Establishment and validation of a mouse model of infection with Plasmodium berghei chimerically expressing P. falciparum circumsporozoite protein

doi:10.12140/j.issn.1000-7423.2026.01.009

Abstract

Abstract:

Objective To establish a mouse model of infection with Plasmodium berghei (Pb) chimerically expressing P. falciparum circumsporozoite protein (PfCSP), and to evaluate the value of the model in evaluation of vaccine-induced immunoprotective effectiveness, so as to provide a reliable proof-of-concept platform for advancing CSP-based malaria vaccines. Methods PCR assay was employed to characterize and verify the transgenic Pb strain chimerically expressing the Pfcsp gene (PfCSP-Pb strain). The proliferation, morphology, and parasitemia were compared between the PfCSP-Pb and wild-type Pb strains during the erythrocytic stage through in vitro culture for 1 to 5 days and in vivo passage in mice for 1 to 6 days. ICR mice that had been infected with either the PfCSP-Pb strain or the wild-type Pb strain for 3 to 4 days were fed to Anopheles stephensi mosquitoes. The oocysts were observed in mosquito midguts using mercurochrome staining 14 days post-infection with An. stephensi, and green fluorescent protein (GFP) fluorescence signals of sporozoites were detected in mosquito midguts and salivary glands using fluorescence microscopy. Salivary gland sporozoites from Anopheles mosquitoes infected with either the PfCSP-Pb strain or the wild-type Pb strain were collected and intravenously injected into ICR mice via tail vein. Mice were sacrificed 42 hours post-infection, and mouse liver tissues were sampled. Total RNA was extracted from mouse liver tissues, and the relative expression of sporozoite 18S rRNA was detected in mice during the liver stage using real-time quantitative real-time reverse transcription PCR (qRT-PCR) assay. Following passive immunization with the PfCSP-specific mAB317 monoclonal antibody (100 μg) in mice, mice were infected with salivary gland sporozoites from Anopheles mosquitoes infected with either the PfCSP-Pb strain or the wild-type Pb strain. Mouse liver parasite load was detected using qRT-PCR assay to verify the blocking effect of the mAB317 monoclonal antibody on the infection of hepatocytes with sporozoites of the PfCSP-Pb strain. Results PCR assay confirmed the stable integration and expression of the pf-csp gene and the gfp-luciferase reporter gene by the PfCSP-Pb strain. No significant differences were observed in proliferation rates between the PfCSP-Pb strain and the wild-type Pb strain either in in vitro (F = 0.86, P > 0.05) or in vivo (F = 2.18, P > 0.05) during the erythrocytic stage. The PfCSP-Pb strain exhibited identical morphology to the wild-type strain across the ring, trophozoite, and schizont stages, with stable GFP fluorescence protein expression observed in the cytoplasm. Mercurochrome staining revealed that both the PfCSP-Pb strain and the wild-type Pb strain produced a large number of oocysts in the midguts of Anopheles mosquitoes infected with both the PfCSP-Pb strain and the wild-type Pb strain on day 14 post-infection, which contained sporozoite precursor cells, and GFP fluorescence signals were detected in the salivary glands and midgut tissues of Anopheles mosquitoes infected with the PfCSP-Pb strain. In the liver stage, qRT-PCR assay showed that the relative 18S rRNA expression was 1.26 ± 0.49 in the PfCSP-Pb strain infection group, which was comparable to that in the wild-type Pb infection group (1.16 ± 0.67) (t = 0.27, P > 0.05). Following immunization of the mAB317 monoclonal antibody, the relative liver parasite load was lower in the PfCSP-specific monoclonal antibody treatment group (0.25 ± 0.48) than in the control group (1.10 ± 0.51) (t = 2.70, P < 0.05) in mice infected with salivary gland sporozoites from Anopheles mosquitoes infected with the PfCSP-Pb strain, while no significant difference was seen in the relative liver parasite load between the PfCSP-specific monoclonal antibody treatment group (0.92 ± 0.44) and the control group (1.28 ± 0.69) (t = 0.99, P > 0.05) in mice infected with salivary gland sporozoites from Anopheles mosquitoes infected with the wild-type Pb strain. Conclusion This study successfully established a chimeric Pb infection model stably expressing chimeric PfCSP. The PfCSP-Pb strain maintains stable biological characteristics both in vitro and in vivo, and is specifically able to be used to evaluate antibody-mediated immunoprotection against PfCSP, which provides a key technical platform and evaluation tool for preclinical researches on development of P. falciparum malaria vaccines.

Key words: Chimeric Plasmodium berghei, Plasmodium falciparum circumsporozoite protein, Infection model, Immunoprotective efficacy, Transmission-blocking strategy

CLC Number:

382.31

WANG Jiji, YUAN Yajie, ZHENG Jian, HAN Lu, GYAWU Stephen Baffour, ZHANG Qinglong, FENG Gaoqian. Establishment and validation of a mouse model of infection with Plasmodium berghei chimerically expressing P. falciparum circumsporozoite protein[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(1): 57-63.

Figures/Tables 4

Table 1

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基因名 Gene name	引物序列（5'➝3'） Primer sequence (5'➝3')	扩增长度/bp Amplication length/bp
pb18s	F：AAGCATTAAATAAAGCGAATACATCCTTAC	210
	R：GGAGATTGGTTTTGACGTTTATGTG
pb-csp-C	F：CAGCCAGGTGGTAATAACAATAACAAAAATAATAATAATG	245
	R：TTAATTAAAGAATACTAATACTAATAATATTACAAATCCTAATGAATTGC
pf-csp-C	F：AATCCTAATAAAAACAATCAAGGTAATGGACAAGG	245
	R：CTAATTAAGGAACAAGAAGGATAATACCATTATTAATCCTATTGAAC
intgr pb-p230p	F：GCAAAGTGAAGTTCAAATATGTG	1 500
	R：AGTGACTTTCAGTGAAATCGC
gfp	F：TGACGGGAACTACAAGACAC	1 500
	R：CTCGCAAAGCATTGAACACC