DOI: https://doi.org/10.21203/rs.3.rs-845729/v1
Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa of MSP1 has long been considered as one of the major candidate antigens for malaria blood-stage vaccine in Plasmodium falciparum. However, there are limited information on the C-terminal 19-kDa region of Plasmodium ovale merozoite surface protein 1 (PoMSP119). This study, therefore, aims to analyse genetic diversity and immunogenicity of PoMSP119.
A total of 37 clinical P. ovale isolates including P. ovale curtisi (Poc) and P. ovale wallikeri (Pow) imported from Africa to China and collected between 2012 and 2016 were used. Genomic DNA were used to amplify pocmsp119 and powmsp119 genes by polymerase chain reaction (PCR). Genetic diversity of pomsp119 was analyzed using the MegAlign and GeneDoc v.6 programs. Recombinant PoMSP119-GST proteins were expressed in an Escherichia coli expression system and analyzed by Western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assays (ELISA). In addition, antigen-specific T-cell responses were performed by lymphocyte proliferation assays. A total of 49 serum samples from P. ovale infections and healthy people were used to evaluate natural immune responses through protein microarray assays.
Sequences of pomsp119 were found thoroughly conserved in all clinical isolates. Recombinant PoMSP119 proteins were efficiently expressed and purified as ~ 37 kDa proteins. High antibody responses in immunized mice with rPoMSP119-GST were observed. The rPoMSP119-GST induced high avidity indexes with an average of 92.57% and 85.32% for Poc and Pow, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found during splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for natural immune responses detection in patients infected with P. ovale were 89.96% and 75%, respectively.
This study revealed high conservation in sequences of pomsp119 and high immunogenicity of rPoMSP119. The rPoMSP119 proteins detected humoral immune responses in patients with P. ovale infection. Such informative results advance our understanding of natural immunity to P. ovale infection and contribute to the knowledge base for the development of a PoMSP119-based vaccine.
Malaria is one of the most severe infectious diseases threatening human health. According to the World Health Organization (WHO), malaria caused 229 million clinical cases and 490,000 deaths in 2019 [1]. Plasmodium ovale is one of the five species of Plasmodium that regularly infect humans and, is geographically distributed in sub-Saharan Africa and the Western Pacific [2]. Plasmodium ovale (P. ovale) was classified into two subspecies including P. ovale curtisi and P. ovale wallikeri in 2010 [3]. Plasmodium ovale shares similar morphology and life-cycle with P. vivax [2]. The prevalence of P. ovale malaria is underestimated because of the low density of parasites in infected subjects, mild clinical symptoms, and mixed infections with other species of Plasmodium [2, 4–5]. However, P. ovale infection could evolve in cases of severe malaria, especially in areas where malaria is endemic [6–7]. Although massive research achievements have been obtained for falciparum malaria vaccines [8–10], no widely effective vaccine exists for P. ovale malaria. It is therefore essential to pay more attention to P. ovale in malaria detection and to engage in studies for the development of effective ovale vaccines. Since 2017, there have been no local malaria cases reported for nearly four consecutive years in China, which have reached the WHO’s malaria elimination standard [11]. Meanwhile, with economic growth and deepening of the global trade, the number of imported malaria cases to China has increased in recent years and, cases of P. ovale infection have been proven to be no exception [12–14].
During the invasion of human erythrocytes by malaria parasites, merozoites surface proteins (MSPs) are exposed to the immune system with the coat of surface proteins shedding from the merozoite surface, in which the merozoite surface protein 1 (MSP1) is predominant [15]. Moreover, antibodies targeting MSP1 have been observed in individuals from malaria-endemic areas and have been shown to confer immunity [16–18]. The molecular weight of MSP1 is approximately 200 kDa [19]. The MSP1 protein undertakes two proteolytic cleavage steps during the invasion of erythrocytes. In a first processing step, MSP1 is cleaved into four polypeptides (83 kDa, 30 kDa, 38 kDa, and 42 kDa). Subsequently, the 42 kDa-fragment is cleaved into 33 kDa (MSP133) and 19 kDa (MSP119) fragments which remains attached to the merozoite surface and enters into erythrocyte [20–22]. In particular, the limited sequence polymorphism of the C-terminal 19 kDa-region of Plasmodium falciparum merozoite surface protein 1 (PfMSP119) has been identified in some of the earlier studies [23–25]. Several studies have investigated that sequences polymorphism of the C-terminal 19-kDa region of P. vivax and also found limited genetic diversity [26–27]. Limited genetic polymorphism is advantageous for vaccine development to ensure the inability to escape from the host immune response [27–28]. Moreover, antibodies against P. falciparum MSP119 (PfMSP19) can prevent invasion of merozoites into erythrocytes [29]. Antibodies to PfMSP119 have been linked with protection in pregnant women, infants and older children from clinical malaria cases [30–32]. These finding have an implication that MSP119 is a promising candidate antigen for blood stage vaccine.
Although genetic polymorphisms of MSP1 in Thailand P.ovale isolates has been previously analyzed and showed low level in sequence diversity [33]. Little is known on genetic diversity and immunogenicity of PoMSP119. In this study, sequences of pomsp119 in clinical P. o. curtisi and P. o. wallikeri isolates from cases of ovale malaria patients imported into China from Africa were investigated. In addition, immunogenicity of PoMSP119 was assessed in mice model, as well as levels of immune responses against PoMSP119 in serum samples of patients infected with P. ovale.
Blood samples of P. ovale-infected febrile patients who had returned from work in malaria endemic areas of sub-Saharan Africa between 2012 and 2016, were obtained from local hospitals in Jiangsu Province, China [34–35]. PCR was used to identify the isolates and parasite species were differentiated by real-time Taq Man PCR [13]. Genomic DNA was extracted from the blood samples to serve as template for PCR. For protein microarray assays, serum samples of 29 P. ovale-infected patients who returned from Africa to China between 2012 and 2016 were used. In addition, serum samples of 20 healthy individuals from China were also obtained from local hospitals of Jiangsu Province for use.
PCR amplification and sequencing of the pomsp119
A total number of 37 clinical P. ovale isolates (P. o. curtisi, n = 20 and P. o. wallikeri, n = 17) were randomly selected for PCR amplification (Additional file 1: Table S1). The gene sequences of pocmsp1 (KC137343) and powmsp1 (KC137341) from the GenBank database of National Centre for Biotechnology Information (NCB1) were used as reference sequences [33–34]. The 258 bp- sequences of pomsp119 were identified via matching with similar sequences was as previously reported [36–37], and were amplified by nested PCR. The first round primers were designed as: pomsp119 forward (5′-AGT AAG GAA AAA GAT TTG ACA A -3′) and pomsp119 reversed (5′-AAG TAA GTT AAA TAG GAT GAT-3′). The primers for nested PCR were as follows: pomsp119 forward (5'-ATG GGA TCT AAA CAT AAA TGT − 3') and pomsp119 reversed (5'-GAA AAC ACC TTC GAA GAA TGG − 3'). Both amplification reactions used the same reaction conditions as follows: 98°C for 3 minutes, followed by 35 cycles of 98°C for 10 seconds, 45°C for 1 minute, and 72°C for 1 minute, and final extension at 72°C for 5 minutes. PCR products were electrophoresed on a 1.2 % agarose gel, analyzed under an ultraviolet transilluminator (Bio-Rad ChemiDoc MP), and sequenced by GENEWIZ (Suzhou, China). PCR amplified fragments were cloned into pUC57 vector and sequenced by Genewiz using universal primers (M13F: 5′-TGT AAA ACG ACG GCC AGT-3′, M13R: 5′-CAG GAA ACA GCT ATG AC-3′). To evaluate the diversity within sequences of different isolates, the pocmsp119 and powmsp119 sequences were aligned using GeneDoc v.2.7.0.
The genes of pomsp119 were subcloned into pGEX-6p-1 expression vector which contained a GST-tag fusion protein (Talen-bio Scientific). Escherichia coli strain BL21 pLysS cells were used to express recombinant pGEX-6p-1pomsp119 plasmids, which were cultured in Luria Bertani (LB) containing ampicillin 50 µg/ml by shaking at 250 rpm, 37°C, until optical density (OD) at 600 nm reached 0.6–0.8. To induce the expression of rPoMSP119 proteins, isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.1 mM) (TransGen Biotech, Beijing, China) was added and the culture were allowed to grow for another eight hours. Proteins were purified by Tanlen-bio Scientific (Wuxi, China). The rPoMSP119-GST proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and detected via Western Blot and Coomassie brilliant blue staining (Beyotime Biotech, China). For Western blot analysis, the separated proteins from SDS-PAGE were electrophoresed on polyvinylidene difluoride (PVDF) membrane (Immobilon, Millipore Sigma). Thereafter, nonspecific binding was blocked by incubating with 5% skimmed milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) at room temperature for two hours. The membranes were incubated with anti-GST rabbit monoclonal antibody (CWBio Biotech) as primary antibody at 1:2000 dilution overnight at 4°C, following by three times wash with 0.1% TBST. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (CWBio Biotech) was used as secondary antibody for incubation at 1:5000 dilution for one hour. Finally, the membranes were analyzed with a ChemiDoc MP imaging system (Bio-Rad) for detection.
Female BALB/c mice (Cavens, Changzhou, China) at six to eight-week-old were intraperitoneally injected with 50 µg of rPocMSP119-GST, rPowMSP119-GST, GST, or phosphate-buffered saline (PBS) mixed with Freund's complete adjuvant (Sigma, San Francisco, USA) as primary immunization. The same amount of antigen was mixed with incomplete Freund's adjuvant then injected on days 21 and 42 after the initial injection as booster immunization. Mouse serum samples were collected and stored at -80 ℃ on days 0, 7, 14, 28, 35, and 49 after the initial injection.
To detect antibodies directed against rPoMSP119-GST from immunized mice, Western blot analysis was carried out. Concretely, rPoMSP119–GST and GST proteins were transferred from SDS-PAGE onto PVDF membranes. The membranes were incubated with serum from mice immunized with rPoMSP119-GST as primary antibody, GST immunized group or PBS as negative control group, and then incubated with HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution.
Levels of mice immunoglobulin (Ig)G antibodies against rPoMSP119-GST and GST were investigated through enzyme-linked immunosorbent assays (ELISA) as previously described [35, 38]. For the assays, 50 ng of rPoMSP119-GST and GST were immobilized on 96-well plates overnight at 4°C in coating buffer (15 mM sodium carbonate and 35 mM sodium bicarbonate in distilled water) and blocked with 5% (w/v) non-fat milk in TBST at room temperature for two hours. Thereafter, 100 µl of a twofold serial dilution (1:10000 to 1:5120000) of anti-rPoMSP119-GST and anti-GST mouse sera was added to each well and incubated for one hour at room temperature. The plates were washed three times with PBS containing 0.1% of Tween-20 (PBST) then, incubated with HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotech) at 1:5000 dilution for one hour at room temperature. Finally, the plates were washed again and incubated with 100 µl of 3,3’,5,5’- tetramethylbenzidine (TMB, Invitrogen) substrate for height minutes in the dark and the reaction was stopped with 50 µl 2 M H2SO4 in each well. The absorbance was read at 450 nm. All samples were tested in duplicate and the mean absorbance was calculated.
Affinity test for anti-rPoMSP119-GST IgG antibody was carried out following the ELISA test as described above, except that here, the test was duplicated in 96-well plates coated with same antigens. Then, sera were incubated for 90 minutes at room temperature, following by washing of one of the plates with PBST while the other was incubated with 100 µl of TBST containing 6 M urea for 10 minutes at room temperature before final washing with PBST. Finally, all the plates were incubated with HRP-conjugated goat anti-mouse IgG antibodies at 1:5000 dilution for one hour at room temperature, the reaction was stopped, and the absorbance was measured at 450 nm. Avidity index (AI) was calculated as follows [39]:
AI = (OD450 of a sample treated with 6 M urea/OD450 of a sample not treated with 6 M urea) × 100.
Assays for lymphocyte proliferation were performed using the Cell Counting Kit-8 (CCK-8, Beyotime Biotech) as previously described [35, 40]. Briefly, lymphocytes from mice immunized with rPoMSP119-GST, GST and PBS (5 × 105 cells/well) were treated with 10 µL of rPocMSP119-GST (5 µg/mL), 10 µL of rPowMSP119-GST (5 µg/mL), 10 µL of GST (5 µg/mL) or 10 µL of concanavalin A (Con A, 2 µg/mL) as positive control in 96-well flat bottom microtiter plates 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 at 37°C for two hours. Finally, cells proliferation was measured at 450 nm through a microplate reader.
Sera from 29 cases of P. ovale-infected and 20 healthy individuals were screened by well-type amine arrays. The microarray screening was performed as previously described [41–42]. Briefly, modified glass slides (75 × 25 mm,) were prepared for protein arrays (CapitalBio, Beijing, China) and warmed at room temperature before use. Teflon tapes with holes were pasted on the glass slides to make well-type amine arrays. One microliter of rPocMSP119-GST, rPowMSP119-GST and GST solution in PBS (100 ng/µl) were spotted to each well of the arrays and incubated for two hours at 37°C. This was followed by three times wash with PBST (PBS containing 0.1% Tween 20) for 10 minutes and the arrays were blocked with 5% of BSA in PBST at 37°C for two hours. Thereafter, the arrays were washed again and probed with 1 µl of serum samples from P. ovale-infected patients or healthy individuals at 1:200 dilution. Finally, 1 µl of Alexa Fluor 546 goat anti-human IgG (10 ng/µl, Invitrogen) in PBS-T was added to the arrays for detection. The intensity of serological responses on the arrays was measured in a fluorescent microarray scanner (CapitalBio, Beijing, China). The positive cut-off value was calculated as the mean plus two standard deviations of mean intensity of the negative controls. Mann-Whitney U test was performed to compare differences in mean fluorescence intensity (MFI) between groups.
The GraphPad Prism software version 5.0 was used for statistical analysis and graphing (GraphPad Software Inc.). Unpaired, two-tailed Students t-test was used and differences were considered statistically significant if p-values < 0.05.
Genetic description and PCR analysis of pomsp19
The predicted full length of PocMSP1 (KC137343) and PowMSP1 (KC137341) were 1727 and 1672 amino acids, respectively [34]. Given that MSP1 undergoes proteolytic processing during merozoite invasion process, the schematic diagram of PocMSP1 and PowMSP1 were divided into seven domains as follows: signal peptide (SP), 83 kDa; 30 kDa; 38 kDa; 33 kDa; 19 kDa; and glycolphosphatidylinositol (GPI) (Fig. 1a and 1b). Alignment of the amino acid sequences between PocMSP119 and PowMSP119 showed that only one amino acid was different. Two different amino acids located at positions 1640 and 1585 in PocMSP1 and PowMSP1, respectively (Fig. 1c). Clinical P. o. curtisi and P. o. wallikeri isolates (n = 20 and n = 17, respectively) were used as source of genomic DNA for PCR amplification (Additional file 1: Table S1) and, the 258 bp-size of pomsp119 genes was successfully amplified. The PCR products were tested by electrophoresis and showed a single band on the electropherograms for pomsp119 (Fig. 2a). Alignment of pomsp119 sequences in all isolates showed that no amino acid mutation occurred (Additional file 2: Fig S1). This suggested that pomsp119 was completely conserved across the isolates.
The molecular weight of rPoMSP119-GST was estimated at approximately 35 kDa (including the molecular weight of PocMSP119/PowMSP119 estimated at approximately 9 kDa and that of the pGEX-6p-1 expression vector which contained a GST-tag fusion protein at approximately 26 kDa). Recombinant proteins were productively expressed and purified as shown in Fig. 2b. The rPoMSP19-GST protein presented in SDS-PAGE as a single band of approximately 37 kDa. Western Blot analysis that used anti-GST tag antibody confirmed expression of rPoMSP119-GST (Fig. 2c).
To verify whether mice immunized could generate anti-rPoMSP119-GST antibodies and also recognize the rPoMSP119-GST proteins, we performed an immunoblot for a specific ~ 37 kDa band of purified proteins. Obviously, sera from mice immunized with rPocMSP119-GST and rPowMSP119-GST detected rPocMSP119-GST and rPowMSP119-GST, respectively, as expected (Fig. 3a and 3b). In addition, sera from mice immunized with GST could also recognize the recombinant proteins (Fig. 3c). No band was found the recombinant proteins were treated with sera from mice immunized with PBS (negative control). These results indicated that rPoMSP119-GST could induce anti–rPoMSP119-GST antibodies in mice.
Moreover, in addition to the detection of rPocMSP119-GST, rPowMSP119-GST, and GST proteins by specific antisera (anti-rPocMSP119, anti-rPowMSP119, and anti-GST sera, respectively) from immunized mice, the antisera also cross-reacted with the rPoMSP119 proteins and GST as well (Fig. 3a, 3b, and 3c). The sera from mice immunized with rPocMSP119-GST could recognize rPowMSP119-GST and GST as shown in Fig. 3a. The sera from mice immunized with rPowMSP119-GST could also recognize rPocMSP119-GST and GST (Fig. 3b). All these observations showed that anti–rPocMSP119-GST and anti–rPowMSP119-GST antibodies had the ability to cross-react with PocMSP119 and PowMSP119 antigens.
Immune response in mice immunized with rPoMSP1 19 -GSTTo measure levels of immune responses against rPoMSP119-GST or PBS (negative control) in mice, ELISA were performed using the rPoMSP119-GST and GST proteins as the coating antigens. The results demonstrated that both rPocMSP119-GST and rPowMSP119-GST were immunogenic (Fig. 4). The average of serum antibody titers were determined by ELISA at 49 days after the first intraperitoneal injection. The rPocMSP119-GST and rPowMSP119-GST proteins induced comparable antibody responses with end-point titers ranging from 1:10000 to 1:2560000 (Fig. 4a). After three consecutive immunizations, the high IgG antibody responses were induced in mice immunized with rPoMSP119-GST (Fig. 4b and 4c). Meanwhile, the titration curves of the GST were indicative of lower responses compared to those of the rPoMSP119-GST proteins. In addition, mice immunized with GST or PBS failed to induce high or no IgG antibody responses, respectively, compared to those immunized with the rPoMSP119-GST proteins. This result suggested that mice immunized with rPoMSP119-GST induced high-avidity IgG antibodies (rPocMSP119-GST mean: 92.57%, p = 0.0024; and rPowMSP119-GST mean: 85.32%, p = 0.014) compared to mice immunized with GST (mean: 37.84%) (Fig. 4d). However, there was no statistically significant difference of immune responses between rPocMSP119-GST and rPowMSP119-GST mice groups (p > 0.05).
Cellular immune responses induced by rPoMSP119-GST were assessed through spleen lymphocyte proliferation assays. The effects of the splenocyte proliferative in vitro under rPocMSP119-GST, rPowMSP119-GST, GST, and ConA (positive control) stimulations were determined. The rPocMSP119-GST protein-induced cell proliferation was 69.51% and that induced by the rPowMSP119-GST protein was 52.17%. Collectively, the rPoMSP119-GST proteins induced a stronger proliferation effect on spleen cells (rPocMSP119-GST: p = 0.0471 and rPowMSP119-GST: p = 0.026) when compared to ConA, and more importantly, GST failed to induced cell proliferation (p < 0.00001; Fig. 4e).
Cross‑reactivity of rPoMSP1 19 -GST proteins with anti‑rPoMSP1 19 -GST antibodiesBecause of the similarity in the amino acid sequences of PocMSP119 and PowMSP119, we examined cross-reactivity between rPocMSP119-GST and rPowMSP119-GST through ELISA. The rPocMSP119-GST could recognize and combine IgG of sera from mice immunized with rPowMSP119-GST protein (Fig. 5a and 5d) and, no significant difference was observed in cross-reactivity and avidity indices of cross-reaction (p > 0.05). However, IgG of anti-GST protein showed significantly lower level of cross-reactivity and avidity indices of cross-reaction with rPocMSP119-GST protein compared with reaction of rPocMSP119-GST to IgG of antirPocMSP119-GST (p < 0.001, Fig. 5a and 5d). In addition, cross-reaction and affinity of anti-rPocMSP119-GST and rPowMSP119-GST showed no significance difference compared with reaction of rPowMSP119-GST to anti-rPowMSP119-GST sera (Fig. 5b and 5e). Unexpectedly, rPocMSP119-GST and rPowMSP119-GST showed significantly higher cross-reaction and affinity than GST to anti-GST sera (p < 0.05, Fig. 5c and 5f). These results suggested that either rPocMSP119-GST or rPowMSP119-GST had the ability to recognize and bind with high affinity the antibodies induced by mice immunized with rPoMSP119-GST.
Humoral immune responses to rPoMSP1 19 -GST in Plasmodium ovale infectionsTo further assess humoral immune responses against rPoMSP119-GST in patients with P. ovale, we used protein microarray technology to screen antibodies against rPoMSP119-GST protein. Antibody responses against rPoMSP119-GST were analyzed from 29 patients infected with P. ovale and 20 serum samples from healthy individuals (Additional file 3: Table S2). We observed that P. ovale-infected patients showed significantly higher MFI of total IgG against rPocMSP119-GST and rPowMSP119-GST than that from healthy individuals (Fig. 6a and 6b, p < 0.0001). The prevalence for anti-rPocMSP119-GST antibodies showed a sensitivity of 89.96% (MFI value of 26 out of 29 patient samples > cut-off value 5752.4) and specificity of 75% (MFI value of 15 out of 20 healthy samples < 5752.4). In addition, the prevalence for anti-rPowMSP119-GST antibodies showed a sensitivity of 89.96% (MFI value of 26 out of 29 patient samples > cut-off value 7092) and specificity of 75% (MFI value of 15 out of 20 healthy samples < cut-off value 7092). However, no significance was observed in MFI of total IgG against GST protein between P. ovale-infected or healthy individuals (p = 0.5964, Fig. 6c). These results showed that both rPocMSP119-GST and rPowMSP119-GST are targets of signatures of exposure and immunity.
The complex life cycle of malaria parasites is a major challenge for the development of an effective malaria vaccine. Malaria vaccines targeting attractive blood-stage molecules of the parasites is therefore a research priority [43]. Several merozoite surface proteins have been regarded as promising malaria vaccine candidates given they are targets of host immune system and play essential roles in erythrocyte invasion [28, 44]. Antigenic diversity of malaria vaccine targets increased the complexity of the prediction effect as it helps parasites escape from host immune responses [45]. We blasted amino acid sequences between PocMSP119 and PowMSP119 as template and analyzed sequences of C-terminal of pomsp1 (pomsp119) in clinical samples. Interestingly, we found that sequences of pomsp119 were completely conserved in 20 P. o. curtisi and 17 P. o. wallikeri isolates. From the perspective of reference amino acid sequences from PocMSP119 and PowMSP119, the 20th amino acid of PocMSP119 is Serine, whereas that of PowMSP119 is Proline. These results were consistent with previous reports [33, 36–37]. Immune-mediated selection pressure is an important mechanism that may cause antigenic diversity [46]. This study found the C-terminal region of PoMSP119 under relatively less selective pressure compared with the N-terminal of PoMSP1 which showed limited genetic diversity [34].
Antibody play an important role in clinical protection against blood-stage malaria parasites. Early studies suggested that antibody responses to PfMSP119 might protect children from high levels of blood-stage parasitemia and clinical malaria [32]. In this study, cross-reaction between rPocMSP119-GST and rPowMSP119-GST was detected. In addition, sera from mice immunized with GST protein as nonspecific control, could detect rPocMSP119-GST and rPowMSP119-GST considering that rPoMSP119-GST contained GST tag. These evidences indicated that rPocMSP119 and rPowMSP119 shared similar antigenic determinants and, therefore, PoMSP119 might possess species-specific efficacy for a vaccine candidate. Of the five classes of immunoglobulins, IgG is well-known in playing a critical role in malaria immunity [47]. The significantly higher levels of IgG antibodies response to rPocMSP119-GST and rPowMSP119-GST compared to GST (Fig. 4), showed that rPoMSP119-GST induced immune responses in mice. Avidity indices of anti–rPocMSP119-GST IgG and anti–rPocMSP119-GST IgG were not significantly different in cross-reactivity, whereas avidity indices of rPoMSP119-GST IgG were higher than those of GST group either in crossreactivity or in auto-antigen binding test (Fig. 5). Such findings suggest that antibodies produced by mice immunized with rPoMSP119-GST could bind tightly to antigens and tend to mature. Although antibodies were induced in mice immunized with GST, it could not promote affinity maturation. High-affinity antibodies are advantageous in a number of biological functions [48]. A better understanding of cellular responses can help develop more effective blood-stage malaria vaccine candidates. Lymphocytes play key roles in the immune system given that they have been shown implied in the specificity of immune responses to infectious micro-organisms and other foreign substances [49]. To determine T cell immune responsiveness to an antigenic stimulus, Lymphocyte proliferation assays are widely being used to determine T cell immune responsiveness to an antigenic stimulus. Results of the current study showed that rPocMSP119-GST could stimulate proliferation of T cells compared to GST (Fig. 4f). Such an instructive result proved that rPocMSP119-GST could induce cellular immune responses in mice and more importantly, demonstrated the immunogenicity of rPocMSP119-GST in mice model.
Furthermore, serologic analyses that investigated IgG antibodies against P. falciparum and P. vivax specific antigens have been reported in a number of studies to assess infection incidence and immunity levels [17, 50–51]; however, the question of response specificity has not been fully explored. In this study, we also analyzed humoral immune responses in P. ovale infections and most of the samples with ovale infection showed positive antibody responses to rPoMSP119-GST. The rPoMSP119-GST showed high sensitivity (89.96%) and specificity (75%). This significant difference in immune responses was not related to the presence of GST protein because no significance was observed in MFI of total IgG against GST protein between P. ovale-infected or healthy individuals (Fig. 6). These data confirmed the antigenicity of rPoMSP119-GST, which may indirectly reflect or contribute to protection against ovale malaria infection, because humoral immune responses are partly involved in preventing from clinical malaria [52]. Sero-epidemiological studies have been particularly effective in areas of low transmission where the sensitivity of surveys for prevalence of parasite was impacted by the number of parasite-infected individuals [53]. Previous studies used PfMSP119 and P. vivax MSP119 as serological markers to detect the prevalence of malaria parasites [54–55]. Due to the limitation in number of serum samples, this study was not able to compare differences in signal of serological responses following other criteria such gender and parasite density. This study characterized immune responses to PoMSP119 which has proven to be immunogenic. Investigation on PoMSP119 as biomarker of exposure is worthy of further study.
This study demonstrated that pomsp119 sequences from clinical P. ovale curtisi and P. ovale wallikeri isolates imported from Africa to China were completely conserved. Furthermore, high immunogenicity of rPoMSP119−GST was observed in mice model and P. ovale infections. In addition, cross-reactivity between rPocMSP119-GST and rPowMSP119-GST was observed and indicated that these proteins shared similar antigenic determinants. Collectively, these findings advanced our knowledge of the immunogenicity of MSP119 in ovale infections and more importantly, contributed to the basis for the rational design of PoMSP119-based vaccine.
MSP1: Merozoite surface protein 1; PCR: Polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assays; SP: signal peptide; GPI: glycophosphatidylinositol.
Acknowledgements
The authors thank all study participants, local health officials and doctors for
their participation and support.
Funding
This work was supported by the National Natural Science Foundation of China [81871681, 81971967]; the Jiangsu Provincial Department of Science and Technology [No. BM2018020]; the National First-class Discipline Program Of Food Science and Technology (JUFSTR20180101).
Availability of data and materials:
The data supporting the conclusions of this article are included within the article and its additional file. New sequences identified in this study are deposited in GenBank with the accession numbers MZ766552-MZ766553.
Authors’ contributions
YC and JC conceived and designed the study. YBL, GDZ, JC and QBW collected the samples. QWX, SHL, BY and JCL conducted the laboratory work. QWX and KK wrote the manuscript. YFS reviewed the manuscript. WLZ and MSZ analysed the data. All authors read and approved the final manuscript.
Ethics approval and consent to participate:
This study was approved by the Ethics Committee, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases (JIPD) (IRB00004221), Wuxi, China. Informed consent was obtained from all of the participants, and the animal trial was approved by the Animal Ethics Committee, Jiangnan University (JN. No20180615t0900930[100]).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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