Dis.

    (2002)
  • A.R. Williams et al.

    Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens

    PLoS Pathog.

    (2012)
  • G.J. Wright et al.

    Plasmodium falciparum erythrocyte invasion: combining function with immune evasion

    PLoS Pathog.

    (2014)
  • V.K. Goel et al.

    Band 3 is a host receptor binding merozoite surface protein 1 during the Plasmodium falciparum invasion of erythrocytes

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • A.M. Dreyer et al.

    Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen

    J. Immunol.

    (2012)
  • K.S. Reddy et al.

    Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion

    Proc. Natl. Acad. Sci. U. S. A.

    (2015)
  • K.E. Wright et al.

    Structure of malaria invasion protein RH5 with erythrocyte basigin and blocking antibodies

    Nature

    (2014)
  • T.M. Tran et al.

    Naturally acquired antibodies specific for Plasmodium falciparum reticulocyte-binding protein homologue 5 inhibit parasite growth and predict protection from malaria

    J. Infect. Dis.

    (2014)
  • D.C. Mayer et al.

    Polymorphism in a Plasmodium falciparum erythrocyte-binding ligand changes its receptor specificity

    J. Exp. Med.

    (2002)
  • A. Zerka et al.

    Studies on immunogenicity and antigenicity of baculovirus-expressed binding region of plasmodium falciparum EBA-140 merozoite ligand

    Arch. Immunol. Ther. Exp.

    (2016)
  • Cited by (0)

    View full text