Limits...
A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites.

van Schaijk BC, Ploemen IH, Annoura T, Vos MW, Foquet L, van Gemert GJ, Chevalley-Maurel S, van de Vegte-Bolmer M, Sajid M, Franetich JF, Lorthiois A, Leroux-Roels G, Meuleman P, Hermsen CC, Mazier D, Hoffman SL, Janse CJ, Khan SM, Sauerwein RW - Elife (2014)

Bottom Line: In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development.The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection.These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands.

ABSTRACT
A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Show MeSH

Related in: MedlinePlus

Genotype analysis of the generated PfΔslarp and PfΔb9Δslarp parasites.(A) Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B) and PCR products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δslarp-a, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δslarp-b, 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δb9-b, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (B) Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the 5′ slarp targeting region (P: 5′ slarp-T; see A) on the left side of the slarp Southern or probed with the 3′slarp targeting region (P: 3′ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9, integration DNA was digested with restriction enzymes (E: RcaI) and probed with the 5′ b9 targeting region (P: 5′ b9-T; see A) on the right panel. The expected fragment sizes are indicated in panel (A). (C) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B) and generated the expected 130 bp fragment.DOI:http://dx.doi.org/10.7554/eLife.03582.009
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4273440&req=5

fig2s2: Genotype analysis of the generated PfΔslarp and PfΔb9Δslarp parasites.(A) Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B) and PCR products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δslarp-a, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δslarp-b, 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δb9-b, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (B) Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the 5′ slarp targeting region (P: 5′ slarp-T; see A) on the left side of the slarp Southern or probed with the 3′slarp targeting region (P: 3′ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9, integration DNA was digested with restriction enzymes (E: RcaI) and probed with the 5′ b9 targeting region (P: 5′ b9-T; see A) on the right panel. The expected fragment sizes are indicated in panel (A). (C) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B) and generated the expected 130 bp fragment.DOI:http://dx.doi.org/10.7554/eLife.03582.009

Mentions: Next, we generated double gene-deletion PfΔb9Δslarp mutants using the FRT/FLPe recombinase methodology (van Schaijk et al., 2010). This methodology employs FLPe recombinase to remove a FRT-site flanked drug resistance marker cassette introduced into the Pf genome when the target gene has been removed by double cross-over homologous recombination as shown for PfΔslarp-b parasites in Figure 2—figure supplement 1,2. After cloning, this ‘marker-free’ line was subsequently transfected with the Pfb9 gene-targeting construct pHHT-FRT-GFP-b9 (Annoura et al., 2014) to delete the b9 locus from the PfΔslarp-b genome (Figure 2—figure supplement 1,2). Subsequently two ‘marker-free’ clones, PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9, were obtained containing the correct genotype that is removal of the slarp and b9 gene loci as well as both respective drug selection cassettes (Figure 2—figure supplement 2). In addition, we confirmed the loss of expression of both slarp and b9 by RT-PCR analysis by demonstrating the absence of transcripts in mRNA collected from PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 salivary gland sporozoites (Figure 2—figure supplement 2). We then examined the phenotype of PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 mutants during blood stage and mosquito development. Asexual blood stage growth of PfΔb9Δslarp parasites was normal as both clones reached an asexual parasitemia between 0.5 and 5% during cloning within 21 days and PfΔb9Δslarp clones produced WT-like numbers of gametocytes, oocysts, and sporozoites (Figure 2).


A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites.

van Schaijk BC, Ploemen IH, Annoura T, Vos MW, Foquet L, van Gemert GJ, Chevalley-Maurel S, van de Vegte-Bolmer M, Sajid M, Franetich JF, Lorthiois A, Leroux-Roels G, Meuleman P, Hermsen CC, Mazier D, Hoffman SL, Janse CJ, Khan SM, Sauerwein RW - Elife (2014)

Genotype analysis of the generated PfΔslarp and PfΔb9Δslarp parasites.(A) Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B) and PCR products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δslarp-a, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δslarp-b, 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δb9-b, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (B) Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the 5′ slarp targeting region (P: 5′ slarp-T; see A) on the left side of the slarp Southern or probed with the 3′slarp targeting region (P: 3′ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9, integration DNA was digested with restriction enzymes (E: RcaI) and probed with the 5′ b9 targeting region (P: 5′ b9-T; see A) on the right panel. The expected fragment sizes are indicated in panel (A). (C) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B) and generated the expected 130 bp fragment.DOI:http://dx.doi.org/10.7554/eLife.03582.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273440&req=5

fig2s2: Genotype analysis of the generated PfΔslarp and PfΔb9Δslarp parasites.(A) Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B) and PCR products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δslarp-a, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δslarp-b, 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δb9-b, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (B) Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the 5′ slarp targeting region (P: 5′ slarp-T; see A) on the left side of the slarp Southern or probed with the 3′slarp targeting region (P: 3′ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9, integration DNA was digested with restriction enzymes (E: RcaI) and probed with the 5′ b9 targeting region (P: 5′ b9-T; see A) on the right panel. The expected fragment sizes are indicated in panel (A). (C) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B) and generated the expected 130 bp fragment.DOI:http://dx.doi.org/10.7554/eLife.03582.009
Mentions: Next, we generated double gene-deletion PfΔb9Δslarp mutants using the FRT/FLPe recombinase methodology (van Schaijk et al., 2010). This methodology employs FLPe recombinase to remove a FRT-site flanked drug resistance marker cassette introduced into the Pf genome when the target gene has been removed by double cross-over homologous recombination as shown for PfΔslarp-b parasites in Figure 2—figure supplement 1,2. After cloning, this ‘marker-free’ line was subsequently transfected with the Pfb9 gene-targeting construct pHHT-FRT-GFP-b9 (Annoura et al., 2014) to delete the b9 locus from the PfΔslarp-b genome (Figure 2—figure supplement 1,2). Subsequently two ‘marker-free’ clones, PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9, were obtained containing the correct genotype that is removal of the slarp and b9 gene loci as well as both respective drug selection cassettes (Figure 2—figure supplement 2). In addition, we confirmed the loss of expression of both slarp and b9 by RT-PCR analysis by demonstrating the absence of transcripts in mRNA collected from PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 salivary gland sporozoites (Figure 2—figure supplement 2). We then examined the phenotype of PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 mutants during blood stage and mosquito development. Asexual blood stage growth of PfΔb9Δslarp parasites was normal as both clones reached an asexual parasitemia between 0.5 and 5% during cloning within 21 days and PfΔb9Δslarp clones produced WT-like numbers of gametocytes, oocysts, and sporozoites (Figure 2).

Bottom Line: In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development.The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection.These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands.

ABSTRACT
A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Show MeSH
Related in: MedlinePlus