Research ArticleMalaria

Prime and target immunization protects against liver-stage malaria in mice

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Science Translational Medicine  26 Sep 2018:
Vol. 10, Issue 460, eaap9128
DOI: 10.1126/scitranslmed.aap9128
  • Fig. 1 Prime and target immunization protects mice against challenge with Tg OVA-spz.

    C57BL/6 mice (n = 6 per group) were primed with Ad.OVA-i.m., followed by Np.OVA-i.v. (Ad.OVA-i.m./Np.OVA-i.v.), Np.OVA-i.m. (Ad.OVA-i.m./Np.OVA-i.m.), or no Np.OVA (Ad.OVA-i.m.). Mice were challenged with (A) OVA::Hep17 P. berghei spz or (B) OVA::mCherryPbhsp70 P. berghei spz. Data shown are representative of two to three independent experiments (log-rank Mantel-Cox test). C57BL/6 mice were primed with either Ad.OVA-i.m. or Np.OVA-i.v. Ad.OVA-i.m.–primed animals either were subsequently vaccinated with OVA protein (Ad.OVA-i.m./OVA-i.v.), Np.OVA-i.m. (Ad.OVA-i.m./Np.OVA-i.m.), or Np.OVA-i.v. (Ad.OVA-i.m./Np.OVA-i.v.) or were left untreated (Ad.OVA-i.m.). Three weeks after Np or Ad.OVA-i.m. vaccination, animals were examined by flow cytometry for (C) to (E) total Pen+ CD8+ T cells in the liver, dLN, and spleen; frequency of (F) Pen+ and (G) IFN-γ+ CD8+ T cells of total CD8+ T cells in the liver; and (H) total cell count of IFN-γ+ CD4+ T cells in the liver. Data shown are pooled from two to four independent experiments (n = 7 to 12 per group). Median shown. Data were analyzed with a linear mixed model (LMM), with experiment and mouse as random effects and vaccination and boost as fixed effects. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. ns, not significant.

  • Fig. 2 Localization and quantification of immune response to spz challenge.

    Immunofluorescence (IF) images depicting spz in the liver 12 hours after challenge (ch:) with an intravenous injection of 5 × 104 OVA::mCherryPbhsp70 P. berghei spz (OVA-spz) or mcherryPbhsp70 (spz). Mice had been previously vaccinated (v:) with Ad.OVA-i.m./Np.OVA-i.v. or Ad.OVA-i.m. only and challenged 3 weeks after Np.OVA-i.v. injection. (A) IF images of liver sections [maximum intensity projection (MIP) of a 20-μm z-stack] stained for CSP protein, CD8, CD4, and F4/80; arrowheads denote spz. (B) IF image depicting a liver section from Ad.OVA-i.m./Np.OVA-i.v. challenged with OVA-spz. Section stained for CD8, CD4, Ki67, GrzB, IFN-γ, and Jojo (nucleus). Right: Single-color panels. (C) Quantification of IF images: Mean cell count per infectious site (IS) of CD8+ and CD4+ T cells (equal numbers of IS imaged per condition). In situ frequency of (D) CD8+ and (E) CD4+ T cells expressing Ki67, GrzB, or IFN-γ from mice vaccinated with Ad.OVA-i.m./Np.OVA-i.v. and challenged with OVA-spz. IF images are representative of two independent experiments, for a total of n = 4 animals per condition (two liver sections imaged per animal). Median shown from all pooled data. Data were analyzed with LMM, with experiment and liver section as random effects and vaccination and spz as fixed effects. (F) C57BL/6 mice (n = 5 per group) were vaccinated with Ad.OVA-i.m./Np.OVA-i.v. Three weeks after Np.OVA-i.v. vaccination, mice were administered anti-CD4, anti-CD8–depleting antibodies (Abs), or a combination of both antibodies. Alternatively, immunoglobulin G2b isotype control (Iso ctrl) antibody was administered. Mice were subsequently challenged with OVA::Hep17 P. berghei spz. Data shown are representative of two independent experiments (log-rank Mantel-Cox test). ****P < 0.0001, **P < 0.01.

  • Fig. 3 Prime and target vaccination induces liver TRM stably maintained in the tissue and sufficient in mediating protection.

    (A) Representative IF images depicting liver tissue of mice vaccinated with Ad.OVA-i.m./Np.OVA-i.v. 5 weeks after Np.OVA-i.v. vaccination. (i) CD8, CXCR6, CD4, CD69, CD44, CD3, CD31, and JoJo (nucleus). (ii) F4/80, MHC-II, CD8, CD4, and JoJo. Bottom: Single-color panels. Arrowhead denotes examples of TRM (CD8+ CXCR6+ CD69+ CD44+ T cell), whereas asterisk-arrowhead denotes TEM (CD8+ CXCR6 CD69 CD44+ T cell) (MIP of a 20-μm z-stack). (B) Quantification of IF images: Total cell count/100 μm3 of TRM or TEM in animals vaccinated with Ad.OVA-i.m. or Ad.OVA-i.m./Np.OVA-i.v. Data were analyzed with LMM, with experiment and tissue slice as random effects and cell type and vaccination as fixed effects. (C) Total number of clusters/100 μm3 in animals vaccinated with Ad.OVA-i.m. or Ad.OVA-i.m./Np.OVA-i.v. Data were analyzed with LMM, with experiment and cluster as random effects and vaccination as fixed effect. (D) Frequency of TEM, TRM, and CD4+ T cells of total CD3+ cells in clusters from mice vaccinated with Ad.OVA-i.m./Np.OVA-i.v. Data were analyzed with LMM, with experiment and cluster as random effects and vaccination as fixed effect. (A to D) Quantification of IF images is from two independent experiments for a total of n = 4 to 6 animals per condition (two liver sections imaged per animal); median shown. (E) C57BL/6 mice were vaccinated with Ad.OVA-im/Np.OVA-i.v. or Ad.OVA-i.m. only. Total number of TCRv+ (Vα2/Vβ5) liver CD8+ T cells over time as determined by flow cytometry. Median shown from two pooled experiments. Data were analyzed with LMM, with experiment as random effect and vaccination and time as fixed effects. (F) At day 36 after Np.OVA-i.v. administration, total Pen+ T cells were determined in the liver and spleen. Data shown are pooled from two independent experiments, each symbol representative of an individual mouse (n = 8 to 9 per group). Data were analyzed with LMM, with experiment and mouse as random effects and vaccination as fixed effect. (G) C57BL/6 mice (n = 6 per group) were vaccinated with Ad.OVA-i.m./Np.OVA-i.v. or Ad.OVA-i.m. Mice were challenged with OVA::Hep17 P. berghei spz 2 or 6 months after Np.OVA-i.v. vaccination. (H) C57BL/6 mice (n = 6 per group) were vaccinated with Ad.OVA-i.m./Np.OVA-i.v. Three weeks after Np.OVA administration, mice were either splenectomized or sham-operated, and FTY720 was administered. Alternatively, mice were treated with FTY720- and/or anti-CD8–depleting antibodies. Mice were subsequently challenged with OVA::Hep17 P. berghei spz. Data shown are representative of two independent experiments (log-rank Mantel-Cox test). ****P < 0.0001, ***P < 0.001, **P < 0.01.

  • Fig. 4 Viral vectors as targeting agents induce strong protective immunity against challenge with Tg OVA-spz.

    BALB/c mice (n = 5 per group) were vaccinated intravenously with Ad, ChAd63, or MVA expressing luciferase (Luc) and imaged at different days (D) after vaccination. (A) Bioluminescence image indicating the location of luciferase. In red, region of interest (ROI) is shown, with (B) ROI flux analysis over time. (C) C57BL/6 mice were all primed with Ad.OVA-i.m. and followed, 2 weeks later, by MVA.OVA-i.m. (Ad.OVA-i.m./MVA.OVA-i.m.), Ad.OVA-i.v. (Ad.OVA-i.m./Ad.OVA-i.v.), MVA.OVA-i.v. (Ad.OVA-i.m./MVA.OVA-i.v.), and Np.OVA-i.v. (Ad.OVA-i.m./Np.OVA-i.v.) or were left untreated (Ad.OVA-i.m.). Total Pen+ T cells in the liver or spleen. Data shown are pooled from two independent experiments, each symbol representative of individual mouse (n = 8 to 12 per group). Median shown. Data were analyzed with LMM model, with experiment and mouse as random variables and vaccination and boost as fixed variables. All statistical comparisons were made with only statistically significant comparisons shown. (D) C57BL/6 mice (n = 6 per group) were vaccinated with Ad.OVA-i.m./Ad.OVA-i.m., Ad.OVA-i.m./MVA.OVA-i.m., Ad.OVA-i.m./Ad.OVA-i.v., or Ad.OVA-i.m./MVA.OVA-i.v. Mice were challenged 3 weeks after final immunization or 2 months after final immunization with OVA::Hep17 P. berghei spz. Data shown are representative of two independent experiments (log-rank Mantel-Cox test). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

  • Fig. 5 Prime and target approach sterilely protects mice against challenge with Tg spz expressing prominent clinical candidate P. falciparum vaccine Ags.

    BALB/c mice (n = 6 per group) were primed intramuscularly with ChAd63 viral vectors expressing the following Ags: (A) ME-TRAP, (B) PfLSA1, or (C) PfLSAP2. Mice were subsequently vaccinated with either MVA-i.m. (ChAd63-i.m./MVA-i.m.) or ChAd63-i.v. (ChAd63-i.m./ChAd63-i.v.), each expressing the respective cognate Ag. For the ME-TRAP–vaccinated group (A), an additional group of BALB/c mice (n = 6 per group) received ChAd63.ME-TRAP-i.m., followed by Np.Pb9-i.v. (ChAd63-i.m./Np.Pb9-i.v.). (D) CD-1 mice (n = 6 per group) were primed with ChAd63 viral vectors expressing PfTRAP-i.m. Mice received a subsequent immunization of either MVA-i.m. or ChAd63-i.v. vector, each expressing PfTRAP Ag. Protective efficacy of immunization was determined after challenge with (A) WT or (B to D) Tg P. berghei spz expressing the cognate P. falciparum Ag. Data shown are representative of two independent experiments. Statistical comparison (log-rank Mantel-Cox test) between ChAd63-i.m./ChAd63-i.v. and ChAd63-i.m./MVA-i.m. vaccinations. *P < 0.05, **P < 0.01.

  • Fig. 6 Safety and immunogenicity data after ChAd63.ME-TRAP-i.v. vaccination in healthy human volunteers.

    (A) After vaccination with ChAd63.ME-TRAP by intravenous peripheral cannula, safety was assessed by active and passive collection of local and systemic AEs. The percentage of volunteers with local (left) and systemic (right) AEs are shown for all tested dose groups: group 1, 5 × 108 vp; group 2, 5 × 109 vp; group 3, 5 × 1010 vp (all n = 3). (B) Median with interquartile range of ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) responses to ME-TRAP peptides for each dose group from volunteers’ peripheral blood mononuclear cells (PBMCs) over time, measured in spot-forming cells (SFCs) per 106 PBMCs. (C) Individual ELISPOT responses for each participant at day 14 after immunization, compared with data from a previous study with the same vaccine construct and dose, administered intramuscularly (54). Notably, a subject in group 3 (marked in red) was incubating mumps at time of immunization.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/460/eaap9128/DC1

    Materials and Methods

    Fig. S1. Intravenous nanoparticle localization and OTI activation in liver tissue after intravenous and intramuscular Np.APC-OVA administration.

    Fig. S2. Schematic representation of prime and target vaccination regimen, efficacy, and immunogenicity.

    Fig. S3. Total Pen+ CD8+ T cell numbers in the liver are not affected by FTY720 administration.

    Fig. S4. T cell recruitment to the liver is dependent in part on the dose of PLGA-OVA administered.

    Fig. S5. A prime and target approach protects mice against challenge with Tg P. berghei OVA-spz only when mice are vaccinated against OVA.

    Fig. S6. Flow plots showing representative lymphocyte depletion after administration of depleting antibodies.

    Fig. S7. Higher numbers of liver Pen+ CD8+ T cells correlated with greater protection after spz challenge.

    Fig. S8. Histocytometric analysis of liver sections.

    Fig. S9. Liver Pen+ CD8+ T cells preferentially home back to the liver in recipient mice.

    Fig. S10. Gating strategy showing TRM phenotype of Pen+ T cells in the liver after nanoparticle administration and long-term efficacy data.

    Fig. S11. Flow plots showing representative lymphocyte depletion and inhibition of egress from lymphatics after administration of depleting antibody and FTY720.

    Fig. S12. A prime and target approach with viral vectors as targeting agents shows similar immunization profile to PLGA nanoparticle targeting.

    Fig. S13. A prime and target approach with viral vectors generates high numbers of HBsAg CD8+ T cells in the liver.

    Fig. S14. Safety and human immune responses to immunization with ChAd63.ME-TRAP-i.v.

    Movie S1. Video showing the internalization of Np.APC-OVA within Kupffer cells.

    Table S1. Primary data file.

    Data file S1. Methodological information regarding clinical trial (NCT03084289).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Intravenous nanoparticle localization and OTI activation in liver tissue after intravenous and intramuscular Np.APC-OVA administration.
    • Fig. S2. Schematic representation of prime and target vaccination regimen, efficacy, and immunogenicity.
    • Fig. S3. Total Pen+ CD8+ T cell numbers in the liver are not affected by FTY720 administration.
    • Fig. S4. T cell recruitment to the liver is dependent in part on the dose of PLGA-OVA administered.
    • Fig. S5. A prime and target approach protects mice against challenge with Tg P. berghei OVA-spz only when mice are vaccinated against OVA.
    • Fig. S6. Flow plots showing representative lymphocyte depletion after administration of depleting antibodies.
    • Fig. S7. Higher numbers of liver Pen+ CD8+ T cells correlated with greater protection after spz challenge.
    • Fig. S8. Histocytometric analysis of liver sections.
    • Fig. S9. Liver Pen+ CD8+ T cells preferentially home back to the liver in recipient mice.
    • Fig. S10. Gating strategy showing TRM phenotype of Pen+ T cells in the liver after nanoparticle administration and long-term efficacy data.
    • Fig. S11. Flow plots showing representative lymphocyte depletion and inhibition of egress from lymphatics after administration of depleting antibody and FTY720.
    • Fig. S12. A prime and target approach with viral vectors as targeting agents shows similar immunization profile to PLGA nanoparticle targeting.
    • Fig. S13. A prime and target approach with viral vectors generates high numbers of HBsAg CD8+ T cells in the liver.
    • Fig. S14. Safety and human immune responses to immunization with ChAd63.ME-TRAP-i.v.
    • Legend for movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Video showing the internalization of Np.APC-OVA within Kupffer cells.
    • Table S1 (Microsoft Excel format). Primary data file.
    • Data file S1 (.pdf format). Methodological information regarding clinical trial (NCT03084289).

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