As of 2025, malaria vaccine development is based on candidates targeting all parasite life stages. The WHO-approved RTS,S/AS01 and R21/Matrix-M (showing 75% efficacy in Phase 3) both target circumsporozoite protein, while blood-stage candidates like RH5.1/Matrix-M demonstrate 55% six-month efficacy in African children [22–24]. However, challenges persist with blood-stage vaccines due to antigenic polymorphism (e.g., AMA-1 variants showing strain-specific efficacy) and redundant invasion pathways. Other promising approaches include whole-sporozoite vaccines (PfSPZ) achieving 90% sterile protection in challenge trials, and transmission-blocking candidates like ProC6C targeting mosquito-stage antigens. In the case of the RTS,S/AS01 malaria vaccine represents a significant advancement but faces several challenges, including moderate efficacy (36–50% in clinical trials) and waning protection over time. Vaccine efficacy declines from approximately 55% in the first year to 28% by four years post-vaccination, necessitating booster doses to maintain protection [22–25]. Furthermore, co-administration with other candidates like FMP1/AS02 revealed immunological interference, reducing anti-RTS,S antibody titers by 30–40% without compromising efficacy in controlled human malaria infection models [26]. The FMP1/AS02 vaccine itself showed no significant protection in Kenyan children despite promising sporozoite challenge data, underscoring the difficulty of translating immunogenicity to real-world efficacy in endemic settings.

MSP-1 is the most abundant P. falciparum MSP, comprising 40% of the GPI-anchored coat [5]. Its essential role in merozoite survival is supported by the fact that attempts to disrupt the MSP-1 gene have failed [12]. MSP-1 mediates merozoite attachment to RBCs, initiating invasion through weak receptor-ligand interactions and heparin-like receptors. The carboxyl-terminus of MSP-1 interacts with RBC Band 3, while the amino-terminus binds to GPA. Mapping of the binding domains has confirmed a direct interaction between MSP-1 and GPA within a region known to strongly inhibit P. falciparum invasion of human RBCs. Additionally, a genetically modified mouse model lacking the GPA-Band 3 complex in RBCs exhibits complete resistance to malaria infection in vivo. These findings suggest that the MSP-1-GPA-Band 3 complex plays a crucial role in the malaria parasite invasion [27]. On the other hand, MSP-1 forms invasion complexes with MSP-3, MSP-6, MSP-7, MSPDBL1, and MSPDBL2, which bind independently but contribute to erythrocyte recognition and invasion, and directly bind to human erythrocytes [28].

MSP-1 undergoes proteolytic processing, generating fragments of 83, 38, 33, and 19 kDa, with MSP-119 remaining membrane-anchored and entering RBCs during invasion [12]. These fragments remain assembled within the MSP-1 structure, which features coiled-coil helical domains with a central cavity, structurally resembling proteins involved in membrane and cytoskeletal interactions [29]. MSP1-42, a key protein fragment in merozoite invasion, binds specifically to heparin, with similar interactions observed for its MSP1-33 fragment but not in MSP1-19. Notably, heparin-like molecules optimally inhibit invasion when highly sulfated (≥ 2 sulfate groups) [30]. Additionally, a 115-amino acid region (p115MSP-1) within the p38 domain of MSP-1 binds specifically to human erythrocytes, independent of GPA, and significantly inhibits P. falciparum invasion in vitro. This region is recognized by sera from individuals in malaria-endemic regions of Africa, and polyclonal antibodies against p115MSP-1 prevent parasite invasion by nearly 90% in vitro [31].

MSP-1 is a major target for opsonizing antibodies [32]. Moreover, MSP-1 contains epitopes that elicit antibodies capable of inhibiting merozoite invasion, which are distributed across its processing products: p83, p30, p38, and p42. These antibodies can act synergistically to enhance their inhibitory effects. Additionally, rabbit antibodies against MSP-1 processing products can inhibit both erythrocyte invasion and intraerythrocytic parasite growth by targeting multiple MSP-1 epitopes [33]. Antibodies targeting MSP-1 and its complexes inhibit P. falciparum invasion by preventing the essential protein shedding required for erythrocyte entry [33]. In particular, antibodies targeting the MSP-1 p83 fragment disrupt all MSP-1 complexes, blocking merozoite attachment and significantly inhibiting parasite growth in vitro [29]. On the other hand, antibodies specific to MSP-119 inhibit parasite growth through multiple mechanisms, including blocking MSP-1 secondary processing and directly preventing invasion. Invasion-inhibitory antibodies targeting MSP-119 significantly reduce infection risk [34]. Interestingly, antibodies against 3D7 MSP-1 cross-inhibit the replication of the FCB-1 strain [33].

Nine high-activity binding peptides (HABPs) within MSP-1 that bind to erythrocytes were identified at the Instituto de Inmunología, San Juan de Dios Hospital (Table 1). These include peptides 1513 and 1522 in the 83 kDa fragment; 1577 and 1582 in the 38 kDa fragment; 1585, 1589, 1590, and 1591 in the 33 kDa fragment; and 5501 in the 19 kDa fragment. These peptides were discovered from a library of 78 synthetic peptides covering the entire MSP-1 molecule. They exhibit binding affinities to RBCs ranging from 140 nM to 250 nM, and some have been shown to inhibit merozoite invasion of erythrocytes [35]. Additionally, certain peptides may interact with erythrocyte receptors such as Band 3 and GPA [36].

HABPs 1513, 1522, 1577, 1585, 1589, 1590, and 1591 are predominantly located in α-helices that are largely surface-exposed in the three-dimensional structure of MSP-1 [35] (PDB 6ZBH). In contrast, HABP-5501 is situated within an exposed loop in the EGF-1 domain structure [37, 38] (PDB 6XQW and 1OB1). Notably, HABPs 1585, 1589, 1590, and 1591 form a contiguous surface-exposed region that could serve as a target for antibodies (Figure 2). Certain HABPs exhibit immunogenic properties, inducing antibodies that recognize both MSP-1 and the merozoite surface. For instance, peptide 1513 elicits antibodies capable of recognizing native MSP-1 and inhibiting merozoite invasion [39–42]. Additionally, a segment of peptide 1513’s sequence was incorporated into the SPf66 vaccine, which has demonstrated protective efficacy ranging from 31% to 60.2%, depending on the population and geographic factors [43].

The first EGF-like domain of the 19 kDa fragment, where peptide 5501 is located, is a critical target of MSP-1-specific monoclonal antibodies (mAbs). These mAbs are classified as inhibitory (preventing MSP-142 processing and erythrocyte invasion), blocking (interfering with the binding and function of inhibitory mAbs), or neutral (neither inhibitory nor blocking). Inhibitory mAbs 12.8 and 12.10, as well as blocking mAbs 1E1 and 7.5, recognize key residues within peptide 5501 (QGMLNISQHQCVKKQCPQNS; inhibitory residues underlined, blocking antibody targets in bold). Specifically, peptide CPQNSGCFR is recognized by mAb 12.8, while FQDMLNIS is targeted by mAb 1E1 (underlined and bold residues corresponding to peptide 5501) [48]. The mAbs 12.8 and 12.10 specifically target the 19 kDa subunit of MSP-1 (MSP-119) and have been shown to prevent both proteolytic cleavage of MSP-1 and erythrocyte invasion [49, 50]. Interestingly, human chimeric antibodies derived from mouse mAbs 12.8 and 12.10 retain their ability to effectively inhibit merozoite invasion and can elicit an immune response in humans [50].

Moreover, the structure of the fragment antigen binding (Fab) G17.12, which does not inhibit merozoite invasion, in complex with the 19 kDa fragment, reveals VKKQ as part of the contact region [46] (PDB 1OB1). In contrast, the MaliM03 Fab, which effectively inhibits P. falciparum merozoite invasion, interacts with the 19 kDa fragment through the QHQCVKKQCP region [37] (PDB 6XQW). The potency of inhibition depends on the nature of these interactions. For instance, the potently neutralizing human monoclonal antibody 42D6 Fab, in complex with MSP1-19, recognizes the QHQC motif within this peptide sequence, leading to strong inhibition of merozoite invasion [51] (PDB 8DFG) (Figure 2).

MSP-2 is a 30-kDa GPI-anchored protein on P. falciparum merozoites and a potential blood-stage vaccine candidate [51, 52]. PfMSP2 is essential for parasite growth and directly involved in merozoite invasion of erythrocytes, as it binds to human RBCs with high affinity facilitating merozoite entry and it is crucial for successful invasion during malaria infection [53, 54]. Synthetic peptides targeting MSP-2 can inhibit invasion, further supporting its role in this process [9].

MSP-2 is an intrinsically unstructured protein (IUP) that forms amyloid fibrils in its recombinant form, probably being part of a possible fibrillar merozoite surface coat. However, its native conformation may differ due to membrane anchoring and protein interactions [55]. Once inside the erythrocyte, PfMSP2 degrades within 10 min, suggesting a role during and immediately after invasion [56]. Monomeric and fibrillar forms of MSP-2 may elicit distinct immune responses [57]. Indeed, antibodies targeting PfMSP2 inhibit parasite growth in vitro and significantly block erythrocyte invasion in both Babesia bovis and P. falciparum [58, 59].

Due to its key role in invasion and its ability to generate immune responses against the parasite, MSP-2 is an important target for vaccine development [60]. However, the antibody response to MSP-2 is short-lived without re-exposure [53], suggesting that MSP-2-specific antibodies can serve as sensitive markers of recent parasite exposure [57]. MSP-2 is highly polymorphic, with conserved N- and C-terminal domains and two major allelic families 3D7 and FC27 [61]. Additionally, genetic variations in MSP-2 have been associated with disease severity [61].

Neutralizing anti-MSP-2 antibodies are associated with malaria resistance [62], and higher antibody affinity for the 3D7 allele correlates with a reduced risk of malaria [63]. Additionally, chimeric MSP-2 constructs have been shown to induce broad immune protection [64]. Evidence also supports strain-transcending opsonizing immunity mediated by anti-MSP-2 antibodies [65]. However, some anti-MSP-2 antibodies are internalized rather than inhibiting invasion, suggesting a complex interplay between immune recognition and parasite evasion [56].

A screening assay using peptides spanning the entire PfMSP2 sequence identified three HABPs (4044, 4045, and 4053) (Table 2), with significant specific binding to human erythrocytes [57]. HABPs 4044 and 4045 are located in the N-terminal region, while HABP 4053 is in the central region. Peptides 4044 and 4045 share the sequence INNAYNMSIR, with binding affinities of 72 nM and 49 nM, respectively. Their binding is affected by neuraminidase and trypsin but remains resistant to chymotrypsin degradation. Both peptides inhibit in vitro erythrocyte invasion by up to 95%, and immunization with a related octapeptide has demonstrated protective effects.

Immunization of rabbits with recombinant MSP-2 elicits high-titer anti-PfMSP2 antibodies capable of opsonizing P. falciparum merozoites for phagocytosis. These antibodies recognize rPfMSP2 from both 3D7 and FC27 alleles, as well as native PfMSP2 and previously identified linear epitopes within these three HABPs [63]. Additionally, mAbs 6D8 and 11E1 recognize the epitopes FINNAYNMSIRRS (present in HABPs 4044 and 4045) and NPKGKGEVQEPNQ (present in HABP 4053), respectively, of MSP-2 [66]. The three-dimensional structure of monoclonal antibody 6D8 in complex with MSP-2 specifically binds to the sequence contained in HABP 4044 [67] (PDB 4R3S0). Moreover, the N-terminal region of MSP-2 (MSP-2_1–25), where HABP 4044 is located, binds lipids and adopts an extended helical conformation spanning at least residues 10–22 [68] (Figure 3). These findings strongly suggest that most of the sequences of these HABPs are exposed not only on the native protein but also on the merozoite surface, where they can be targeted by neutralizing antibodies, as previously reported [66–68].

The P. falciparum ABRA, also known as MSP-9, is located on the merozoite surface and within the PV of infected erythrocytes. MSP-9 plays a crucial role in erythrocyte invasion and is physically associated with other essential invasion proteins [70, 71]. It directly interacts with the erythrocyte Band 3 receptor, binding specifically through two regions (MSP-9/Δ1 and MSP-9/Δ2) to the 5ABC region of Band 3, thereby facilitating P. falciparum merozoite invasion of erythrocytes [72]. Additionally, native MSP-9 forms a stable complex with MSP1-42, which targets erythrocyte receptors during invasion [28, 72]. Blocking MSP-9 binding using recombinant polypeptides significantly reduces P. falciparum reinvasion [72].

The immune response against MSP-9 not only inhibits parasite replication but also modulates inflammatory responses, with significant implications for vaccine development [73]. Vaccination with virus-like particles presenting MSP-9 elicits a strong antibody response and enhances T cell activation, generating anti-MSP-9 antibodies that effectively neutralize the parasite during invasion [73].

A series of 20-mer peptides derived from the P. falciparum ABRA/MSP-9, spanning the entire protein, have been evaluated in erythrocyte binding assays, leading to the identification of peptides 2148, 2149, 2150, 2152, and 2153 (Table 3) [74]. These peptides, originating from the N-terminal region of ABRA (residues 121–240), exhibit high-affinity binding to erythrocytes (Kd = 70–180 nM). Peptides 2148 and 2149 significantly inhibited merozoite invasion, while peptide 2149 and its analogs also displayed hemolytic and antimicrobial activity. Synthetic ABRA peptides elicit antibodies capable of blocking in vitro erythrocyte invasion by merozoites. Notably, peptides 2148 and 2149 inhibited invasion by over 90% without affecting intraerythrocytic parasite development. The binding of these peptides is reduced by up to 60% following chymotrypsin treatment of erythrocytes, suggesting that their target receptor is protease-sensitive. Interestingly, peptides 2148–2153 are located within the first of two MSP-9 regions (amino acids 77–183 and 364–528) that directly interact with the 5ABC segment of Band 3 during P. falciparum erythrocyte invasion. These regions form a co-ligand complex with MSP1-42, which binds to the invasion receptor 5ABC [72]. Antibodies against these MSP-9 regions may inhibit merozoite reinvasion, while the hemolytic activity of peptide 2149 suggests a potential interaction with the erythrocyte membrane [72]. Additionally, synthetic ABRA peptides inoculated into rabbits elicited IgG antibodies that blocked in vitro merozoite invasion of human erythrocytes by up to 90% [75]. According to the theoretical structure of MSP-9 predicted by AlphaFold Server, HABPs 2148, 2149, and 2150 form α-helices that are potentially surface-exposed. This structural prediction aligns with experimental evidence suggesting that they are targets of neutralizing antibodies (Figure 4).

AMA-1 is a conserved transmembrane protein located in the apical complex of P. falciparum merozoites. Its role in erythrocyte invasion is supported by its interactions with host cells and its ability to induce neutralizing antibodies. AMA-1 undergoes proteolytic processing into several fragments, with its ectodomain containing critical binding sites [76]. This protein facilitates strong attachment and mediates the formation of a tight junction with the host cell membrane [77].

AMA-1 forms a crucial complex with RON2, RON4, and RON5 (rhoptry neck proteins), collectively known as the AMA-1/RON complex, which is essential for tight junction formation between the merozoite and the erythrocyte [12, 78, 79]. Additionally, AMA-1 associates with MSP-1/6/7, a complex involved in merozoite invasion of erythrocytes; however, these two systems function independently, as antibodies targeting MSP-1 do not affect AMA-1 processing and vice versa [80].

Antibodies against AMA-1 effectively inhibit merozoite invasion by blocking its secondary processing and surface redistribution [81]. Studies have also identified critical residues in AMA-1 that are necessary for antibody binding, which are important for vaccine development [82]. The avidity of antibodies against AMA-1 is influenced by infection frequency, with higher malaria transmission linked to lower avidity levels, which may impact immune protection [83].

From a set of thirty-one non-overlapping synthetic peptides covering the full length of the AMA-1 protein, eight HABPs to erythrocytes have been identified, including 4313, 4321, and 4325 (Table 4) [84]. These peptides exhibit binding constants around 103 nM, and all conserved peptides blocked merozoite invasion and development, suggesting their involvement in P. falciparum invasion. Some of these peptides also share homology with the erythrocyte binding domains of MAEBL.

High-binding activity peptides 4313, 4315, 4316, and 4321 are located in domain I, a major target for antibodies capable of inhibiting erythrocyte invasion (Figure 5). Additionally, peptide 4322 overlaps with a known B-cell epitope [85]. Peptide 4322 is one of the most exposed regions, forming a hairpin structure, which aligns with findings supporting its immunogenic role. Notably, peptide 4322 (LWVDGNCEDIPHVNEFSAID) contains an underlined amino acid sequence previously identified as a B-cell epitope capable of inducing antibodies that block parasite growth and incorporated into a multi-component malaria vaccine candidate [85].

According to the crystal structure of AMA-1, HABPs 4313, 4325, and 4337 form a binding region that is surface-exposed and located opposite the region that interacts with the RON complex proteins [91] (PDB 3ZWZ). Peptides 4325, 4315 and 4316 contribute to the binding site of the inhibitory mAb 1F9 on the AMA-1 surface, as observed in the crystal structure of AMA-1 in complex with the Fab fragment 1F9. Indeed, 1F9 binds to the solvent-exposed hydrophobic patch of AMA-1. Mutations in polymorphic AMA-1 residues within the 1F9 epitope disrupt 1F9 binding and significantly reduce the affinity of human antibodies. Remarkable, 1F9 binding to AMA-1 competes with naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immune recognition [89] (PDBs 2Q8A and 2Q8B) (Figure 5).

Rhoptry organelle antigens have been implicated in the invasion process and proposed as vaccine candidates for P. falciparum-induced malaria. Rhoptry-associated protein 1 (RAP-1) is a merozoite antigen located within P. falciparum rhoptries, with highly conserved nucleotide and amino acid sequences. RAP-1 has been detected on the surface of both normal erythrocytes and ring-stage-infected erythrocytes. Together with RSP-2/RAP-2, RAP-1 forms an integral part of the rhoptry complex involved in tagging normal erythrocytes and erythroid precursor cells, a process linked to erythrocyte destruction. Both RAP-1 and RAP-2 bind to the surface of normal erythrocytes [92].

Antibodies against RAP-1 contribute to natural immunity against malaria, alongside responses to MSPs [93]. A significant proportion (86%) of individuals in Papua New Guinea over the age of 30 possess antibodies recognizing both P. falciparum RAP-1 and RAP-2 [94]. The detection of RAP-1 in sera from protected animals, along with the ability of RAP-1-specific antibodies to inhibit parasite invasion, supports RAP-1 as a promising vaccine candidate [95, 96]. Notably, mAbs targeting RAP-1 have successfully inhibited P. falciparum growth in vitro and conferred protective immunity in monkey models [95]. Furthermore, mAbs developed against RSP-2/RAP-2 and RAP-1 have demonstrated the ability to inhibit parasite invasion in vitro [97].

A set of thirty-seven 20-mer and two 21-mer synthetic peptides corresponding to the complete P. falciparum RAP-1 protein sequence led to the identification of HABPs 26188, 26201, 26202, 26203, and 26204 (Table 5). These HABPs exhibit affinity constants (Kd) ranging between 700 nM and 900 nM. Some of these peptides have been shown to inhibit in vitro merozoite invasion and specifically bind to a 72 kDa protein on the RBC membrane. Two erythrocyte-specific RAP-1 binding regions have been identified: one within the cysteine-rich region (residues C461–K540) and another within the N-terminal region of the p67 product (residues T201–Y220). HABPs 26188 and 26203 significantly inhibit merozoite invasion (Figure 6) [98].

Notably, the underlined sequence within peptide 26188 (TLTPLEELYPT) corresponds to an epitope that elicits inhibitory antibodies against parasite infection [98, 100]. Additionally, two mAbs, SP5.2 and SP8.18, generated through immunization with peptides from the synthetic malaria vaccine SPf66, specifically targeting its building block 35.1, have been found to cross-react with a RAP-1-derived sequence. These mAbs specifically recognize the sequence within peptide 26188 (LEELYPTNVNLFNYK). Cross-reactivity with RAP-1 is blocked by peptide 35.1, and the anti-35.1 mAb SP8.18 exhibits parasite growth-inhibitory activity, which is further enhanced in the presence of non-inhibitory anti-RAP-1 mAbs [101]. According to the AlphaFold ID: AF-Q9U414-F1 theoretical structure, HABPs 26201, 26202, 26203, and 26204 adopt helical conformations and form a surface-exposed patch on the protein. In contrast, HABP 26188 forms a prominent loop on the opposite side of this region, which could explain why this sequence is part of the epitope recognized by antibodies generated through immunization with the intact protein. In conclusion, most of the HABP sequences are exposed in this theoretical protein structure (Figure 6).

P. falciparum binds to host cells, potentially involving GBP-130 [102]. GBP-130 has been identified as a GBP that interacts with human erythrocytes [103]. GBP-130 binds weakly to human erythrocytes, with its binding sites located in the repeat region. Antibodies targeting this region have been shown to block merozoite invasion.

GBP-130 is also targeted by antibodies that prevent merozoite dispersal, leading to the formation of immune clusters of merozoites (ICM) and subsequent inhibition of parasite growth [104]. Additionally, GBP-130 generates neutralizing antibodies that protect against merozoite entry [102]. PfGBP-130 is recognized by antibodies from children who have developed resistance to P. falciparum infection but not by those who remain susceptible. Furthermore, mRNA encoding the full ectodomain of PfGBP-130 (amino acids 89–824) has been shown to generate parasite growth-inhibitory antibodies [105]. Immunization of mice with PfGBP-130-A (amino acids 111–374), formulated as a DNA plasmid or lipid-encapsulated mRNA, or mRNA encoding the full ectodomain of PfGBP-130 (amino acids 89–824), induced antibodies that inhibited RBC invasion [16].

A screening using synthetic peptides spanning the full length of this protein identified a HABP at residues 701–720, named 2220, which binds to human erythrocytes with a dissociation constant of 150 nM (Table 6). Notably, HABP 2220 does not bind to SA, as neuraminidase treatment increases its binding to erythrocytes, while trypsin treatment reduces it. HABP 2220 has been shown to inhibit P. falciparum merozoite invasion by 91% and exhibits higher specific binding to human erythrocytes compared to erythrocytes from other species [105]. Interestingly, peptide 2220 belongs to a repeat region that binds glycophorin and elicits antibodies capable of blocking merozoite invasion of erythrocytes [106]. According to the theoretical structure reported by AlphaFold (ID: AF-P02895-F1), this peptide is located within a beta-sheet region exposed on the protein surface (Figure 7).

Pf-SERA5 is a blood-stage antigen expressed during the late trophozoite and schizont stages as a 120 kDa precursor, which is secreted into the PV following the removal of its signal peptide [108]. Pf-SERA5 is cleaved by an essential P. falciparum protease into 47 kDa, 56 kDa, and 18 kDa fragments [109]. The 47 kDa fragment is linked to the 18 kDa fragment via a disulfide bond and localizes in the merozoite surface [56, 110]. The 56 kDa fragment undergoes further cleavage, generating 50 kDa and 6 kDa fragments just before parasite egress [109–111]. Pf-SERA5 plays a pivotal role in merozoite egress from erythrocytes, where its phosphorylation activates its proteolytic activity [112].

Despite its high transcription levels during blood-stage infection, low seropositivity for Pf-SERA5 in endemic areas suggests that it may bind to host proteins to evade immune recognition rather than directly binding to erythrocytes [113]. However, Pf-SERA5 has been shown to bind directly to human erythrocyte membranes and is synthesized as a 111-kDa antigen between 32 h and 36 h into the erythrocytic cycle.

Antibodies recognizing SERA form ICM, preventing merozoite dispersion and inhibiting parasite multiplication [104]. Moreover, antibodies against the N-terminal region of the SERA protein have been shown to inhibit parasite growth and block merozoite release through agglutination [114, 115]. mAbs targeting the SERA protein have also been demonstrated to inhibit in vitro parasite invasion [116]. According to Inselburg et al. [117], antibodies targeting SERA amino acids 24–285 or the N-terminal region (amino acids 24–506) significantly reduce parasitemia in Aotus monkeys.

From 49 non-overlapping peptides spanning the complete sequence of the SERA-5 protein, seven HABPs with strong RBC-binding activity have been identified (Table 7). Six of these peptides are located in conserved regions and have been demonstrated to inhibit in vitro merozoite invasion of erythrocytes. The affinity constants for these high-binding peptides range from 150 nM to 1,100 nM. Notably, enzyme treatment of erythrocytes does not alter the binding specificity of these peptides. Furthermore, all SERA-derived HABPs inhibit both parasite invasion and intraerythrocytic development in vitro [118]. Serum from mice immunized with the FESNSGSLEKKKYVKLPSNG peptide recognized SERA [12]. This peptide shares a sequence (bolded) with high-binding peptide 6725. Additionally, peptides 6727 and 6733 are recognized by antibodies elicited during natural infection in African patients, as well as by antibodies induced through vaccination with the N-terminal recombinant protein [119]. According to the crystal structure of the central protease-like domain of P. falciparum SERA-5 (which contains only HABPs 6746 and 6754), HABP 6746 is a surface-exposed random coil within this domain, while HABP 6754 adopts a loop-helix structure, with the loop region being surface-exposed (Figure 8).

EBA-140, also known as BAEBL or PfEBP-2, is a P. falciparum ligand that specifically binds to GPC on human erythrocytes, distinguishing it from EBA-175, which binds to GPA [121, 122]. The specificity of EBA-140 for GPC represents an adaptive response to host evolution [123]. EBA-140 interacts cooperatively with reticulocyte-binding-like (RBL) proteins, enhancing merozoite invasion efficiency [124]. This ligand plays a critical role in tight junction formation during invasion, serving as a key binding molecule at the erythrocyte interface [125].

Interestingly, both EBA-140 and EBA-175 can also bind to CD44, indicating that while their primary receptors are GPC and GPA, respectively, they may also interact with additional receptors [126]. The Region II (RII) domain of these EBA proteins is essential for receptor binding, with certain polymorphisms affecting affinity but not specificity [122, 127]. Additionally, RII of EBA-140 has been shown to interact with heparin and possibly with cell surface glycosaminoglycans (GAGs). Heparitinase treatment of erythrocytes inhibits RII-EBA-140 binding [128].

Antibodies targeting conserved regions of EBA-140 have demonstrated strong growth-inhibitory activity against diverse P. falciparum strains [129]. High levels of EBA-140-specific antibodies correlate with lower reinfection rates, suggesting their effectiveness in inhibiting merozoite invasion [125]. However, the ability of EBA-140 and other EBA family members to elicit invasion-inhibitory antibodies varies over time [130].

A total of sixty-one synthetic peptides covering the complete EBA-140 sequence have been analyzed, identifying HABPs 26135, 26144, 26147, 26160, 26170, and 26177, which exhibit high erythrocyte-binding activity, with affinity constants ranging from 350 nM to 750 nM (Table 8). These HABPs have conserved amino acid sequences and are capable of inhibiting invasion by 11–69% at 200 μM, with HABPs 26160 and 26170 showing the highest inhibition rates (69 ± 2% and 39 ± 2%, respectively). These HABPs specifically bind to erythrocyte proteins with apparent molecular weights of approximately 25, 52, and 75 kDa [131].

Notably, HABPs 26135, 26144, and 26147 are located in RII of EBA-140: HABP 26135 is within the F1 domain, while HABPs 26144 and 26147 reside within the F2 domain. Immunization with EBA-140 induced antibodies that recognize the amino acid sequences of these peptides. Specifically, mAbs 4E7, 4B7, and 1G6 recognize peptide 26144 and exhibit moderate neutralization activity, while mAbs 9G3 and 1E2, which recognize HABP-26135, display stronger neutralization activity [125].

According to the crystal structure of domain II from EBA-140, HABPs 26135, 26144, and 26147 are predominantly helical. HABP 26135 forms a fully helical structure that is surface-exposed on this domain, while HABPs 26144 and 26147 contain surface-exposed random coil fragments (Figure 9).

EBA-175 is a microneme protein that functions as a ligand for merozoite binding to erythrocytes. It is highly conserved and binds to GPA on the erythrocyte surface in a SA-dependent manner. Antibodies targeting RII of EBA-175, the domain responsible for binding GPA, inhibit merozoite invasion of erythrocytes [133]. Interestingly, antibodies generated against EBA-175 RII potently block the binding of native EBA-175 to erythrocytes [134–136] and even inhibit the invasion of P. falciparum strains that do not require SAs for erythrocyte invasion in vitro [137].

Moreover, conserved regions of EBA-175 stimulate a strong production of neutralizing antibodies, which can inhibit merozoite invasion across various P. falciparum strains [129]. The presence of invasion-inhibitory antibodies against EBA-175 correlates with a reduced risk of malaria symptoms, demonstrating their protective function during infection [137]. However, variability in antibody responses to EBA-175 over time underscores the challenge of achieving long-lasting immunity [130].

Six HABPs from the EBA-175 CAMP strain, 1758, 1779, 1783, 1814, 1815, and 1818, have been identified using synthetic peptides spanning the entire protein (Table 9). These peptides exhibit affinity constants ranging from 60 nM to 180 nM. Some of these high-binding peptides can inhibit in vitro merozoite invasion and block the binding of recombinant RII-EBA to RBCs [138].

In Region I, the conserved high-binding peptide 1758 (KSYGTPDNIDKNMSLIHKHN) has been identified, which binds independently of SA, suggesting an alternative mechanism of interaction. Interestingly, an EBA-175 recombinant fragment (residues 1054–1319) is recognized by antibodies eluted from ICMs, which preferentially recognize functionally important epitopes exposed on live merozoites. This fragment contains HABPs 1814, 1815, and 1818 (Figure 10) [139].

In RII, peptides 1779 and 1783, which are critical for invasion, have been found to effectively inhibit in vitro merozoite invasion. More precisely, they are located within the F2 region (residues 430–747), which belongs to the N-terminal portion of the 65 kDa fragment and binds independently of SA to RBCs [139, 143]. Notably, HABPs 1779 and 1783 effectively inhibited the binding of recombinant RII-EBA (rRII-EBA) to RBCs [139, 143]. Additionally, antibodies eluted from ICMs recognize a recombinant fragment (residues 501–753), which includes peptides 1779 and 1783, but do not bind to the fragment containing peptide CP-1 [139, 143].

Structural analyses indicate that these two peptides form helical structures that are spatially close in the three-dimensional structure of the RII-EBA fragment. They are also part of the binding site for α(2,3)-sialyllactose, with residues 5 and 6 of the sugar moiety interacting with the KQNDK motif, which corresponds to the KKNDK sequence in HABP 1783 [144] (PDB IDs: 1ZRO, 1ZRL).

A study using five mAbs specific for the F1 and F2 regions of EBA-175 find that three mAbs targeting F2 potently block EBA-175 binding to erythrocytes and inhibit merozoite invasion (IC₅₀: 10 μg/mL to 100 μg/mL IgG in growth inhibition assays) [145]. Four of these mAbs recognize conformation-dependent epitopes within F1 or F2. When used in combination, F1- and F2-specific mAbs synergistically block the binding of native EBA-175 to erythrocytes and inhibit parasite invasion in vitro. Among them, mAb R217 is the most potent [145], and it directly blocks GPA engagement (Figure 10) [146].

Structural analyses reveal that R217 binds to a conformational epitope, which includes the F2 β-finger (residues 477–485, revised) and a loop and helix (residues 561–567, revised). These regions contain residues that are proposed to directly interact with glycans from GpA, playing a critical role in erythrocyte binding [144]. Residues K485, N561, and R566 are proposed glycan-binding sites, and mutations in these residues reduce erythrocyte binding [144]. Furthermore, R217 prevents the N-terminal region of peptide 1783 from interacting with the cell receptor, as observed in the native EBA-175 structure (PDB ID: 4QEX) (Figure 10).

Most HABPs discussed in this review are derived from erythrocyte-binding regions that serve as targets for neutralizing antibodies. These peptides correspond to critical contact regions in reported protein complexes and have been identified through multiple techniques, including: phage display libraries, surface plasmon resonance, flow cytometry, fluorescence microscopy, ELISA, and pull-down assays [147–149]. For instance, optical tweezers studies have demonstrated that while PfEBA and PfRH protein families are essential for erythrocyte attachment, MSP-1 plays a relatively minor role, with deletion experiments showing no significant impact on binding strength [147].

The development of P. falciparum merozoite protein-based vaccines faces significant challenges due to: (1) extensive antigenic polymorphism in key targets such as MSP-1, particularly within immunogenic block 2 regions (K1, MAD20, and RO33 alleles); (2) the parasite’s capacity to utilize alternative invasion pathways when primary routes are blocked; and (3) the transient exposure of critical invasion ligands during erythrocyte entry [125]. These biological features, combined with redundant invasion mechanisms, create substantial obstacles for antibody-mediated neutralization. Consequently, effective vaccine strategies must simultaneously target multiple conserved epitopes to overcome these evasion tactics and achieve broad protection or regionally tailored multivalent formulations to overcome parasite diversity and achieve consistent protection across malaria-endemic regions [150].

The rapid evolution of antigens like AMA-1 leads to strain-specific immunity, while the parasite’s ability to switch invasion mechanisms complicates target selection. To address these challenges, multi-antigen vaccines are being developed that combine targets such as AMA-1-RON2 or MSP-1-MSP-3, demonstrating synergistic protective effects in preclinical trials [151]. These approaches aim to broaden protection by targeting multiple invasion pathways while enhancing both humoral and cellular immune responses [152].

For example, the extensive genetic diversity of merozoite antigens, evidenced by high Shannon entropy scores (> 0.2) in immunogenic regions and binding interfaces, poses major challenges for vaccine design. Current vaccines based on reference strains like 3D7 often target rare haplotypes, potentially explaining their limited efficacy against diverse circulating strains. Geographical variation in antigen diversity suggests local adaptation to human and vector populations, as demonstrated by differential antibody recognition patterns between travelers and endemic populations [153]. In the case of MSP-1 vaccine development has been found strain-specific immunity due to antigenic diversity, limited cross-protection, and variable immunogenicity of different vaccine formulations (subunit vs. full-length). While anti-MSP-1 antibodies correlate with protection, their functional quality involving Fc-mediated effector mechanisms remains poorly characterized. Recent advances are addressing these limitations through structural-guided design of full-length MSP-1 immunogens that preserve native conformation, novel adjuvants like TLR4 agonists to enhance antibody effector functions, and multivalent approaches combining MSP-1 with other blood-stage antigens such as RH5. Clinical evaluation using controlled human malaria infection models and systems serology approaches are helping bridge the gap between preclinical promise and clinical efficacy [154].

An effective blood-stage malaria vaccine should primarily elicit high-affinity neutralizing antibodies targeting conserved erythrocyte-binding regions, with sufficient durability to maintain long-term circulating protection. Current research reveals complex antibody persistence patterns, with some responses being short-lived while others demonstrate extended durability. A significant challenge arises from immunodominant epitopes that are often non-protective and localized to polymorphic antigen regions. For instance, endemic populations develop higher-affinity antibodies against AMA-1 compared to MSP-2, particularly favoring the MSP-2-3D7 allele [63]. Surface plasmon resonance studies demonstrate substantial variation in antibody affinities across antigen types, allelic variants, and individual exposure histories. Longitudinal data indicate 67–79% of individuals maintain stable antibody affinities over time, while others exhibit fluctuation patterns that correlate with parasite exposure intensity [18]. Crucially, anti-PfRh2 antibody affinity serves as a strong predictor of invasion inhibition capacity, highlighting the functional significance of antibody quality beyond mere binding metrics.

HABPs represent a promising approach for malaria vaccine development due to their interspecies conservation and demonstrated invasion-inhibitory properties. These synthetic peptides contain predicted B-cell epitopes that are recognized by SERA from Plasmodium-infected mice [155], with similar immunogenicity observed in other animal models and humans (as discussed below). This cross-species reactivity suggests their potential to elicit broad protective immunity. To optimize vaccine efficacy, development efforts should prioritize: (1) multivalent formulations combining liver- and blood-stage antigens; (2) novel adjuvant systems to enhance balanced Th1/Th2 responses; and (3) advanced delivery platforms that address both antigenic diversity and the need for durable immunity in endemic populations.

Effective peptide vaccines must induce coordinated humoral and cellular immunity, as demonstrated by early viral peptide studies showing MHC-I-restricted CD8⁺ T cell protection. The canine parvovirus peptide vaccine achieved T-cell-dependent efficacy comparable to whole-virus vaccines, while cancer immunotherapy research has shown that longer peptides (> 20 amino acids) enhance antigen-presenting cell (APC) processing and CD4⁺ T cell help compared to minimal epitopes [156]. Current strategies focus on overcoming antigenic variation through conserved multi-epitope designs (e.g., P. falciparum MSP-RH5-AMA-1 combinations) and optimizing delivery systems such as nanoparticle formulations and mRNA conjugates to improve immunogenicity.

Phage display technology has transformed malaria antigen discovery, enabling identification of novel erythrocyte-binding proteins [157] and inhibitory peptides targeting AMA-1 [158]. Proteomic approaches have characterized vital merozoite protein complexes, revealing stage-specific interactions between MSP-1 and rhoptry proteins like PfRhopH3 that are shed during invasion [159]. These methods, combined with structural analyses and functional assays, provide a robust toolkit for vaccine antigen selection and validation. The integration of computational predictions with experimental data is significantly advancing the development of next-generation multi-antigen vaccines against P. falciparum.

Recombinant EBA-140 RII and its F2 domain fragment were successfully characterized using anti-myc monoclonal antibodies in immunoblotting assays. Polyclonal rabbit antibodies generated against full-length EBA RII with MPL adjuvant showed cross-reactivity with both baculovirus-expressed RII and bacterially produced F2 fragments. Crucially, these antibodies effectively blocked EBA-140 binding to erythrocytes, confirming this region as a promising vaccine target [160]. Key blood-stage antigens such as AMA-1, MSPs, and RON2 have been prioritized based on their essential roles in invasion and capacity to induce neutralizing antibodies in preclinical studies. Structural analyses have identified conserved epitopes in these antigens that may help overcome strain-specific immunity limitations.

The development of minimal, subunit-based, multi-epitope vaccines against P. falciparum involves a systematic approach focusing on modified HABPs (mHABPs) derived from conserved regions of key invasion proteins (MSP, RH5, and AMA-1). These engineered mHABPs demonstrate: (1) high immunogenicity in immunoassays; (2) protective efficacy in Aotus monkey challenge models; and (3) confirmed MHC-II binding affinity through computational modeling (NetMHCIIpan 4.0), leveraging the 50% allelic similarity between Aotus and human MHC systems [103, 161]. Structural characterization via ¹H-NMR has revealed that targeted residue modifications significantly alter peptide secondary structure, directly modulating immunological presentation [162]. These findings position mHABPs as promising candidates for multi-stage vaccine development, though clinical validation remains essential.

MHC-II-restricted CD4⁺ T cell activation represents a crucial determinant of vaccine efficacy, driving robust adaptive immunity. Compared to traditional whole-pathogen vaccines, peptide-based approaches offer distinct advantages through:

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Precise 20–30 residue epitopes minimizing allergenic potential.

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The rational design process employs a multidisciplinary framework.

This approach combines the precision of structural biology with advanced immunoinformatics to overcome historical challenges in malaria vaccine development, particularly regarding antigenic diversity and population-level coverage. The integration of conserved mHABPs with optimized delivery systems represents a promising path toward developing effective multi-epitope vaccines against P. falciparum.

These innovative approaches, when combined with next-generation adjuvant systems, aim to achieve durable protection against malaria while maintaining the inherent safety and cost-effectiveness of peptide-based platforms. The integration of conserved HABPs with multivalent designs and advanced delivery technologies represents a promising path toward developing effective malaria vaccines capable of addressing the challenges posed by parasite diversity and immune evasion mechanisms.