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Background <p>The glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) protein is integral to the invasion of erythrocytes by <i>Plasmodium</i> species, with well-documented roles in the merozoite binding of both <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i>. In the malaria endemic areas infections caused by <i>Plasmodium ovale</i> spp. can escalate to severe malaria, characterized by life-threatening anaemia. Nevertheless, <i>P. ovale</i> spp. have been frequently neglected in the study, and the species-specific <i>P. ovale wallikeri</i> (Pow) GAMA (PowGAMA) protein remains uncharacterized. This study investigates the genetic diversity of <i>P. ovale</i> spp. imported from Africa and evaluates the immunogenicity of recombinant PowGAMA (rPowGAMA).</p> Methods <p>The study conducted an analysis of 56 <i>P. ovale</i> isolates, comprising 38 <i>P. ovale wallikeri</i> and 18 <i>P. ovale curtisi</i>, collected from Africa between 2012 and 2016. The <i>powgama</i> gene was subjected to polymerase chain reaction (PCR) amplification, sequencing, and genetic diversity analysis utilizing DnaSP v6 software. The rPowGAMA protein was expressed in <i>Escherichia coli</i>, subsequently purified, and employed to immunize BALB/c mice. The antigenicity of rPowGAMA was evaluated through immunoblotting, while immune responses were assessed utilizing enzyme-linked immunosorbent assay (ELISA) and splenocyte proliferation assays. Additionally, cytokine production was quantified through ELISA. Seroreactivity was determined via protein microarray analysis using sera from 30 individuals infected with <i>P. ovale</i> and 20 healthy controls.</p> Results <p>A genetic analysis of 38 <i>P. ovale wallikeri</i> isolates revealed 10 single nucleotide polymorphism (SNP) sites within the <i>powgama</i> gene, with nucleotide diversity (π) ranging from 0 to 0.00435. The isolates were categorized into 13 distinct haplotypes (<i>Hd</i> = 0.812), indicating moderate genetic diversity. Notably, conserved regions approximately 2.3&#xa0;kb in length (π ≈ 0) were identified, particularly within functional domains. Immunization with the rPowGAMA protein, spanning amino acids 276 to 646, elicited a robust Th1-polarized immune response in mice. This response was characterized by notably high production of IFN-γ and significant lymphocyte proliferation, culminating in an endpoint antibody titer of 1:5,120,000 and an avidity of 78.37%. The protein demonstrated 83.3% sensitivity and 70% specificity in detecting natural infections.</p> Conclusion <p>This study illustrates the significant conservation of <i>powgama</i> gene sequences among <i>P. ovale</i> isolates. The rPowGAMA expressed from this conserved sequence demonstrates substantial immunogenicity, eliciting both high-avidity antibodies and robust T-cell responses. These findings indicate the potential of rPowGAMA to elicit dual protective immunity.</p>

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Immunogenicity and gene polymorphism analysis of Plasmodium ovale wallikeri glycosylphosphatidylinositol-anchored micronemal antigen (GAMA)

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月4日

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  • Xin Ling,
  • Feng Chen,
  • Sui Xu,
  • Yuan Tan,
  • Yifan Sun,
  • Su Han,
  • Qiubo Wang,
  • Yang Cheng

摘要

Background

The glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) protein is integral to the invasion of erythrocytes by Plasmodium species, with well-documented roles in the merozoite binding of both Plasmodium falciparum and Plasmodium vivax. In the malaria endemic areas infections caused by Plasmodium ovale spp. can escalate to severe malaria, characterized by life-threatening anaemia. Nevertheless, P. ovale spp. have been frequently neglected in the study, and the species-specific P. ovale wallikeri (Pow) GAMA (PowGAMA) protein remains uncharacterized. This study investigates the genetic diversity of P. ovale spp. imported from Africa and evaluates the immunogenicity of recombinant PowGAMA (rPowGAMA).

Methods

The study conducted an analysis of 56 P. ovale isolates, comprising 38 P. ovale wallikeri and 18 P. ovale curtisi, collected from Africa between 2012 and 2016. The powgama gene was subjected to polymerase chain reaction (PCR) amplification, sequencing, and genetic diversity analysis utilizing DnaSP v6 software. The rPowGAMA protein was expressed in Escherichia coli, subsequently purified, and employed to immunize BALB/c mice. The antigenicity of rPowGAMA was evaluated through immunoblotting, while immune responses were assessed utilizing enzyme-linked immunosorbent assay (ELISA) and splenocyte proliferation assays. Additionally, cytokine production was quantified through ELISA. Seroreactivity was determined via protein microarray analysis using sera from 30 individuals infected with P. ovale and 20 healthy controls.

Results

A genetic analysis of 38 P. ovale wallikeri isolates revealed 10 single nucleotide polymorphism (SNP) sites within the powgama gene, with nucleotide diversity (π) ranging from 0 to 0.00435. The isolates were categorized into 13 distinct haplotypes (Hd = 0.812), indicating moderate genetic diversity. Notably, conserved regions approximately 2.3 kb in length (π ≈ 0) were identified, particularly within functional domains. Immunization with the rPowGAMA protein, spanning amino acids 276 to 646, elicited a robust Th1-polarized immune response in mice. This response was characterized by notably high production of IFN-γ and significant lymphocyte proliferation, culminating in an endpoint antibody titer of 1:5,120,000 and an avidity of 78.37%. The protein demonstrated 83.3% sensitivity and 70% specificity in detecting natural infections.

Conclusion

This study illustrates the significant conservation of powgama gene sequences among P. ovale isolates. The rPowGAMA expressed from this conserved sequence demonstrates substantial immunogenicity, eliciting both high-avidity antibodies and robust T-cell responses. These findings indicate the potential of rPowGAMA to elicit dual protective immunity.