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. 2018 Aug 24;3:33.
doi: 10.1038/s41541-018-0068-2. eCollection 2018.

A Plasmodium berghei Sporozoite-Based Vaccination Platform Against Human Malaria

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Free PMC article

A Plasmodium berghei Sporozoite-Based Vaccination Platform Against Human Malaria

António M Mendes et al. NPJ Vaccines. .
Free PMC article

Abstract

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

Conflict of interest statement

A.M.M., M.M.M., and M.P. are inventors on a patent or patent application issued, allowed or filed internationally, covering parts of this work. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Rodent P. berghei parasites successfully develop within human hepatocytes but not within human RBCs. a Representative images of developing rodent PbWT parasites in mouse (black square) and human (red square) hepatic cells of liver-humanized FRG mice 48 h post infection (hpi) by iv injection of freshly isolated sporozoites. b Relative proportion of Pb-infected mouse (grey) and human (red) hepatocytes in humanized FRG mice, normalized to the total humanization of the chimeric liver. c PbWT development in mouse (grey) and human (red) hepatocytes 42 and 48 hpi of liver-humanized FRG mice. d Representative flow cytometry plots of peripheral blood from blood-humanized NSG mice infected by iv injection of Pf-infected (left) or Pb-infected RBCs (middle-left) before and after magnetic separation (middle-right and right); Syto-16 for nucleic acids; TER-119 for murine erythroid lineage; imRBCs/ihRBCs: infected mouse or human RBCs; bimRBCs: background signal for infected murine erythroid lineage. e Relative proportion of mouse and human RBCs infected with Pf (left) or Pb (right) parasites; bars indicate standard error. f Representative pictures of Pb parasite forms observed within magnetically separated imRBCs and ihRBCs from the total blood of infected blood-humanized NSG mice after 2 and 20 h of in vitro culture
Fig. 2
Fig. 2
PfCS expression and human hepatic infectivity of PbVac pre-erythrocytic stages. a, b Representative Immunofluorescence microscopy images of PbCS (grey) and PfCS (purple) expressed by PbVac sporozoites a and exoerythrocytic forms in the livers of mice infected by iv injection of freshly isolated sporozoites b. c Comparative infection rates of PbVac and Pf parasites in ex vivo cultures of human hepatocytes assessed by immunofluorescence microscopy. The shading of dots indicates distinct biological replicates obtained employing human hepatocytes from different donors. The boxes correspond to the 25th and 75th percentiles; the lines and bars indicate mean of infection and standard error of the mean, respectively; ***p < 0.001, as determined by Mann–Whitney U test. Scale bars: 10 µm
Fig. 3
Fig. 3
Infection of NZW rabbit hepatocytes with P. berghei sporozoites. a Rodent Pb parasite development 24 and 48 hpi in ex vivo cultures of rabbit primary hepatocytes. b Representative immunofluorescence microscopy image of a Pb merosomes developing ex vivo within rabbit primary hepatocytes 60 hpi and presenting typical markers of late exoerythrocytic development. c qRT-PCR quantification of hepatic infection of rabbits following iv injection of increasing amounts of freshly isolated Pb sporozoites (1 × 105, 5 × 105, and 1 × 106). d Representative immunofluorescence microscopy images of rodent Pb parasites developing in the livers of NZW rabbits at different hpi. e Parasitemia in the peripheral blood of rabbits (orange) or mice (grey) following iv injection of freshly isolated Pb sporozoites. Scale bars: 10 µm
Fig. 4
Fig. 4
Immune responses in NZW rabbits after PbVac sporozoite immunization. a Diagram of the immunization protocol. Immunizations were performed by exposure to the bites of 75-100 mosquitoes. b Total IgG titers against PfCS repeat sequence in serum after 1, 2, and 3 mock immunizations (grey), or immunization with PbWT (orange) and PbVac (purple), or at the time of animal sacrifice (S) 60–90 days after last immunization. c Serum binding capacity to Pf sporozoites after PbVac immunization. d Spleen cell proliferation upon stimulation with peptide pools spanning the entire PbCS or PfCS proteins in immunized rabbits, as indicated by assessment of 3H-thymidine incorporation. e Spleen cell proliferation upon stimulation with PbWT, PbVac or Pf sporozoites, as indicated by assessment of 3H-thymidine incorporation. Stimulation with an extract of uninfected mosquito salivary gland material was used as control. f IFNy production in rabbit spleen cell supernatant after stimulation with PbWT, PbVac or Pf sporozoites. Measurements were taken from distinct samples. The boxes correspond to the 25th and 75th percentiles; the line and bars indicate mean of infection and standard error of the mean, respectively; *p < 0.05; **p < 0.01; ***p < 0.001, as determined by Kruskal–Wallis test, corrected with Dunn’s multiple comparisons test
Fig. 5
Fig. 5
PbVac-mediated protection against Pf challenge. a Diagram of the experimental protocol. b Pf sporozoite infection of HC-04 human immortalized hepatocyte cultures incubated with purified IgG from mock- (grey), PbWT- (yellow) or PbVac (purple)-immunized rabbits. c qRT-PCR quantification of Pf hepatic infection inhibition in liver-humanized FRG mice following passive transfer of purified IgGs from mock (grey)-, PbWT (orange)- or PbVac (purple)-immunized rabbits and challenge by the bites of 20 Pf-infected mosquitoes (n = 3 mice per group). Measurements were taken from distinct samples. The boxes correspond to the 25th and 75th percentiles in B), and the minimum and maximum data range in C); the lines and bars indicate mean of infection and standard error of the mean, respectively; *p < 0.05; **p < 0.01, as determined by one-way ANOVA, corrected with Dunnett’s multiple comparisons test

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References

    1. Malaria vaccine: WHO position paper-January 2016. Wkly Epidemiol Rec91, 33–51 (2016). - PubMed
    1. Prudencio M, Rodriguez A, Mota MM. The silent path to thousands of merozoites: the Plasmodium liver stage. Nat. Rev. Microbiol. 2006;4:849–856. doi: 10.1038/nrmicro1529. - DOI - PubMed
    1. Hill AV. Vaccines against malaria. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2011;366:2806–2814. doi: 10.1098/rstb.2011.0091. - DOI - PMC - PubMed
    1. Gordon DM, et al. Safety, immunogenicity, and efficacy of a recombinantly produced Plasmodium falciparum circumsporozoite protein-hepatitis B surface antigen subunit vaccine. J. Infect. Dis. 1995;171:1576–1585. doi: 10.1093/infdis/171.6.1576. - DOI - PubMed
    1. Olotu A, et al. Seven-year efficacy of RTS,S/AS01 malaria vaccine among young African children. N. Engl. J. Med. 2016;374:2519–2529. doi: 10.1056/NEJMoa1515257. - DOI - PMC - PubMed

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