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. 2017 Sep 4;7(1):10372.
doi: 10.1038/s41598-017-10480-1.

Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

Simone Haeberlein  1   2 , Séverine Chevalley-Maurel  1 , Arifa Ozir-Fazalalikhan  1 , Hester Koppejan  1 , Beatrice M F Winkel  1 , Jai Ramesar  1 , Shahid M Khan  1 , Robert W Sauerwein  3 , Meta Roestenberg  1   4 , Chris J Janse  1 , Hermelijn H Smits  1 , Blandine Franke-Fayard  5
Affiliations

Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load

Simone Haeberlein et al. Sci Rep. .

Abstract

In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53-68%) than IV-I (93-95%). Unprotected mice developed less sporozoite-specific CD8+ and CD4+ effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8+ dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
ID immunization with 50 K sporozoites results in same parasite liver load as 10 K IV but determines different outcome of protection. BALB/c mice were immunized with a liver-attenuated luciferase-expressing P. yoelii mutant (ΔPyFabBF-GFP-Luccon) by intravenous (IV) or intradermal (ID) inoculation of 10 K or 50 K sporozoites, respectively, followed by an IV or ID boost 2 weeks later. 44 h after primary immunization and boost, parasite liver loads were analyzed by in vivo imaging of luciferase activity. (A) In vivo images which show 5 representative mice for each group after primary immunization and boost (parasite load measured as relative luminescence units, RLU, red color indicates high parasite load). (B) Summary of luciferase activity of 48 (10 K IV) and 43 (50 K ID) mice after primary immunization from 6 experiments. ns: no significance as determined by unpaired t-test. ns, not significant. (C–E) BALB/c mice were IV or ID immunized and challenged IV with 10 K luciferase-expressing wild-type (wt) P. yoelii sporozoites (Py-GFP-Luccon) 2 weeks after boost (see treatment scheme in Fig. S2). 44 h after immunization, boost or challenge, parasite liver loads were analyzed by in vivo imaging of luciferase activity. Blood smears were analyzed from day 4–14 after challenge to assess prepatency times. (C) Cumulative results from two separate experiments (n = 18, IV inoculation; n = 20, ID inoculation and n = 8, naïve) of liver load measured by luciferase activity. (D) In vivo images of 5 representative mice per group. (E) Protection rate and days of prepatency summarized for 4 experiments. All naïve mice infected with wt parasites developed blood infections.
Figure 2
Figure 2
Level of protection in IV and ID immunized mice treated with artesunate (ART) after challenge with wild type parasites. BALB/c mice were immunized by IV or ID route. From the day of wt sporozoite challenge, mice were treated with artesunate to prevent development of blood stages in mice (see treatment scheme in Fig. S4). 44 h after primary immunization, boost or challenge, parasite liver loads were quantified by in vivo imaging of luciferase activity. (A) Luciferase activity of 5 representative mice from the ID immunized group visualized after primary immunization, boost and challenge. (B) Summary of luciferase activity during the immunization/challenge protocol of mice immunized by the IV and ID route, and of challenged naïve mice. (C) Protection rates from 2 experiments. Based on the luciferase activity after challenge, mice were grouped into protected (luciferase negative) and unprotected (luciferase positive). ns: no significance, *p < 0.05 as determined by unpaired t-test. (D) Mice were immunized by ID route and challenged with wt sporozoites under artesunate treatment. Development of liver stages was analyzed by in vivo imaging at 44 h after primary immunization, boost, or challenge. Based on the luciferase activity after challenge, mice were grouped into protected and unprotected. Summary of 19 ID immunized mice.
Figure 3
Figure 3
Unprotected mice after ID immunization show reduced liver T cell effector responses compared to protected mice. ID immunized mice were challenged followed by artesunate treatment, and distinguished as protected (p, i.e. luciferase negative) or unprotected (unp, i.e. luciferase positive). Hepatic CD8 and CD4 T cells were analyzed at day 7 after challenge. (A) IFN-γ concentration in supernatant of total leukocytes after culture for 36 h with CSP and sporozoites as measured by ELISA. (B) Representative FACS plots of CD8+ gated T cells for intracellular expression of IFN-γ or TNF after culture for 4 h with CSP and brefeldin A, and for surface expression of CD107a after 4 h culture with CSP, brefeldin A and monensin. Numbers indicate the frequency of the gated cell population. Graphs show a summary of 2 experiments with 8–14 mice per group for frequency of IFN-γ (C), TNF (D), or CD107a (E) -expressing CD8 T cells and CD4 T cells. The dotted line indicates the mean cytokine or expression level for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 (to naïve control group), and #p < 0.05, ##p < 0.01 (between immunized mouse groups).
Figure 4
Figure 4
Unprotected mice have higher regulatory immune responses compared to protected mice. Hepatic CD4 T cells (gated Foxp3−) and Foxp3+CD25+ Treg cells of protected and unprotected ID immunized mice were analyzed for regulatory marker expression at day 7 after challenge under artesunate treatment. (A) Representative FACS plots of Foxp3− gated CD4 T cells for intracellular IL-10 expression after 36 h culture of hepatic leukocytes with CSP and sporozoites and addition of PMA/ionomycin plus brefeldin A in the last 4 h, and for surface expression of CTLA-4 or GITR ex vivo. Graphs show a summary of (B) intracellular IL-10 expression, (C) surface CTLA-4 and (D) GITR expression of Foxp3− CD4 T cells and Treg cells from 2 experiments with 8–14 mice per group. The dotted line indicates the mean frequency for protected IV immunized mice (N = 9–16). Significant difference by Mann-Whitney test is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 (to naïve control group), and #p < 0.05, ###p < 0.001 (between immunized mouse groups). (E) The production of secreted IFN-γ by hepatic leukocytes was correlated with the percentage of hepatic CTLA4+ Foxp3− CD4 T cells. Correlation coefficient and p-value are indicated.
Figure 5
Figure 5
Induction of activated CD44+ CD8 T cells is less by ID compared to IV immunization. BALB/c mice were immunized twice 2 weeks apart with liver-attenuated luciferase-expressing ΔPyFabBF-GFP-Luccon sporozoites (PyGAP) either with 10 K IV or 50 K ID. Expression of the activation and memory markers CD44 and CD45RB on hepatic T cells at day 7 after primary immunization or boost were analyzed by flow cytometry. (A) Representative FACS plots of CD8+ and CD4+ gated T cells after IV or ID boost compared to a naïve control. Numbers indicate the frequency of the gated cell population. (B) Frequencies of CD8+ T cells within total CD3+ T cells, of (C) CD44hi cells and (D) CD45RBlow CD44hi cells within the CD8 T cell population, (E) number of CD44hi CD8 T cells per liver, and (F) of CD44hi cells within the CD4 T cell population. Summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by **p < 0.01, ***p < 0.001 (to naïve control group), and ##p < 0.05, ##p < 0.01 (between immunized mouse groups).
Figure 6
Figure 6
CD8 dendritic cell subset frequencies are smaller after ID compared to IV immunization. Dendritic cell (DC) subsets in liver were analyzed by flow cytometry at 7 days after primary or boost immunization via IV or ID route. (A) MHCII+CD11chi/int DC were gated for CD11cintCD64hi monocyte-derived DC (mo-DC) and CD11chiCD64low conventional DC (cDC) after excluding F4/80-expressing macrophages. Representative FACS plots of both DC subsets for CD8 and intracellular IL-12p40 expression in mice 7 days after IV or ID boost or in naïve control mice are shown. Numbers indicate the frequency of the gated cell population. (B) Frequencies of DC subsets within total leukocytes. (C) Number of CD8-expressing DC per liver. (D) Frequencies of CD8-expressing DC within the mo-DC or cDC subset. (E) Intracellular IL-12p40 expression of DC subsets after culture of leukocytes with brefeldin A for 4 h. Summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 (to naïve control group), and #p < 0.05, ##p < 0.01 (between immunized mouse groups).
Figure 7
Figure 7
ID immunization induces stronger regulatory immune responses in liver compared to IV. Hepatic leukocytes were analyzed 7 days after IV or ID primary immunization or boost for regulatory marker expression by flow cytometry directly ex vivo (A,B) or after 36 h culture with CSP and sporozoites (C–G). (A) Number of Foxp3+CD25+ Treg cells per liver. (B) Frequency of Treg cells within the CD4+ T cell population. (C) Intracellular IL-10 expression of Treg cells after addition of PMA, ionomycin and brefeldin A for 4 h to the culture. (D) Representative FACS plots of CD4+ Foxp3-negative T cells (CD4 T) in one IV or ID immunized and a naïve control mouse. (E,F) Summary of intracellular IL-10 expression of CD4+Foxp3− T cells after addition of PMA, ionomycin and brefeldin A expressed as frequencies (E) and cell number per liver (F). Representative FACS plots (G) and summary (H) of intracellular IL-10 expression in CD19+ gated B cells after addition of PMA, ionomycin and brefeldin A. Graphs show 1 representative out of 2 similar experiments (C) or a summary of 2 experiments with 8–10 mice per group. Significant difference by Mann-Whitney test is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 (to naïve control group), and #p < 0.05, ##p < 0.01 (between immunized mouse groups).
Figure 8
Figure 8
ID but not IV immunization increases regulatory immune responses in the skin-draining lymph node. Inguinal lymph node cells were analyzed 7 days after IV or ID primary immunization for regulatory marker and cytokine expression. Flow cytometry was performed after 36 h culture with CSP and sporozoites and addition of PMA, ionomycin and brefeldin A in the last 4 h (A,B), or directly ex vivo (C). Frequencies calculated relative to naïve control are given for (A) IFN-γ+ CD4 T cells and CD8 T cells, (B) IL-10+ Foxp3− CD4 T cells, IL-10+ Foxp3+CD25+ Treg cells, IL-10+ CD19+ B cells, and (C) CTLA-4+ Foxp3− CD4 T cells and CTLA-4+ Foxp3+CD25+ Treg cells. Summary of 2 experiments with 10 mice per group. Significant difference by unpaired t-test is indicated by *p < 0.05, **p < 0.01, ***p < 0.001 (to naïve control group), and ##p < 0.01, ###p < 0.001 (between immunized mouse groups).
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References

    1. Luke TC, Hoffman SL. Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. The Journal of experimental biology. 2003;206:3803–3808. doi: 10.1242/jeb.00644. - DOI - PubMed
    1. Moorthy VS, Newman RD, Okwo-Bele JM. Malaria vaccine technology roadmap. Lancet. 2013;382:1700–1701. doi: 10.1016/S0140-6736(13)62238-2. - DOI - PubMed
    1. Pinzon-Charry A, Good MF. Malaria vaccines: the case for a whole-organism approach. Expert opinion on biological therapy. 2008;8:441–448. doi: 10.1517/14712598.8.4.441. - DOI - PubMed
    1. Hoffman SL, Vekemans J, Richie TL, Duffy PE. The March Toward Malaria Vaccines. American journal of preventive medicine. 2015;49:S319–333. doi: 10.1016/j.amepre.2015.09.011. - DOI - PMC - PubMed
    1. Seder RA, et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science. 2013;341:1359–1365. doi: 10.1126/science.1241800. - DOI - PubMed

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