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Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.

Obeid M, Franetich JF, Lorthiois A, Gego A, Grüner AC, Tefit M, Boucheix C, Snounou G, Mazier D - EMBO Mol Med (2012)

Bottom Line: However, it has recently been demonstrated that priming also occurs in the skin.We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes.We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver.

View Article: PubMed Central - PubMed

Affiliation: Inserm, UMR-S 945, Paris, France. michel.obeid@inserm.fr

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Protective antigens are probably present in the immunizing sporozoitesAll experiments were repeated three times (n = 3) with five mice per group (A) or by three triplicates (B) at each time.100 × 103 CD11c+-DC were sorted by FACS after co-culture with γ-spz or medium for 1.5 h, and then injected i.v. into recipient naïve mice. One week later, the recipient mice were challenged i.v. with 5 × 103 PyWT and the appearance of blood stage parasites was monitored three times a week by microscopic examination of Giemsa-stained blood smears from day 4 to day 14 post sporozoites infection. Each immunization group had a naïve control group of five mice (n = 5) immunized with PBS.Exo-erythrocytic forms (EEFs) were detected by fluorescence microscopy after double staining with anti-CSP and anti-HSP70, in the FACS-purified DC after co-culture with γ-spz sporozoites (or medium) for 0, 24 or 48 h after the co-culture of DC. The infectiousness of the γ-spz was assessed in control wells containing primary mouse hepaotcyte (Hep). Results are expressed as mean ± standard deviation (SD).Parasites were also detected by RT-nPCR after co-culture of γ-spz and DC, in the DC fraction purified by FACS immediately, 24 or 48 h after the co-culture. The control for sporozoite infectiousness was HepG2-A16-CD81, which are highly receptive to P. yoelii sporozoites (Yalaoui et al, 2008). RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA.
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fig04: Protective antigens are probably present in the immunizing sporozoitesAll experiments were repeated three times (n = 3) with five mice per group (A) or by three triplicates (B) at each time.100 × 103 CD11c+-DC were sorted by FACS after co-culture with γ-spz or medium for 1.5 h, and then injected i.v. into recipient naïve mice. One week later, the recipient mice were challenged i.v. with 5 × 103 PyWT and the appearance of blood stage parasites was monitored three times a week by microscopic examination of Giemsa-stained blood smears from day 4 to day 14 post sporozoites infection. Each immunization group had a naïve control group of five mice (n = 5) immunized with PBS.Exo-erythrocytic forms (EEFs) were detected by fluorescence microscopy after double staining with anti-CSP and anti-HSP70, in the FACS-purified DC after co-culture with γ-spz sporozoites (or medium) for 0, 24 or 48 h after the co-culture of DC. The infectiousness of the γ-spz was assessed in control wells containing primary mouse hepaotcyte (Hep). Results are expressed as mean ± standard deviation (SD).Parasites were also detected by RT-nPCR after co-culture of γ-spz and DC, in the DC fraction purified by FACS immediately, 24 or 48 h after the co-culture. The control for sporozoite infectiousness was HepG2-A16-CD81, which are highly receptive to P. yoelii sporozoites (Yalaoui et al, 2008). RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA.

Mentions: In order to provide unequivocal evidence that the priming leading to sterile protection can be solely due to antigens present in the immunizing sporozoite, in vitro-generated DC were co-cultured with γ-spz for 90 min, after which the CD11c+-DC were FACS sorted and injected into recipient naïve mice. One week later, the recipient mice were challenged with 5 × 103 PyWT i.v. None of the recipient mice developed a blood stage infection, i.e. the transfer of CD11c+-DC primed with γ-spz was sufficient to confer sterile protection against a sporozoite challenge (Fig 4A). Given the observations on extra-hepatic development of exo-erythrocytic forms (EEFs), we analysed the presence of EEFs in DC by a double immunofluorescence staining with anti-CSP and anti-HSP70 (two parasites markers). None were detected in the purified DC co-cultured with γ-spz that were maintained for 0, 24 or 48 h (Fig 4B). Additional confirmation was obtained by RT-nPCR assays on samples collected at the same time intervals, which also proved negative (Fig 4C). In order to demonstrate that the γ-spz batch employed in these experiments was capable of developing into EEFs, the co-culture and incubations were conducted in parallel using primary mouse hepatocytes (Fig 4B) or HepG2-A16-CD81 cells (Fig 4C). These were invaded by γ-spz, which then developed into recognizable and PCR-detectable EEFs (Fig 4B and C). Finally, when DC incubated with PyWT sporozoites were FACS-purified and inoculated into mice, these did not develop a blood stage parasitemia, demonstrating that viable sporozoites are unlikely to be retained with the FACS-purified DC.


Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.

Obeid M, Franetich JF, Lorthiois A, Gego A, Grüner AC, Tefit M, Boucheix C, Snounou G, Mazier D - EMBO Mol Med (2012)

Protective antigens are probably present in the immunizing sporozoitesAll experiments were repeated three times (n = 3) with five mice per group (A) or by three triplicates (B) at each time.100 × 103 CD11c+-DC were sorted by FACS after co-culture with γ-spz or medium for 1.5 h, and then injected i.v. into recipient naïve mice. One week later, the recipient mice were challenged i.v. with 5 × 103 PyWT and the appearance of blood stage parasites was monitored three times a week by microscopic examination of Giemsa-stained blood smears from day 4 to day 14 post sporozoites infection. Each immunization group had a naïve control group of five mice (n = 5) immunized with PBS.Exo-erythrocytic forms (EEFs) were detected by fluorescence microscopy after double staining with anti-CSP and anti-HSP70, in the FACS-purified DC after co-culture with γ-spz sporozoites (or medium) for 0, 24 or 48 h after the co-culture of DC. The infectiousness of the γ-spz was assessed in control wells containing primary mouse hepaotcyte (Hep). Results are expressed as mean ± standard deviation (SD).Parasites were also detected by RT-nPCR after co-culture of γ-spz and DC, in the DC fraction purified by FACS immediately, 24 or 48 h after the co-culture. The control for sporozoite infectiousness was HepG2-A16-CD81, which are highly receptive to P. yoelii sporozoites (Yalaoui et al, 2008). RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569641&req=5

fig04: Protective antigens are probably present in the immunizing sporozoitesAll experiments were repeated three times (n = 3) with five mice per group (A) or by three triplicates (B) at each time.100 × 103 CD11c+-DC were sorted by FACS after co-culture with γ-spz or medium for 1.5 h, and then injected i.v. into recipient naïve mice. One week later, the recipient mice were challenged i.v. with 5 × 103 PyWT and the appearance of blood stage parasites was monitored three times a week by microscopic examination of Giemsa-stained blood smears from day 4 to day 14 post sporozoites infection. Each immunization group had a naïve control group of five mice (n = 5) immunized with PBS.Exo-erythrocytic forms (EEFs) were detected by fluorescence microscopy after double staining with anti-CSP and anti-HSP70, in the FACS-purified DC after co-culture with γ-spz sporozoites (or medium) for 0, 24 or 48 h after the co-culture of DC. The infectiousness of the γ-spz was assessed in control wells containing primary mouse hepaotcyte (Hep). Results are expressed as mean ± standard deviation (SD).Parasites were also detected by RT-nPCR after co-culture of γ-spz and DC, in the DC fraction purified by FACS immediately, 24 or 48 h after the co-culture. The control for sporozoite infectiousness was HepG2-A16-CD81, which are highly receptive to P. yoelii sporozoites (Yalaoui et al, 2008). RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA.
Mentions: In order to provide unequivocal evidence that the priming leading to sterile protection can be solely due to antigens present in the immunizing sporozoite, in vitro-generated DC were co-cultured with γ-spz for 90 min, after which the CD11c+-DC were FACS sorted and injected into recipient naïve mice. One week later, the recipient mice were challenged with 5 × 103 PyWT i.v. None of the recipient mice developed a blood stage infection, i.e. the transfer of CD11c+-DC primed with γ-spz was sufficient to confer sterile protection against a sporozoite challenge (Fig 4A). Given the observations on extra-hepatic development of exo-erythrocytic forms (EEFs), we analysed the presence of EEFs in DC by a double immunofluorescence staining with anti-CSP and anti-HSP70 (two parasites markers). None were detected in the purified DC co-cultured with γ-spz that were maintained for 0, 24 or 48 h (Fig 4B). Additional confirmation was obtained by RT-nPCR assays on samples collected at the same time intervals, which also proved negative (Fig 4C). In order to demonstrate that the γ-spz batch employed in these experiments was capable of developing into EEFs, the co-culture and incubations were conducted in parallel using primary mouse hepatocytes (Fig 4B) or HepG2-A16-CD81 cells (Fig 4C). These were invaded by γ-spz, which then developed into recognizable and PCR-detectable EEFs (Fig 4B and C). Finally, when DC incubated with PyWT sporozoites were FACS-purified and inoculated into mice, these did not develop a blood stage parasitemia, demonstrating that viable sporozoites are unlikely to be retained with the FACS-purified DC.

Bottom Line: However, it has recently been demonstrated that priming also occurs in the skin.We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes.We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver.

View Article: PubMed Central - PubMed

Affiliation: Inserm, UMR-S 945, Paris, France. michel.obeid@inserm.fr

Show MeSH
Related in: MedlinePlus