Background
Eradication of malaria is still among the major priorities in the malaria research agenda as the disease continues to kill thousands of peoples worldwide [
1]. In 2017, the World Health Organization (WHO) estimated 219 million cases of malaria and 435,000 deaths, a figure that was assumed too high [
2]. Effective vaccine development was emergently required to enhance existing malaria control measures because of the moderate spread of drug and insecticide resistance. Recently, some African countries applied one vaccine RTS, S/AS01; however, this vaccine only targets
Plasmodium falciparum [
3‐
5] and might probably be inadequate in areas where a remarkable proportion of patients suffers from
Plasmodium vivax or mixed infections. Moreover, three other parasitic species, namely,
Plasmodium ovale,
Plasmodium malariae, and
Plasmodium knowlesi, can cause malaria infection.
Plasmodium ovale can cause malaria infection in humans but has lower incidence compared with
P. falciparum and
P. vivax [
6,
7]. Some cases of
P. ovale infection can occur in endemic areas of malaria where other species co-exist [
8,
9]; therefore, such evidence should be considered in malaria control strategies.
Plasmodium ovale has been separated into two distinct species (
P. ovale curtisi and
P. ovale wallikeri) in 2010 [
10‐
12]. Similar to other malaria parasites of primates,
Anopheles species are able to transmit
P. ovale; the parasites then invade reticulocytes and begin the erythrocytic cycle that might last approximately 49 h [
7].
Hosts respond to malaria parasites by generating antibodies against parasite-derived antigens, and naturally acquired immunity is developed after repeated exposure to infections [
13]. Notably, antibodies against merozoite antigens play a significant role in conferring immunity against malaria [
14,
15]. Asexual-stage antigens located on apical organelles or on the surfaces of merozoites offer considerable potential as components of vaccines against malaria. Immune responses induced by such vaccines can block the invasion of host erythrocytes through merozoites [
16]. Thus, malaria antigens recognized as candidates for vaccine development are generally grouped as pre-erythrocytic, erythrocytic, and transmission-blocking antigens. Antigens in the asexual stages of malaria parasites represent potential targets for malaria vaccines. Blood-stage vaccines point to target the subsequent disease-causing stage of the
Plasmodium life cycle and may provide protection against disease severity, reducing blood stage asexual parasitaemia and transmission [
17]. Merozoite surface protein 1 (MSP1) and apical membrane antigen (AMA1) are leading blood-stage malaria antigens and considered important vaccine candidates [
15], especially due to their association with protection in pre-clinical studies of mice and non-human primates [
18‐
20]. Protection is associated with the induction of high-titre antibodies. Several studies have investigated immune cross-reactivity of antigens in erythrocytic asexual blood stages. For example, immune sera and monoclonal antibodies against AMA1 manifested only limited cross-reactivity between
P. falciparum and
P. vivax [
21]. Similar studies using sera from people infected with
P. falciparum and
P. vivax showed cross-reactivity of merozoite surface protein 5 (MSP5)-specific antibodies [
22]. Evidence of immune response has been reported for the asexual erythrocytic stages of
P. falciparum and
P. vivax antigens, but there is limited information on antigens of
P. ovale species.
Merozoite surface protein 4 (MSP4) is a glycosylphosphatidylinositol-anchored protein that contains an epidermal growth factor (EGF)-like domain at the carboxyl terminus [
23‐
25]. MSP4 protein has been shown immunogenic in laboratory animals [
25] and crucial for parasite survival [
26]. Murine models of malaria showed that this protein can induce protective immunity against lethal challenge and protect against heterologous challenge by a different species of murine malaria [
27,
28]. Immunization with recombinant
Plasmodium yoelii MSP4/5 in mice, a homolog of MSP4 and its related antigen MSP5, has induced protective immune responses against lethal parasite challenge with
P. yoelii [
27,
28]; protection is enhanced when MSP4/5 is immunized in combination with
P. yoelii MSP1 [
29]. This finding suggested that MSP4 is a potential malaria vaccine candidate, especially in combination with other antigens. MSP4 immunogenicity has been associated with protection in natural infections with
P. falciparum [
30‐
32]. In addition, MSP4 shows a high degree of conservation among
P. falciparum isolates [
16,
33,
34], supporting its potential consideration as a subunit component for malaria vaccine formulations. However, information on
P. ovale MSP4 anti-malarial properties of immunity is scant.
In this study, MSP4 sequences were analysed in clinical isolates of P. o. curtisi and P. o. wallikeri from infected subjects to assess conservation and immunogenicity of P. ovale spp. MSP4. Furthermore, recombinant PoMSP1 and PoAMA1 antigens were tested against anti-PoMSP4 immunoglobulin G (IgG) antibodies to evaluate the specificity of PoMSP4 antigens.
Methods
Malaria samples
P.o. curtisi and
P. o. wallikeri infected blood samples were obtained from local hospitals in Jiangsu Province (China) between 2012 and 2016 from febrile patients who had returned from work in malaria endemic areas of sub-Saharan Africa [
35]. Identification of the isolates was confirmed by polymerase chain reaction (PCR) analysis, and parasite species were distinguished using real-time TaqMan PCR [
36].
PCR amplification and sequencing of pomsp4 genes
Genomic DNA extracted from
P. ovale-infected individual blood samples was previously preserved in our laboratory. A total of 46
P. ovale spp. genomes (
P. o. curtisi, n = 23 and
P. o. wallikeri, n = 23) were randomly selected for amplification. Information on the imported
P. ovale spp. specimens is given in Additional file
1: Table S1. Full nucleotide sequences of
pocmsp4 and
powmsp4 were amplified via PCR using primers designed as follows:
pocmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′),
pocmsp4 reverse (5′-TTA ATT TAT TGA CGC TAA AAT G-3′),
powmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), and
powmsp4 reverse (5′-TTA ATT TAT TGA CGC TAA AAT G-3′).
Pocmsp4 (
Plasmodium Genomics Resource database, PocGH01_04023000) and
powmsp4 (National Centre for Biotechnology Information GenBank database, accession number: LT594508.1) were used as reference gene sequences. Reactions were carried out in a volume of 20 μL, including 1 μL of genomic DNA, 0.8 μL of each primer (10 µM), 7.4 μL of double-distilled water, 0.5 units of DNA polymerase, and 2 mM deoxynucleoside triphosphate within 10 μL of premix (2× Phanta
® Max Master Mix, Vazyme). PCR amplification was performed in a Mastercycler (Eppendorf) as follows: denaturation at 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 51 °C for 30 s, and 72 °C for 30 s; and final extension at 72 °C for 5 min. PCR products were analysed in 1% agarose gel electrophoresis, visualized under an ultraviolet transilluminator (Bio-Rad ChemiDoc MP), and sequenced by Genewiz.
Protein bank
The N-terminal of PoMSP1 [pocmsp1 (GenBank: KC137343) and powmsp1 (GeneBank: KC137341)] and full length of PoAMA1 [pocama1 (PlasmoDB: PocGH01_09039800) and powama1 (GenBank: SBT36045.1)] merozoite surface proteins, which were previously expressed and preserved in our laboratory, were used for specificity tests of PoMSP4 protein-raised antibodies.
Construction of recombinant pomsp4 clones
PoMSP4-predicted open reading frames (ORFs) without the EGF-like domain consisting of 1–119 (PocMSP4) and 1–97 (PowMSP4) amino acids, for which a
P. falciparum ortholog has been reported as highly immunogenic [
30,
31,
37], were selected. Genomic DNA from
P. ovale isolates was used as template for PCR amplification of
pomsp4 ORFs. Primers were as follows:
pocmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′),
pocmsp4 reverse (5′-AGG CGA TGC TAT CGG TTT TG-3′),
powmsp4 forward (5′-ATG AGG GTA CTC CAA TTT TTA TTA C-3′), and
powmsp4 reverse (5′-TGC TAT ACC TAG GAC ATT TTT ACC C-3′). The reaction was performed in a 20 µL volume as described above on a Mastercycler (Eppendorf) with the following temperature profile: initial denaturation at 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. PCR products were also analysed as described above.
The amplified fragments were cloned into the pUC57 vector, sequenced by Genewiz on an ABI 3730xl DNA analyzer (Thermo Fisher Scientific) using universal primers (M13F: 5′-TGT AAA ACG ACG GCC AGT-3′, M13R: 5′-CAG GAA ACA GCT ATG AC-3′), and subcloned into pET32a expression plasmid vector (YouLong Biotech). Recombinant plasmids were transformed into expression host Escherichia coli strain BL21 (DE3) pLysS and sequenced using universal primers T7 through Genewiz.
Protein expression and purification
Escherichia coli BL21 (DE3) pLysS cells containing recombinant plasmid pET32apomsp4 were cultured in Luria–Bertani (supplemented with 50 mg/mL ampicillin) at 37 °C with shaking until optical density (OD) of 600 nm reached 0.6–0.8. The culture was induced with 0.5 mM isopropyl β-d-1 thiogalactopyranoside and allowed to grow for another 3 h at 37 °C. Cells were harvested through centrifugation at 4000×g for 30 min. Protein purification was performed by YouLong Biotech using the following technique. Thawed cells were suspended in purification buffer [50 mM Tris–HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole] and lysed by sonication. The insoluble fraction was separated by centrifugation at 15,000×g for 15 min at 4 °C. The soluble fraction was applied to a column containing 1.0 mL of Ni-nitrilotriacetic acid-agarose (Qiagen) and then washed with 10 mL of purification buffer containing 20 mM imidazole. Recombinant proteins were eluted from the column with purification buffer containing 250 mM imidazole and then exchanged into Tris–HCl storage buffer [50 mM Tris–HCl (pH 8.5), 100 mM NaCl, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 10% (v/v) glycerol] using a 30 kDa ultrafiltration tube (Millipore). Proteins were stored at − 80 °C until use.
Analyses of protein
The concentration of recombinant proteins (rPoMSP4) was determined through the Bradford method using bovine serum albumin (BSA) as standard (Bradford protein assay kit, Solarbio). Purified proteins were analysed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Beyotime Biotech) to assess the expression level and immunoreactivity. The separated proteins from SDS-PAGE were electrophorectically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon) and blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk at 4 °C. The membranes were probed with anti-His antibody (ABclonal) at 1:5000 dilution along with primary antibody dilution buffer (Meilunbio) overnight at 4 °C. Membranes were washed three times with 0.1% TBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for 90 min. Finally, the membranes were analysed with a ChemiDoc MP imaging system (Bio-Rad).
Antibody raising and immunodetection
Six- to eight-week-old female BALB/c mice were used for immunizations as follows. Mice were grouped into the rPoMSP4-immunized (n = 5 per group) and negative control groups (n = 3 per group). Each mouse was intraperitoneally injected with 50 µg of rPocMSP4, rPowMSP4, or PBS, all of which were diluted in PBS with complete Freund’s adjuvant (Sigma). An equal volume of antigen with incomplete Freund’s adjuvant (Sigma) was used for subsequent boosters, which were administered on days 21 and 42 post-immunization intraperitoneally. The control group was administered an equal amount of PBS and adjuvant. Mouse blood samples were collected from the tip of the tail on days 0, 7, 14, 28, 35, and 49. Sera were obtained via centrifugation for 20 min at 2000 × rpm and stored at − 80 °C.
Purified rPoMSP4 was tested against sera from rPocMSP4- or rPowMSP4-infected mice to assess anti-PoMSP4 IgG antibodies through Western blot analysis. During the assays, PVDF membranes were incubated with antisera (1:2000 dilutions) from the rPoMSP4-immunized group or negative control group, followed by HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution.
Specificity of anti-PoMSP4 antibodies
Levels of IgG antibodies targeting PoMSP4 in mouse sera were detected via enzyme-linked immunosorbent assays (ELISA). In brief, 96-well ELISA plates were coated with 50 ng of rPoMSP4 antigen dissolved in coating buffer solution (15 mM sodium carbonate and 35 mM sodium bicarbonate in distilled water) overnight at 4 °C. After three times wash with PBS containing 0.1% of Tween-20 (PBST), the plates were blocked with 1% BSA in PBS and incubated at room temperature for 2 h. Thereafter, individual mouse sera (100 µL) diluted at different dilutions were added on the plate and incubated at room temperature for 2 h. The plates were washed again three times with PBST and HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotech) at 1:5000 dilution and incubated for 1 h 30 min at room temperature. The plates were finally washed three times with PBST and incubated with 3,3′,5,5′-tetramethylbenzidine (Invitrogen) substrate for a few minutes in the dark, and 2 M H2SO4 was added to stop the reaction. The absorbance at OD of 450 nm was measured using a microplate reader (Synergy, BioTeK). Furthermore, anti-PoMSP4 IgG antibodies were tested against rPoMSP1 and rPoAMA1 antigens via ELISA to test for the specificity of antibodies.
Lymphocyte proliferation assays
Lymphocyte proliferation was measured using a cell counting kit-8 (CCK-8, Beyotime Biotech). A certain amount of 5 × 105 cells/well of PoMSP4- and PBS-immunized cells was treated with 10 µL of PocMSP4 (5 µg/mL), 10 µL of PowMSP4 (5 µg/mL), or 10 µL of concanavalin A (Con A, 2 μg/mL), as positive control, in 96-well flat-bottom microtitre plates and then incubated for 72 h at 37 °C with 5% CO2. Thereafter, 10 µL of CCK-8 was added to each well, and the plates were incubated for 2 h at 37 °C and measured at 450 nm using a microplate reader.
Sequence alignment and analysis of data
Full nucleotide sequences of pomsp4 genes from all clinical isolates were translated to the deduced amino acid sequences using the MegAlign module of Lasergene 7 software package (DNAstar) and then aligned with reference sequences to assess conservation within PoMSP4. Amino acid sequences of the segments of PocMSP4 and PowMSP4 isolates were aligned with those of the PocGH01_04023000 and LT594508.1 reference strains, respectively. Sequence alignment for all P. ovale isolates was performed using MEGA v.7.0 software.
Statistical analysis and graphing were conducted using GraphPad Prism software version 5.0 (Graph Pad software, Inc.). SPSS v.16.0 was performed to analyse cross-reaction and antibody responses. Student’s t-test with probability (P) value of < 0.05 indicated a significant difference.
Discussion
The effectiveness of malaria vaccines mostly depends on the high degree of conservation and excellent level of immunogenicity. The present study analysed 46
P. ovale clinical isolates imported from Africa. Amplification and sequencing results showed that the full lengths of
pomsp4 nucleotide and PoMSP4 amino acid sequences (from 23
P. o. curtisi isolates and 23
P. o. wallikeri isolates) were completely conserved. These results were in accordance with other reports that showed a high degree of conservation of
msp4 orthologs in
P. falciparum isolates [
33,
34,
37]. In addition, the high degree of conservation suggested that
pomsp4 can be useful in PCR-based diagnostic testing for
P. ovale spp.
The development of blood-stage vaccines against malaria focuses on surface-exposed proteins of erythrocytes infected with the malaria parasites, which are accessible for antibodies. Thus, humoral immune responses are important against malaria parasites because they play essential roles in protecting against blood-stage malaria [
38,
39]. Antibody responses against blood-stage antigens are known for their importance in protecting against malaria [
40], and this study showed that recombinant PoMSP4 proteins were immunogenic in mice. IgG antibody titres elicited by PocMSP4 and PowMSP4 fragments were substantially higher than those found in the control group (PBS), and sera from immunized mice showed positive reactivity with rPocMSP4 and rPowMSP4 proteins. Cross-reaction between rPocMSP4 and rPowMSP4 was detected through ELISA and Western blot analyses. These results suggested that rPoMSP4 shared similar antigenic determinants that could enable the measurement of species-specific efficacy in vaccine trials. Such high cross-reaction between the two proteins addressed the possibility that both proteins may be used together for vaccine design. However, this result would need future functional studies to address the possibility of vaccine development. Antibody avidity seems to play an important role in protective immunity [
41]. Results of this study indicated that rPocMSP4 and rPowMSP4 induced high-avidity antibodies (PocMSP4: 80.50% and PowMSP4: 92.32%). These findings suggested that MSP4 is a valid model for understanding the immune response in mice. MSP1 and AMA1 proteins are currently being developed as subunits for the development of malaria vaccines [
19,
42,
43]. In the present study, cross-antisera reaction between PoMSP4 and PoAMA1 or PoMSP1 was observed, that is, anti-PoMSP4 antiserum cross-reacted with recombinant PoAMA1 and PoMSP1 in ELISA. This ability of PoMSP4 antigens to generate antibodies with broad immune responses against such important proteins make them attractive candidates to induce a polyclonal immune response.
Lymphocytes play an important role in the immune system because these cells determine the specificity of the immune response to infectious microorganisms and other foreign substances. Lymphocyte proliferation assays are widely used to measure cell-mediated T-cell immunity and responses to specific antigens [
44,
45]. Besides humoral responses, the cellular immune response is also an important indicator for evaluating the immunogenicity of a vaccine candidate. As demonstrated by the results, of this study PocMSP4 and PowMSP4 proteins could induce cellular immune responses in mice, suggesting that humoral and cellular immune responses play crucial roles for PoMSP4 in protection.
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