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

ABSTRACT:

Background

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.

Methods

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.

Results

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.

Conclusions

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.

SUBMITTER: Xu Q

PROVIDER: S-EPMC8611641 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThere is an urgent need for an effective vaccine to control and eradicate malaria, one of the most serious global infectious diseases. Plasmodium merozoite surface protein 4 (MSP4) has been listed as a blood-stage subunit vaccine candidate for malaria. Infection with Plasmodium ovale species including P. ovale wallikeri and P. ovale curtisi, 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.MethodsThe msp4 gene was amplified through polymerase chain reaction using genomic DNA extracted from blood samples of 46 patients infected with P. ovale spp. and amplified products were 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, analysed, and immunized in BALB/c mice. The specificity of anti-MSP4-immunoglobulin (Ig) G antibodies was evaluated by Western blot and enzyme-linked immunosorbent assays, and cellular immune responses were analysed via lymphocyte proliferation assays.ResultsFull 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. o. wallikeri isolate. Further, truncated PoMSP4 segments were successfully expressed and purified as ~ 32 kDa proteins. Importantly, high antibody responses with end-point titres ranging from 1:10,000 to 1:2,560,000 in all immunized mouse groups were observed, 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. Additionally, anti-PoMSP4 IgG antibodies showed 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%) during splenocyte proliferation assays.ConclusionFindings from this study suggest conservation in PoMSP4 protein sequences and high immunogenicity was observed in rPoMSP4. Furthermore, induction of immune responses in PocMSP4- and PowMSP4-immunized mice informed that both humoral and cellular immune responses play crucial roles for PoMSP4 in protection.

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