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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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Sterile protection acquired by γ-spz immunization regimens associated with undetectable hepatic parasitesRT-nPCR analysis of cDNA derived from total RNA purified from liver or spleen samples collected from mice inoculated with γ-spz s.c. (250 × 103), i.d. (100 × 103), or i.v. (5 × 102 or 5 × 103), 4 h post inoculation for the spleen and 40–44 h post-inoculation for the liver.Subcutaneous with 100 × 103 γ-spz 1, 6, 12 or 40 h post inoculation or i.v. with 5 × 103 γ-spz 40–44 h post inoculation. RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. The + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA. Control mice received PBS.BALB/c, Cd81+/+, or Cd81−/− mice were immunized s.c. or i.v. with PBS (controls) or three doses of 100 × 103 γ-spz at 1-weekly interval. Five days after the last immunization, 30 × 106 splenocytes were transferred into each naïve mice, and these were challenged 2 days later by i.d. inoculation with 5 × 103 PyWT. The naïve recipients adoptively transferred with the splenocytes of control mice became patent at day 4–5 after PyWT challenge. Blood stage parasites could not be detected in any of the naïve recipients of splenocytes from immunized animals in Giemsa-stained blood smears collected three times a week from days 4 to 14 post sporozoite challenge. Results expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with 10 mice per group at each time. *p = 0.0005 by Kruskal–Wallis test with Dunn's post-test.
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fig01: Sterile protection acquired by γ-spz immunization regimens associated with undetectable hepatic parasitesRT-nPCR analysis of cDNA derived from total RNA purified from liver or spleen samples collected from mice inoculated with γ-spz s.c. (250 × 103), i.d. (100 × 103), or i.v. (5 × 102 or 5 × 103), 4 h post inoculation for the spleen and 40–44 h post-inoculation for the liver.Subcutaneous with 100 × 103 γ-spz 1, 6, 12 or 40 h post inoculation or i.v. with 5 × 103 γ-spz 40–44 h post inoculation. RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. The + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA. Control mice received PBS.BALB/c, Cd81+/+, or Cd81−/− mice were immunized s.c. or i.v. with PBS (controls) or three doses of 100 × 103 γ-spz at 1-weekly interval. Five days after the last immunization, 30 × 106 splenocytes were transferred into each naïve mice, and these were challenged 2 days later by i.d. inoculation with 5 × 103 PyWT. The naïve recipients adoptively transferred with the splenocytes of control mice became patent at day 4–5 after PyWT challenge. Blood stage parasites could not be detected in any of the naïve recipients of splenocytes from immunized animals in Giemsa-stained blood smears collected three times a week from days 4 to 14 post sporozoite challenge. Results expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with 10 mice per group at each time. *p = 0.0005 by Kruskal–Wallis test with Dunn's post-test.

Mentions: It is possible that some of s.c.-inoculated attenuated sporozoites escape the skin and reach the liver, where they, or antigens derived from them, would also induce immune responses leading to sterile protection. Therefore, we used a sensitive amplification protocol to establish whether parasites could be detected in the spleens or livers of mice inoculated with 100 × 103 γ-spz i.d., or with a substantially larger dose (250 × 103) s.c. The spleens and livers of some experimental animals were harvested 4 or 40 h post-inoculation, respectively. In other s.c. inoculated animals, the spleens and livers were harvested at 1, 6, 12 and 40 h post-inoculation. Total RNA was isolated from these livers and spleens and used in an RT-nested PCR assay to detect small amounts of the parasite's small subunit ribosomal RNA gene. No amplification product was obtained in any of the liver or spleen samples collected from mice inoculated s.c. (Fig 1B), in contrast to robust amplification in the samples collected from i.d.- and i.v.-inoculated mice (Fig 1A). The sensitivity of the method is attested by the positive results observed for the liver samples obtained from mice inoculated i.v. with only 500 γ-spz (assuming most of the sporozoites invaded hepatocytes and that each parasite harbours a conservative 500 ribosomes, the aliquot assayed would correspond to about 150 target copies). Thus, it is likely that none, or substantially <0.2% of the 250 × 103 γ-spz inoculated s.c. would have reached, or persisted in, the liver. Given that an i.v. immunization schedule based on 500 γ-spz does not lead to sterile protection against normal challenge, a minute proportion of the s.c.-inoculated γ-spz that might have made their way out of the skin to liver would not account for the protective immunity we obtained in our model.


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)

Sterile protection acquired by γ-spz immunization regimens associated with undetectable hepatic parasitesRT-nPCR analysis of cDNA derived from total RNA purified from liver or spleen samples collected from mice inoculated with γ-spz s.c. (250 × 103), i.d. (100 × 103), or i.v. (5 × 102 or 5 × 103), 4 h post inoculation for the spleen and 40–44 h post-inoculation for the liver.Subcutaneous with 100 × 103 γ-spz 1, 6, 12 or 40 h post inoculation or i.v. with 5 × 103 γ-spz 40–44 h post inoculation. RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. The + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA. Control mice received PBS.BALB/c, Cd81+/+, or Cd81−/− mice were immunized s.c. or i.v. with PBS (controls) or three doses of 100 × 103 γ-spz at 1-weekly interval. Five days after the last immunization, 30 × 106 splenocytes were transferred into each naïve mice, and these were challenged 2 days later by i.d. inoculation with 5 × 103 PyWT. The naïve recipients adoptively transferred with the splenocytes of control mice became patent at day 4–5 after PyWT challenge. Blood stage parasites could not be detected in any of the naïve recipients of splenocytes from immunized animals in Giemsa-stained blood smears collected three times a week from days 4 to 14 post sporozoite challenge. Results expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with 10 mice per group at each time. *p = 0.0005 by Kruskal–Wallis test with Dunn's post-test.
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fig01: Sterile protection acquired by γ-spz immunization regimens associated with undetectable hepatic parasitesRT-nPCR analysis of cDNA derived from total RNA purified from liver or spleen samples collected from mice inoculated with γ-spz s.c. (250 × 103), i.d. (100 × 103), or i.v. (5 × 102 or 5 × 103), 4 h post inoculation for the spleen and 40–44 h post-inoculation for the liver.Subcutaneous with 100 × 103 γ-spz 1, 6, 12 or 40 h post inoculation or i.v. with 5 × 103 γ-spz 40–44 h post inoculation. RT− is a control for DNA contamination prepared in the absence of the reverse transcriptase. The + is a positive control for the amplification reaction from genomic P. yoelii 265BY DNA. Control mice received PBS.BALB/c, Cd81+/+, or Cd81−/− mice were immunized s.c. or i.v. with PBS (controls) or three doses of 100 × 103 γ-spz at 1-weekly interval. Five days after the last immunization, 30 × 106 splenocytes were transferred into each naïve mice, and these were challenged 2 days later by i.d. inoculation with 5 × 103 PyWT. The naïve recipients adoptively transferred with the splenocytes of control mice became patent at day 4–5 after PyWT challenge. Blood stage parasites could not be detected in any of the naïve recipients of splenocytes from immunized animals in Giemsa-stained blood smears collected three times a week from days 4 to 14 post sporozoite challenge. Results expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with 10 mice per group at each time. *p = 0.0005 by Kruskal–Wallis test with Dunn's post-test.
Mentions: It is possible that some of s.c.-inoculated attenuated sporozoites escape the skin and reach the liver, where they, or antigens derived from them, would also induce immune responses leading to sterile protection. Therefore, we used a sensitive amplification protocol to establish whether parasites could be detected in the spleens or livers of mice inoculated with 100 × 103 γ-spz i.d., or with a substantially larger dose (250 × 103) s.c. The spleens and livers of some experimental animals were harvested 4 or 40 h post-inoculation, respectively. In other s.c. inoculated animals, the spleens and livers were harvested at 1, 6, 12 and 40 h post-inoculation. Total RNA was isolated from these livers and spleens and used in an RT-nested PCR assay to detect small amounts of the parasite's small subunit ribosomal RNA gene. No amplification product was obtained in any of the liver or spleen samples collected from mice inoculated s.c. (Fig 1B), in contrast to robust amplification in the samples collected from i.d.- and i.v.-inoculated mice (Fig 1A). The sensitivity of the method is attested by the positive results observed for the liver samples obtained from mice inoculated i.v. with only 500 γ-spz (assuming most of the sporozoites invaded hepatocytes and that each parasite harbours a conservative 500 ribosomes, the aliquot assayed would correspond to about 150 target copies). Thus, it is likely that none, or substantially <0.2% of the 250 × 103 γ-spz inoculated s.c. would have reached, or persisted in, the liver. Given that an i.v. immunization schedule based on 500 γ-spz does not lead to sterile protection against normal challenge, a minute proportion of the s.c.-inoculated γ-spz that might have made their way out of the skin to liver would not account for the protective immunity we obtained in our model.

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