Genetic diversity and immunogenicity of the merozoite surface protein 1 C-terminal 19-kDa fragment of Plasmodium ovale imported from Africa into China

Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119. A total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012–2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (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 assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays. Sequences of pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, 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 in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively. This study revealed highly conserved gene sequences of pomsp119. In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design.


Background
Malaria is one of the most severe infectious diseases that threaten human health. According to the World Health Organization, there were 229 million clinical cases of malaria and 490,000 deaths from this disease in 2019 [1]. Plasmodium ovale is one of the five species of Plasmodium that regularly infect humans, and accounts for 0.5-10.5% of all malaria cases [2]. It is geographically distributed in sub-Saharan Africa and the Western Pacific Region and is classified into two subspecies: Plasmodium ovale curtisi and Plasmodium ovale wallikeri [2,3]. Plasmodium ovale has a similar morphology and life cycle to Plasmodium vivax [2]. The prevalence of P. ovale malaria is underestimated because of the low density of these parasites in infected subjects, mild clinical symptoms (e.g. fever), and mixed infections with other species of Plasmodium [2,4,5]. However, P. ovale infection can evolve into severe malaria with severe anemia and may become fatal, especially in areas where malaria is endemic [6,7]. Although there have been impressive achievements in research for P. falciparum malaria vaccines [8][9][10], no widely effective vaccine exists for P. ovale malaria. China has eliminated malaria within its borders [11], and was very recently certified as officially free of malaria by the World Health Organization. However, as a consequence of economic growth and deepening of the global trade the number of cases of malaria imported into China, including cases of P. ovale infection, increased in recent years [12][13][14].
During the invasion of human erythrocytes by malaria parasites, the merozoite surface proteins (MSPs) (including MSP1) are exposed to the host immune system [15]. Moreover, antibodies targeting MSP1 have been observed in individuals from malaria-endemic areas and have been shown to confer immunity [16][17][18]. The molecular weight of MSP1 is approximately 200 kDa [19]. The MSP1 protein undergoes two proteolytic cleavage steps during invasion of the erythrocytes. In the first step, MSP1 is cleaved into four polypeptides (with molecular weights of 83 kDa, 30 kDa, 38 kDa, and 42 kDa). Subsequently, the 42-kDa fragment is cleaved into 33-kDa (MSP1 33 ) and  fragments; the latter remains attached to the merozoite surface and enters the erythrocytes [20][21][22]. Several studies have identified limited polymorphism in the C-terminal 19-kDa region of both P. falciparum MSP1 (PfMSP1 19 ) [23][24][25] and P. vivax MSP1 (PvMSP1 19 ) [26,27]. Genetic conservation within vaccine candidate antigens is advantageous for vaccine development as it helps to reduce antigenic escape [27,28]. Moreover, antibodies against PfMSP1 19 can prevent the invasion of merozoites into erythrocytes [29]. Antibodies to PfMSP1 19 have been associated with protection from malaria in pregnant women, infants and older children naturally infected with P. falciparum [30][31][32]. These instructive findings imply that MSP1 19 is a promising candidate antigen for a blood-stage vaccine.
Genetic polymorphisms of MSP1 in clinical P. ovale isolates from Thailand were analyzed and showed low sequence diversity [33]. However, there is a paucity of information on PoMSP1 19 . In the present study, sequences of pomsp1 19 from clinical P. ovale curtisi and P. ovale wallikeri isolates from patients with P. ovale malaria imported into China from Africa were investigated. In addition, the immunogenicity of PoMSP1 19 was assessed in mice, and the levels of immune responses against PoMSP1 19 assessed in serum samples of patients infected with P. ovale.

Collection of samples
Blood samples of febrile patients who had returned from work in malaria-endemic areas of sub-Saharan Africa during the period 2012-2016, and who were confirmed for P. ovale curtisi or P. ovale wallikeri infection by polymerase chain reaction (PCR) tests [13], were obtained from local hospitals in Jiangsu Province, China [34,35]. Genomic DNA was extracted from the blood samples for PCR amplification of pomsp1 19 genes. In addition, serum samples of the P. ovale-infected patients (n = 29) who had returned from Africa and those of healthy individuals (n = 20) from China were also obtained from the local hospitals of Jiangsu Province.

Recombinant expression and purification of proteins
Genes including pocmsp1 19 and powmsp1 19 were subcloned into the pGEX-6p-1 expression vector which contained a glutathione S-transferase (GST)-tag fusion protein. Recombinant pGEX-6p-1 pomsp119 were transferred into Escherichia coli strain BL21 pLysS cells to express PoMSP1 19 proteins. Colonies of recombinants were cultured in Luria Bertani broth supplemented with ampicillin 50 µg/ml by shaking at 250 r.p.m. at 37 °C until the optical density (OD) at 600 nm reached 0.6-0.8. To induce the expression of recombinant PoMSP1 19 (rPoMSP1 19 ) proteins, isopropyl β-D-1thiogalactopyranoside (0.1 mM) (TransGen Biotech, Beijing, China) was added and the culture was allowed to grow for another eight hours. Proteins were purified by Tanlen-bio Scientific (Wuxi, China). The rPoMSP1 19 -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 proteins were electrophoresed on a polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore Sigma). Nonspecific bindings were blocked by incubation with 5% skimmed milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) at room temperature for 2 h. The membranes were incubated with anti-GST rabbit monoclonal antibody (CWBio Biotech) as the primary antibody at 1:2000 dilution overnight at 4 °C, and then washed three times with 0.1% TBST. Horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G (IgG) (CWBio Biotech) was used as the secondary antibody for detection (at 1:5000 dilution for 1 h). Finally, the membranes were visualized using the ChemiDoc MP imaging system (Bio-Rad).

Immunization of mice
Female BALB/c mice (Cavens; Changzhou, China) aged 6-8 weeks were intraperitoneally injected with 50 μg of rPocMSP1 19 -GST, rPowMSP1 19 -GST, GST, or phosphate-buffered saline (PBS) mixed with Freund's complete adjuvant (Sigma, San Francisco, CA) as the primary immunization. The same amount of recombinant protein was mixed with incomplete Freund's adjuvant then injected at days 21 and 42 after the initial injection to boost immunization. Mouse serum samples were collected and stored at − 80 ℃ on days 0, 7, 14, 28, 35, and 49 after the initial injection.
Western blot analysis was performed to detect antibodies directed against rPoMSP1 19 -GST from the sera of immunized mice. Concretely, rPoMSP1 19 -GST and GST proteins were transferred from SDS-PAGE onto PVDF membranes. The membranes were incubated with the sera of mice immunized with rPoMSP1 19 -GST as the primary antibody, with sera of the GST immunized group, or with the sera of those injected with PBS, the negative control group, and then incubated with HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for detection.

Enzyme-linked immunosorbent assay
Levels of IgG antibodies against rPoMSP1 19 -GST and GST in the sera of immunized mice were evaluated through enzyme-linked immunosorbent assay (ELISA) as previously described [35,38]. For the assays, 50 ng of rPoMSP1 19 -GST or GST was 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% (weight/volume) non-fat milk in TBST at room temperature for 2 h. A 100-µl volume of a twofold serial dilution (1:10,000 to 1:5,120,000) of anti-rPoMSP1 19 -GST or anti-GST mouse sera was added to each well and incubated for 1 h 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 1 h at room temperature. Finally, the plates were washed again and incubated with 100 µl of 3,3' ,5,5'-tetramethylbenzidine (Invitrogen) substrate for 8 min in the dark, and the reaction was stopped with 50 µl of 2 M H 2 SO 4 in each well. The absorbance was read at 450 nm. All samples were tested in duplicate and the mean absorbance was calculated.
The affinity test of the anti-rPoMSP1 19 -GST IgG antibody was performed in accordance with the abovementioned ELISA test with the exception that the test here was repeated in a 96-well plate coated with the same recombinant proteins. More specifically, sera were incubated for 90 min at room temperature, following by washing of one of the plates with PBST while the other plate was first incubated with 100 µl of TBST containing 6 M urea for 10 min 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 1 h at room temperature. The reaction was stopped with 50 µl of 2 M H 2 SO 4 in each well and absorbance was measured at 450 nm. The avidity index (AI) was calculated as follows [39]:

Screening of serum samples
Serum samples from 29 cases of P. ovale-infected and 20 healthy individuals were screened by well-type amine arrays. The screening was performed as previously described [41,42]. Briefly, modified glass slides (75 × 25 mm,) were prepared for the protein arrays (Cap-italBio, Beijing, China) and warmed to room temperature before use. Teflon tapes with holes were pasted on array glass slides to ensure integrity of the array. One microliter of rPocMSP1 19 -GST, rPowMSP1 19 -GST and GST solution in PBS (100 ng/µl) was spotted into each well of the arrays and incubated for 2 h at 37 °C. Array slides were washed three times with PBST for 10 min and were blocked with 5% of bovine serum albumin in PBST at 37 °C for 2 h. The arrays were washed again and probed with 1 µl of serum samples at 1:200 dilution. Finally, 1 µl of Alexa Fluor 546 goat anti-human IgG (10 ng/µl; Invitrogen) in PBST was added to the arrays for antibody detection. The intensity of the serological responses was measured by a fluorescent microarray scanner (Capi-talBio). The positive cut-off value was calculated as the mean fluorescence intensity (MFI) of negative controls plus 2 SD. A Mann-Whitney U-test was performed to compare differences in MFI between groups. AI = (OD450 of a sample treated with 6Murea/OD450 of a sample not treated with 6Murea) × 100

Statistical analysis
GraphPad Prism software (version 5.0; GraphPad Software) was used for the statistical analyses and graphing. An unpaired two-tailed Student t-test was used for the comparison of mean values between samples; differences were considered statistically significant when P-values < 0.05.

Expression, purification, and analysis of rPoMSP1 19 -GST proteins
The molecular weight of rPoMSP1 19 -GST was estimated as approximately 35 kDa (including the molecular weights of PocMSP1 19 /PowMSP1 19 and pGEX-6p-1 expression vector containing a GST-tag fusion protein, estimated as 9 and 26 kDa, respectively). Recombinant proteins were efficiently expressed and purified, as shown in Fig. 2b, and presented in SDS-PAGE as a single band of approximately 37 kDa. Western blot analysis confirmed the expression of rPoMSP1 19 -GST proteins (Fig. 2c).

Mice-derived antibodies against rPoMSP1 19 -GST recognized the recombinant proteins
To verify whether anti-rPoMSP1 19 -GST antibodies were produced in the sera of immunized mice, we performed an immunoblot for a specific ~ 37-kDa band of purified rPoMSP1 19 -GST proteins. As expected, sera from mice immunized with rPocMSP1 19 -GST and rPowMSP1 19 -GST detected rPocMSP1 19 -GST and rPowMSP1 19 -GST, respectively (Fig. 3a, b). In addition, sera from mice immunized with GST could also recognize the recombinant proteins (Fig. 3c). No band was found for the recombinant proteins treated with sera from mice immunized with PBS (negative control). These results indicated that rPoMSP1 19 -GST could induce anti-rPoMSP1 19 -GST antibodies in mice.

Levels of immune responses in mice immunized with rPoMSP1 19 -GST
To measure levels of immune responses against rPoMSP1 19 -GST or PBS (negative control) in mice, ELISA was performed using rPoMSP1 19 -GST and GST proteins as the coating antigens. The results showed that both rPocMSP1 19 -GST and rPowMSP1 19 -GST were immunogenic (Fig. 4). The average antibody titers were determined by ELISA at 49 days after the first intraperitoneal injection. The proteins rPocMSP1 19 -GST and rPowMSP1 19 -GST induced comparable antibody responses, with end-point titers ranging from 1:10,000 to 1:2,560,000 (Fig. 4a). After three consecutive immunizations, high IgG antibody responses were induced in mice immunized with rPoMSP1 19 -GST (Fig. 4b, c). The titration curves for GST were indicative of low responses compared to those for rPoMSP1 19 -GST proteins. In addition, in comparison to immunization with rPoMSP1 19 -GST proteins, GST immunization failed to induce high IgG antibody responses in mice, and PBS induced no IgG antibody response at all. These results suggested that immunization of mice with rPoMSP1 19 -GST proteins induced higher levels of IgG antibodies (rPocMSP1 19 -GST, mean avidity, 92.57%, P = 0.0024; rPowMSP1 19 -GST, mean avidity, 85.32%, P = 0.014) than immunization of mice with GST (mean avidity, 37.84%) (Fig. 4d). However, there was no statistically significant difference in immune response between the rPocMSP1 19 -GST and rPowMSP1 19 -GST groups of mice (P > 0.05). Cellular immune responses induced by rPoMSP1 19 -GST were assessed through spleen lymphocyte proliferation assays. The proliferation of splenocytes in vitro under rPocMSP1 19 -GST, rPowMSP1 19 -GST, GST, and ConA (positive control) stimulations was determined. Cell proliferation induced by rPocMSP1 19 -GST was 69.51% and that induced by rPowMSP1 19 -GST was 52.17%. Collectively, rPoMSP1 19 -GST proteins induced a stronger proliferation response of spleen cells (rPocMSP1 19 -GST, P = 0.0471; rPowMSP1 19 -GST, P = 0.026) than ConA. GST failed to induce cell proliferation (P < 0.00001; Fig. 4e).

Cross-reactivity of rPoMSP1 19 -GST proteins with anti-rPoMSP1 19 -GST antibodies
We examined cross-reactivity between rPocMSP1 19 -GST and rPowMSP1 19 -GST using ELISA. rPocMSP1 19 -GST could recognize IgG of sera from mice immunized with rPowMSP1 19 -GST (Fig. 5a, d), with no significant difference observed in cross-reactivity and avidity indices of cross-reaction (P > 0.05). However, anti-GST IgG antibodies showed a significantly lower level of crossreactivity and avidity indices of cross-reaction with rPocMSP1 19 -GST than in reaction of rPocMSP1 19 -GST with anti-rPocMSP1 19 -GST IgG (P < 0.001; Fig. 5a, d). In addition, the cross-reactivity and affinity of anti-rPoc-MSP1 19 -GST antibodies and rPowMSP1 19 -GST showed no significance difference in comparison to the reaction of rPowMSP1 19 -GST with anti-rPowMSP1 19 -GST antibodies (Fig. 5b, e). Unexpectedly, rPocMSP1 19 -GST and rPowMSP1 19 -GST showed a significantly higher crossreactivity and affinity than those of GST with anti- GST   Fig. 4 a-e Immune responses in mice immunized with rPoMSP1 19 -GST. a Sera from immunized mice were multiply diluted (from 1:10,000 to 1:5,120,000), and the data are expressed as average optical density (OD) values. b Increasing trend of immunoglobulin G (IgG) levels detected using ELISA. c Specific immune responses in sera from mice immunized with different proteins confirmed by testing the interaction between antibodies and rPocMSP1 19 -GST, rPowMSP1 19 -GST and GST. The mean OD shows the strength of specific immune responses. d Avidity indices of anti-rPoMSP1 19 -GST IgG determined by ELISA. e Splenocytes from immunized mice were stimulated with recombinant proteins, and concanavalin A (ConA) was used as a positive control. * P < 0.05, ** P < 0.01, *** P < 0.001; not significant (ns) P > 0.05. For other abbreviations, see Figs. 1 and 2 antibodies (P < 0.05) (Fig. 5c, f ). These results suggested that either rPocMSP1 19 -GST or rPowMSP1 19 -GST had the ability to recognize and bind with high-affinity antibodies induced in mice immunized with rPoMSP1 19 -GST.

Humoral immune responses to rPoMSP1 19 -GST in P. ovale infections
Naturally acquired humoral immune responses in patients infected with P. ovale were assessed against rPoMSP1 19 -GST. Protein microarrays were used to screen anti-rPoMSP1 19 -GST antibodies in 29 serum samples from P. ovale-infected individuals and 20 serum samples from healthy individuals (Additional file 3: Table S2). Patients infected with P. ovale showed significantly higher MFI of total IgG against rPocMSP1 19 -GST and rPowMSP1 19 -GST than healthy individuals (P < 0.0001) (Fig. 6a, b). Anti-rPocMSP1 19 -GST antibodies showed sensitivity and specificity of 89.96% (MFI value of 26/29 patient samples > cut-off value of 5752.4) and 75% (MFI value of 15/20 healthy samples < 5752.4), respectively. Anti-rPowMSP1 19 -GST antibodies showed sensitivity and specificity of 89.96% (MFI value of 26/29 patient samples > cut-off value 7092) and 75% (MFI value of 15/20 healthy samples < cut-off value 7092), respectively. However, no statistically significant difference was observed in the MFI of total IgG against GST protein between P. ovaleinfected and healthy individuals (P = 0.5964; Fig. 6c). These results showed that both rPocMSP1 19 -GST and rPowMSP1 19 -GST are targets of signatures of exposure and immunity.

Discussion
Molecules of blood-stage malaria parasites are components of interest for the development of effective malaria vaccines [43]. Several merozoite surface proteins are regarded as promising malaria vaccine candidates because they are targets of host immune selection and play essential roles in erythrocyte invasion [28,44].
In the first rodent model used to study a malaria vaccine, MSP1 was proven to provide protection against challenge infection [45]. That study showed that after MSP1 undergoes proteolytic cleavage only MSP1 19 is carried into the invaded erythrocytes. In a later study, antibodies against MSP1 19 were shown to prevent merozoites from invading erythrocytes in vitro [20]. Antigenic diversity increases the difficulty of developing an effective malaria vaccine, as it helps parasites to escape from host immune responses [46]. In the present study, sequences of pomsp1 19 were completely conserved in 20 P. ovale curtisi and 17 P. ovale wallikeri clinical isolates. These results are consistent with those from previous studies which reported genetic conservation of msp1 19 across malaria parasites [33,36,37]. Immune-mediated selection pressure is an important mechanism as it may lead to antigenic diversity [47]. In the present study, we found that the C-terminal region of PoMSP1 19 is under relatively lower selective pressure than the N-terminal of PoMSP1, which has also been shown to have low genetic diversity [34].
Antibodies play an important role in protection against clinical disease. Earlier studies suggested that antibody responses to PfMSP1 19 might protect children from high levels of blood-stage parasitemia and clinical malaria [32]. In the present study, cross-reaction between rPocMSP1 19 -GST and rPowMSP1 19 -GST was detected. This cross-reaction indicates that rPocMSP1 19 and rPowMSP1 19 share similar antigenic determinants and, therefore, PoMSP1 19 might possess species-specific efficacy as a vaccine candidate. In addition, as rPoMSP1 19 -GST protein contains the GST tag, rPocMSP1 19 -GST and rPowMSP1 19 -GST could be detected by using the sera of mice immunized with GST protein as a non-specific control. Of the five classes of immunoglobulins, IgG is well-known as one that plays a critical role in malaria immunity [48]. A significantly higher level of IgG antibodies was detected in the sera of mice immunized with rPoMSP1 19 -GST proteins than in the sera of the control group of mice immunized with GST protein (Fig. 4), which showed that rPoMSP1 19 -GST induced immune responses in mice. There was no significant difference in cross-reactivity between anti-rPocMSP1 19 -GST IgG and anti-rPowMSP1 19 -GST IgG antibodies, while the affinity index of anti-rPoMSP1 19 -GST IgG was higher than that of the GST group in the cross-reactivity and autoantigen binding test (Fig. 5). These findings imply that antibodies induced in mice immunized with rPoMSP1 19 -GST can bind tightly to antigens and tend to mature. Although anti-GST antibodies were induced in mice immunized with GST protein, affinity maturation was not promoted. High-affinity antibodies are advantageous in a number of biological functions [49]. A better understanding of cellular responses can help in the development of effective blood-stage vaccine candidates for malaria. Lymphocytes play a key role in the immune system and have been shown to be specific for immune responses to infectious microorganisms and other foreign substances [50]. Lymphocyte proliferation assays are widely used to determine T cell immune responsiveness to an antigenic stimulus. The current study showed that rPocMSP1 19 -GST could stimulate the proliferation of T cells (Fig. 4f ). This result is particularly instructive as it proves that rPocMSP1 19 -GST can induce cellular immune responses in mice, and more importantly, proves the immunogenicity of rPoMSP1 19 -GST in mice. A number of studies have reported serologic analyses that investigated IgG antibodies against specific antigens of P. falciparum and P. vivax to assess infection incidence and immunity levels [17,51,52]; however, the question of response specificity has not been fully explored. In this study, we also analyzed humoral immune responses in P. ovale infections. Most of the P. ovale-infected serum samples showed positive antibody responses to rPoMSP1 19 -GST, with a high sensitivity (89.96%) and specificity (75%). The differences in immune responses were not related to the presence of GST protein as no significant difference was observed in the MFI of total IgG against GST protein between P. ovale-infected and healthy individuals (Fig. 6). These data confirmed the antigenicity of rPoMSP1 19 -GST, which may indirectly reflect or contribute to protection against P. ovale malaria infection as humoral immune responses are partly involved in preventing clinical malaria [53]. Seroepidemiological studies have been particularly informative in areas of low transmission where the sensitivity of surveys for prevalence of malaria parasites is impacted by the low number of parasite-infected individuals [54]. Previous studies have used PfMSP1 19 and PvMSP1 19 as serological markers to detect the prevalence of malaria parasites [55,56]. Due to the limited number of serum samples in the present study we were not able to compare differences in the signal of serological responses with respect to other criteria such as gender and parasite density. However, PoMSP1 19 as a biomarker of exposure is worthy of further study.

Conclusions
This study demonstrated that pomsp1 19 sequences were completely conserved in clinical P. ovale curtisi and P. ovale wallikeri isolates. Furthermore, the immunogenicity of rPoMSP1 19 in mice was comprehensively analyzed. The informative results presented here contribute to the knowledge base for the development of a PoMSP1 19 -based vaccine. In addition, the naturally acquired antibody responses against rPoMSP1 19 shown here indicate that further study of PoMSP1 19 could provide new insights for serological research. The crossreactivity of PoMSP1 19 with sera from patients infected with P. falciparum or P. vivax needs to be explored in future studies, as does the clinical protection against malaria afforded by anti-PoMSP1 19 antibodies.