Immunogenicity analysis of conserved fragments in Plasmodium ovale merozoite surface protein 4

Background : An effective vaccine to control and eradicate malaria, one of the most serious global infectious diseases, is an urgent need. Plasmodium merozoite surface protein 4 (MSP4) has been listed as a blood-stage subunit vaccine candidate for malaria. Plasmodium ovale infection is also a source of malaria burden in tropical regions where it is sometimes mixed with other Plasmodium species. However, little is known about P. ovale MSP4. Methods : The msp4 gene was amplified through polymerase chain reaction using genomic DNA extracted from blood samples of 46 patients infected with P. ovale and then sequenced. Open reading frames predicted as immunogenic peptides consisting of 119 and 97 amino acids of P. ovale curtisi MSP4 (PocMSP4) and P. ovale wallikeri MSP4 (PowMSP4), respectively, were selected for protein expression. Recombinant proteins (rPoMSP4) were expressed in Escherichia coli , purified, analyzed, and immunized in BALB/c mice. The specificity of anti-MSP4-immunoglubulin (Ig) G antibodies was evaluated by Western blot and enzyme-linked immunosorbent assays, and cellular immune responses were analyzed via lymphocyte proliferation assays. Results : Full peptide sequences of PocMSP4 and PowMSP4 were completely conserved in all clinical isolates, except in the epidermal growth factor-like domain at the carboxyl terminus where only one mutation was observed in one P. ovale wallikeri isolate. Furthermore, we successfully expressed the truncated pocmsp4 and powmsp4 and purified as ~32 kDa proteins. Our results showed that PocMSP4 and PowMSP4 induced high antibody responses with end-point titers ranging from 1:2,560,000 to 1:10,000 in all immunized mouse groups and with high IgG avidity to PocMSP4 (80.5%) and PowMSP4 (92.3%). Furthermore, rPocMSP4 and rPowMSP4 cross-reacted with anti-PowMSP4-specific or anti-PocMSP4-specific antibodies. broad immuno-specificity in reacting against rPoMSP1 and rPoAMA1. Lastly, PocMSP4- and PowMSP4-immunized mice induced cellular immune responses with PocMSP4 (36%) and PowMSP4 cells (15.8%) as observed through splenocyte proliferation assays. Conclusion : Our study suggested conservation in PoMSP4 protein sequences with high immunogenicity. Furthermore, PocMSP4- and PowMSP4-immunized mice induced cellular immune responses, suggesting that both humoral and cellular immune responses play crucial roles in the protection against any antigen. amino Genomic P. ovale isolates PCR amplification of pomsp4 ORFs. Primers 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 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 analyzed as described


Conclusion:
Our study suggested conservation in PoMSP4 protein sequences with high immunogenicity. Furthermore, PocMSP4-and PowMSP4-immunized mice induced cellular immune responses, suggesting that both humoral and cellular immune responses play crucial roles in the protection against any antigen.

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, 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][4][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 malaria, and Plasmodium knowlesi, can cause malaria infection. P. 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. This species was first distinguished as a distinct species (P. ovale curtisi and P. ovale wallikeri) in 2010 [10][11][12]. Similar to other malaria parasites of primates, this parasite can only be transmitted via the bites of infected Anopheles mosquitoes, which invade reticulocytes and begin the erythrocytic cycle that might last approximately 49 h [13].
Hosts respond to malaria parasites by generating antibodies against parasite-derived antigens, and naturally acquired immunity is developed after repeated exposure to infections [14]. Notably, antibodies against merozoite antigens play a significant role in conferring immunity against malaria [15,16]. 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 [17]. 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 urgent 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 parasitemia and transmission [18]. Merozoite surface protein 1 (MSP1) and apical membrane antigen (AMA1) are leading blood-stage malaria antigens and considered important vaccine candidates [16], especially due to their association with protection in pre-clinical studies of mice and non-human primates [19][20][21]. Protection is associated with the induction of high-titer antibodies. Several studies have investigated immune crossreactivity 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 [ 22]. Similar studies using sera from people infected with P. falciparum and P. vivax showed the cross-reactivity of merozoite surface protein 5 (MSP5)specific antibodies [23]. Evidence of cross-reactivity has been reported for the asexual erythrocytic stages of P. falciparum and P. vivax antigens, but cross-reactivity of P. ovale antigen has limited information. In this study, MSP4 sequences were analyzed in clinical isolates of P. ovale curtisi and P. ovale wallikeri species from infected subjects to assess the conservation and immunogenicity of P. ovale MSP4. Furthermore, recombinant PoMSP1 and PoAMA1 antigens were tested against anti-PoMSP4 immunoglobulin G (IgG) antibodies to evaluate the specificity of PoMSP4 antigens.

Malaria samples
P. ovale curtisi-and P. ovale wallikeri-infected blood samples were obtained from local hospitals in Jiangsu Province (China) between 2012 and 2016 from febrile patients who had recently returned from work in malaria endemic areas of sub-Saharan Africa [38].
Identification of the isolates was confirmed by polymerase chain reaction (PCR) analysis, and parasite species were distinguished using real-time TaqMan PCR [39].

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 genomes (P. ovale curtisi, n=23 and P. ovale wallikeri, n=23) were randomly selected for amplification. Information on the imported P. ovale specimens is given in ( Additional file 1: Table S1). Full nucleotide sequences of pocmsp4 and powmsp4 were amplified via PCR using primers designed as

Protein bank
The N-terminal of PoMSP1 and full length of PoAMA1 merozoite surface proteins, which were previously expressed and preserved in our laboratory, were used for specificity tests of PoMSP4 protein-raised antibodies.

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 analyzed by 12% sodium dodecyl sulfatepolyacrylamide 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 analyzed 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 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.

Anti-PoMSP4 antibody specificity
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 washing three times 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 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 H 2 SO 4 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.
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.

Analysis of amplified pomsp4 genes revealed the conservation of amino acids in isolate sequences
The full length of pomsp4 genes was successfully amplified from the genomic DNA of 23 P.
ovale curtisi-and 23 P. ovale wallikeri-infected individuals. A phylogenetic tree was reconstructed through the neighbor-joining method based on human, non-human primate, murine, and avian malaria species to infer genetic relationships of pocmsp4 and powmsp4.
Phylogenetic analysis of gene sequences of pocmsp4 and powmsp4 revealed 99% similarity (Fig. 1). The alignments of PoMSP4 amino acid sequences from both subspecies showed that no amino acid mutation occurred among all isolate samples from 17 different countries in Sub-Saharan Africa (Additional file 2: Figure S1), suggesting the complete conservation of PoMSP4, which was consistent with previous findings, especially in P.

Expression, purification, and analysis of recombinant PoMSP4 proteins
The selected pomsp4 fragments were successfully amplified in isolates through PCR, generating single PCR products with the expected size of 360 bp for pocmsp4 and 294 bp for powmsp4. Direct sequencing of purified PCR fragments showed no superimposed signal on the electropherograms for pomsp4 (Fig. 2a).
The molecular weight of expressed PocMSP4 and PowMSP4 ORFs was estimated to be approximately 32 kDa, which was consistent with that obtained in purified proteins as shown in SDS-PAGE analysis with Coomassie blue staining (Fig.2b). Immunoblotting analysis that used anti-His tag antibody confirmed that rPoMSP4 was expressed (Fig. 2c).

Mice-derived antibodies against PoMSP4 recognized the recombinant proteins
We elaborated an immunoblot specific to 32 kDa bands corresponding to the sizes of the two rPoMSP4 proteins to determine whether mice anti-rPoMSP4 antibodies can identify rPoMSP4 (Fig. 3a). The antibody responses of the sera of immunized mice were potent against rPoMSP4, suggesting that PoMSP4 could induce immune responses and was immunogenic in mice. As a negative control, no reactivity was noted in the normal and PBS-immunized mice (Fig. 3b). Furthermore, cross-reactivity was tested using anti-rPoMSP4-immune mouse sera with each of the rPoMSP4 proteins (Fig. 3c). These results showed that mice anti-rPocMSP4 and anti-rPowMSP4 antibodies could recognize rPowMSP4 and rPocMSP4 antigens, respectively.

Immune responses against rPoMSP4 in mice
Antibody responses from all groups of mice were measured through ELISA using the same recombinant proteins constructed as the solid phase coating antigen. ELISA results showed that antibodies against PoMSP4 in mice were detected 1 week after the primary booster. A high response was detected at day 14 post-immunization and continued to rise until day 28 post-immunization (Fig. 4a). Mean serum antibody titers were evaluated through ELISA at 49 days after the first immunization. Similarly, PocMSP4 and PowMSP4 induced a high antibody response with end-point titers ranging from 1:2,560,000 to 1:10,000 (Fig. 4b). Anti-rPocMSP4 and anti-rPowMSP4 antibodies in all immunized mouse groups exhibited high avidity indexes (AIs), and the average AIs of anti-rPocMSP4 and anti-rPowMSP4 IgGs were 80.5% and 92.3%, respectively (Fig. 4c).

Cross-reactivity of rPoMSP4 proteins with anti-rPoMSP4 antibodies
ELISA was used to investigate whether the antibodies from rPocMSP4-immunized mice can combine with PowMSP4 protein and whether the antibody of rPowMSP4-immunized mice can combine with PocMSP4 protein. The results showed that mouse anti-rPocMSP4 or anti-rPowMSP4 antibodies could recognize rPocMSP4 and rPowMSP4, with no significant difference (p=0.5758; Fig. 4d), suggesting the high specificity of both anti-PoMSP4 antibodies.

Lymphocyte proliferation assays
PocMSP4 and PowMSP4 protein antigens were able to induce high antibody levels in mice.
Therefore, we used spleen lymphocyte proliferation assays to test whether these antigens can induce cellular immune responses. The proliferation assay was performed to assess the influence of the splenocyte proliferative response to rPoMSP4 proteins in vitro under rPocMSP4, rPowMSP4, and ConA (positive control) stimulations. Results showed that rPoMSP4-induced cell proliferation was estimated at 36% for PocMSP4 and 15.8% for rPowMSP4 (Fig. 4e).

Specificity of PoMSP4-derived antisera
The specificity and reactivity of antibodies raised against PoMSP4 proteins were evaluated by testing rPoAMA1 or rPoMSP1 proteins with mouse anti-rPoMSP4 sera from each immunized group using ELISA. All sera from mice immunized with rPoMSP4 recognized the antigen when the ELISA plate was sensitized with rPoAMA1 proteins (Figs. 5a and 5b), and no significant difference in cross-reactivity was observed (p>0.05). However, significant differences in cross-reaction were detected between rPocMSP1 to anti-rPocMSP4 sera and rPocMSP4 to anti-rPocMSP4 sera (p<0.0001) whereas no significant difference was observed between rPocMSP4 and PowMSP1 to anti-rPocMSP4 sera (p=0.9063; Fig. 5c). In addition, cross-reactivity was observed between rPocMSP1 to anti-rPowMSP4 sera and rPowMSP4 to anti-rPowMSP4 sera (p<0.0001) and between rPowMSP4 and PowMSP1 to anti-rPowMSP4 sera (p<0.0430; Fig. 5d). In summary, anti-rPoMSP4 sera cross-reacted against rPocAMA1 or rPowAMA1, and rPowMSP1 proteins by contrast, they showed low reactivity with rPocMSP1 proteins. This result suggested that rPoMSP4 antigens were able to generate antibodies with broad immune responses against such important proteins (known to be associated with red blood cell invasion [16]), making them attractive candidates to induce a polyclonal immune response.

Discussion
The effectiveness of malaria vaccines mostly depends on the high degree of conservation and excellent level of immunogenicity. Here, we analyzed 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. ovale curtisi isolates and 23 P. ovale 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 [37,41,42]. In addition, the high degree of conservation suggested that pomsp4 can be useful in PCR-based diagnostic testing for P. ovale.
The development of blood-stage vaccines against malaria focuses on infected erythrocytes or proteins, which are accessible for antibodies, expressed on the surface of the parasites.
Thus, humoral immune responses are important against malaria parasites because they play essential roles in protecting against blood-stage malaria [43]. Antibody responses against blood-stage antigens are known for their importance in protecting against malaria [44], and our results showed that recombinant PoMSP4 proteins were immunogenic in mice. IgG antibody titers 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. This high cross-reaction between the two proteins addressed the possibility that both proteins may be used

Conclusion
Immunization with conserved rPoMSP4 protein fragments resulted in a remarkable humoral immune response. Cross-reaction between rPocMSP4 and rPowMSP4 proteins or between rPoAMA1 and rPoMSP1 proteins to anti-rPoMSP4 was detected, suggesting that these proteins shared similar antigenic determinants. PoMSP4 could induce cellular immune responses, suggesting that these fragments of P. ovale may be more widely evaluated as potential vaccine candidates, although further study needs be carried out to validate its potential and limitations.

Availability of data and materials
The data supporting the conclusions of this article are included within the article and its additional files.

Competing interests
The authors declare that they have no competing interests.

Acknowledgment
We would like to acknowledge all study participants and we are particularly grateful to all patients who donated their blood samples.    Immune response in mice immunized with rPoMSP4. a IgG was detected at day 7 post-immunization, and high response was detected at day 14 post-immunization and continued to rise until day 28 post-immunization. b Immunized mice sera were diluted from 1:10,000 to 1:2,560,000. Data are presented as the geometric mean OD obtained at different concentrations. Numbers on the X-axis indicate the dilutions tested. Antigen specificity was confirmed using pre-immune serum samples as control. c Antibody avidity of rPoMSP4 was verified through ELISA.  Specificity and reactivity of the sera of rPoMSP4-immunized mice using PoAMA1

Authors' information
and PoMSP1 proteins. The reactivity of sera from mice immunized with rPoMSP4 was evaluated using rPoAMA1 and rPoMSP1 proteins. a, b. Cross-reaction of rPoAMA1 proteins with antisera from mice immunized with rPoMSP4. No significant differences were observed between PocMSP4, PocAMA1, or PowAMA1 and the anti-PocMSP4 antisera and between PowMSP4, PocAMA1, or PowAMA1 and the anti-PowMSP4 antisera (p>0.05). c, d Cross-reaction of rPoMSP1 proteins with antisera from mice immunized with rPoMSP4. Significant differences between