A conserved protein of Babesia microti elicits partial protection against Babesia and Plasmodium infection

Abstract

Background: The protozoan parasite Babesia microti that causes the zoonotic disease babesiosis resides in the erythrocytes of its mammalian host during its life-cycle. No effective vaccines are currently available to prevent Babesia microti infections.

Methods: We previously identified a highly seroactive antigen, named Bm8, as a B. microti conserved erythrocyte membrane-associated antigen, by high-throughput protein chip screening. Bioinformatic and phylogenetic analysis showed that this membrane-associated protein is conserved among apicomplexan hemoprotozoa, such as members of genera Babesia, Plasmodium and Theileria. We obtained the recombinant protein Bm8 (rBm8) by prokaryotic expression and purification.

Results: Immunofluorescence assays confirmed that Bm8 and its Plasmodium homolog were principally localized in the cytoplasm of the parasite. rBm8 protein was specifically recognized by the sera of mice infected with B. microti or P. berghei. Also, mice immunized with Bm8 polypeptide had a decreased parasite burden after B. microti or P. berghei infection.

Conclusions: Passive immunization with Bm8 antisera could protect mice against B. microti or P. berghei infection to a certain extent. These results lead us to hypothesize that the B. microti conserved erythrocyte membrane-associated protein Bm8 could serve as a novel broad-spectrum parasite vaccine candidate since it elicits a protective immune response against Babesiosis and Plasmodium infection.

Keywords: Antigens; Babesia microti; Bioinformatic analysis; Conserved protein; Vaccine.

Fig. 1

Bioinformatics analysis of the conserved membrane-associated antigen, Bm8. a Conserved domain search indicated that the protein has a homolog with a known structure at 274–423; two transmembrane regions were detected by the TMHMM Server(https://dtu.biolib.com/DeepTMHMM). Numbers from 0 to 500 represent the length of the protein sequence. The transmembrane region is shown in blue. b Multiple sequence alignment by ClustalW of the conserved membrane-associated sequences of Babesia microti with other apicomplexan protozoa (Plasmodium berghei, Plasmodium falciparum, Plasmodium vivax, Babesia bovis, Theileria annulata and Toxoplasma gondii). The green box shows the synthetic conservative polypeptide regions (i.e. all amino acids are identical); areas marked in yellow represent incompletely conserved protein regions (i.e. at least 4 of the 6 samples have identical amino acids). The green box shows the conserved polypeptides among Babesia, Plasmodium, Theileria and Toxoplasma with the synthesized peptides of B. microti selected in this study. c Phylogenetic tree of the sequence of the conserved membrane-associated sequences with other apicomplexan protozoa species. The scale bar represents the nucleotide substitutions per position. Branch lengths represent the amount of genetic distance change between the strains. d Three-dimensional structural models of erythrocyte membrane-associated conserved protein of Bm8 predicted by SWISS-MODEL program. The conserved membrane-associated sequences of other apicomplexan protozoa (P. berghei, P. vivax, B. bovis, and T. annulata) were predicted simultaneously. The green regions represent the high antigenic conservative polypeptides applied in this study. Bm8, B. microti protein 8

Fig. 2

Antigenicity of rBm8 and its subcellular localization. a Evaluation of antigenicity by ELISA with pooled sera from 5 mice infected with B. microti or P. berghei, respectively. b Localization of Bm8 in iRBCs of B. microti detected by IFA: i, ii iRBCs were co-incubated with anti rBm8 sera; iii co-incubation of iRBCs infected with B. microti, with pooled sera of normal mice as the control groups. c Localization of Bm8 in iRBCs of P. berghei detected by IFA: i, ii iRBCs were co-incubated with anti-rBm8 sera; iii co-incubation of iRBCs infected with P. berghei with pooled sera of normal mice as the control groups. Scale bars: 5 μm. iRBCs, red blood cells infected with parasites; IFA, immunofluorescent assay; rBm8, recombinant B. microti protein 8

Fig. 3

Active immunization protects against B. microti infection in BALB/c mice induced by the Bm8 polypeptide. a Detection of the copy number of the B. microti gene in BALB/c mice on days 3, 6, 9 and 12 post-infection by qRT-PCR. Asterisks indicate significant difference at **P < 0.01 vs the adjuvant group (t-test). b Parasitemia comparison in BALB/c mice infected with B. microti on days 3, 6, 9 and 12 post-infection detected by microscopy. Asterisk indicates a significant difference at *P < 0.05 vs the adjuvant group (t-test). c Severity of babesiosis in mice receiving active immunization with Bm8 polypeptide versus the control group, on days 3, 6, 9 and 12 post-infection: i–iii changes in Hb level (i), body weight (ii) and body temperature (iii). Bm8, B. microti protein 8; Hb, hemoglobin; mRNA, messenger RNA; qRT-PCR, real-time quantitative PCR

Fig. 4

Comparison of body indicators between immunized mice and control mice infected with P. berghei after active immunization with Bm8 polypeptide or adjuvant, respectively. a Detection of copy number of P. berghei gene in BALB/c mice on days 3, 6, 9 and 12 post infection by qRT-PCR. Asterisk indicates a significant difference at *P < 0.05 vs the adjuvant group (t-test). b Parasitemia comparison in BALB/c mice infected with P. berghei versus control mice on days 3, 6, 9 and 12 post infection detected by microscopy. c Severity of babesiosis in mice receiving active immunization with Bm8 polypeptide versus the control group: i–iii changes in Hb level (i), body weight (ii) and body temperature (iii) in BALB/c mice challenge infection with P. berghei on days 3, 6 and 9 post infection. Asterisk indicates a significant difference at *P < 0.05 vs the adjuvant group (t-test). Bm8, B. microti protein 8; Hb, hemoglobin; mRNA, messenger RNA; qRT-PCR, real-time quantitative PCR

Fig. 5

Passive immunization protects against B. microti infection in BALB/c mice induced by Bm8 antisera. a Detection of the copy number of B. microti gene in BALB/c mice on days 3, 6, 9 and 12 post infection by qRT-PCR. Asterisk indicates significance at *P ＜ 0.05 vs control serum group (t-test). b Parasitemia comparison in BALB/c mice infected with B. microti on days 3, 6, 9 and 12 post infection versus control group detected by microscopy. c Severity of mice babesiosis in mice receiving passive immunization with Bm8 polypeptides antisera versus those receiving control sera: i–iii changes in Hb level (i), body weight (ii) and body temperature (iii) in BALB/c mice challenge infection with B. microti on days 3, 6, 9 and 12 post infection. Asterisks indicate a significant difference vs control serum group at *P < 0.05 and **P < 0.01 (t-test). Bm8, B. microti protein 8; Hb, hemoglobin; qRT-PCR, real-time quantitative PCR; mRNA, messenger RNA; real-time quantitative PCR

Fig. 6

Passive immunization protects against P. berghei infection in BALB/c mice induced by Bm8 antisera. a Detection of the copy number of P. berghei gene in BALB/c mice on days 3, 6, 9 and 12 post infection by qRT-PCR. Asterisk indicates significant difference at *P < 0.05 vs adjuvant group (t-test). b Parasitemia comparison in BALB/c mice infected with P. berghei on days 3, 6 and 9 post infection versus control group detected by microscopy. Asterisk indicates significant difference at *P < 0.05 vs adjuvant group (t-test). c Severity of mice babesiosis in mice passive immunization with Bm8 polypeptides antisera group verus those receiving control sera: i, ii, iii changes in Hb level (i), body weight (ii) and body temperature (iii) in BALB/c mice challenge infection with P. berghei days 3, 6 and 9 post infection. a, b, c (i). Asterisks indicate a significant difference at ***P < 0.001 (t-test). Bm8, B. microti protein 8; Hb, hemoglobin; qRT-PCR, real-time quantitative PCR; mRNA, messenger RNA; real-time quantitative PCR