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To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Copyright © 2020 Miyazaki, Marin-Mogollon, Imai, Mendes, van der Laak, Sturm, Geurten, Miyazaki, Chevalley-Maurel, Ramesar, Kolli, Kroeze, van Schuijlenburg, Salman, Wilder, Reyes-Sandoval, Dechering, Prudencio, Janse, Khan and ̂ Franke-Fayard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP., CM-M was, in part, supported by Colciencias Ph.D. fellowship (Call 568 from 2012 Resolution 01218 Bogotá, Colombia). TI was, in part, supported by Uehara Memorial Foundation grant. Work performed at IMM was supported by Fundação para a Ciência e Tecnologia (FCT-Portugal)’s grants PTDC/BBB-BMD/2695/2014 and PTDC-SAU-INF-29550-2017. AR-S is supported by the MRC-DPFS grant MR/N019008/1.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11ahiCD44hi) CD8+ and CD4+ T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4+ T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

Additional file 7. Generation and genotyping a P. falciparum mutant line lacking expression of CSP (PfΔcsp). A. The PfΔcsp line was generated using CRISPR/Cas9 methodology. The pfcsp gene was replaced by insertion of a mCherry expression cassette (mCherry under control of the of the constitutive gapdh promoter) using donor-DNA plasmids pLf0086. A schematic representation of the pfcsp locus before and after insertion of the construct showing the location of the restriction sites (A: AvaII), sizes (in bp) of restriction fragments (red for Southern blot analysis), location of primers (p), PCR amplicons and sizes of the fragments (in black) used to analyse correct disruption of the pfcsp and insertion of the mCherry cassette (B, C). HR1, HR2: pfcsp homology (targeting) regions. The figure is not shown to scale. Primer sequences can be found in Additional file 8. B. Diagnostic PCR confirming correct integration of the mCherry cassette into the PfCSP locus. 5′ integration PCR (lane 2; primers p15/p19); pfcsp open reading frame (lane 3; primers p17/p18); P. falciparum sequestrin as a control gene (lane 1; primers p22/p23); mCherry gene (lane 4; primers P20/P21) of cloned parasites of PfΔcsp (cl3) and WT. C. Southern blot analysis of AvaII/XhoI restricted DNA of WT and PfΔcsp parasites confirms the specific integration of the mCherry cassette into the pfcsp gene locus. DNA was hybridized with a probe targeting the homology region 2 (upper panels; HR2; primers p3/p4; see (A) of pfcsp. The hybridization pattern observed with the HR2 probe identified the expected different-sized DNA fragments in WT and pf-pvcsp parasites (2057 bp and 4105 bp). In addition to show absence of donor-DNA plasmid and presence of single cross-over events DNA was hybridized with a probe for the ampicillin gene (lower panels; intermediate donor-DNA plasmid pLf0040 digested with AatII and PvuI).

Introduction Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

Additional file 1. Vector maps of the P. vivax csp genes introduced into P. falciparum. A. Vector maps of the different plasmids used to generate two chimeric P. falciparum parasite lines (pf-pvcsp) expressing P. vivax CSP(VK210) of CSP(VK247) and a P. falciparum line lacking expression of CSP (PfÎ csp). See â Methodsâ section for description and details of the generation of these plasmids.

Additional file 5. Degenerated oocysts of two chimeric P. falciparum parasite lines (pf-pvcsp) and a PfCSP knockout line (PfΔcsp). Light microscope pictures of degenerated oocysts at day 10 after feeding gametocytes to Anopheles stephensi mosquitoes. These oocysts are classified as degenerate based on the absence of sporozoite formation and vacuolated cytoplasm. See Fig. 2 for pf-pvcsp and wild type P. falciparum (WT) oocyst in which sporozoite formation occurred. No sporozoite formation was observed in PfΔcsp oocysts. Scale bar, 20 µm.

Additional file 8. Chimeric rodent parasite lines pb-pvcsp(vk210) and pb-pvcsp(vk247) are able to produce salivary gland sporozoites that are infectious to mice. A. Upper panel, left: Light microscope pictures of wild type P. berghei (WT) and pb-pvcsp oocyst at days 7, 10 and 14 after feeding of An. stephensi mosquitoes. Black arrows indicate oocyst with sporozoite formation and white arrows indicate degenerated, vacuolated oocyst without sporozoite formation (scale bar, 10 µm). Upper panel, right, Percentage of degenerated oocyst in An. stephensi mosquitoes (n = 5) at day 14 after feeding (***P =

Additional file 8. Chimeric rodent parasite lines pb-pvcsp(vk210) and pb-pvcsp(vk247) are able to produce salivary gland sporozoites that are infectious to mice. A. Upper panel, left: Light microscope pictures of wild type P. berghei (WT) and pb-pvcsp oocyst at days 7, 10 and 14 after feeding of An. stephensi mosquitoes. Black arrows indicate oocyst with sporozoite formation and white arrows indicate degenerated, vacuolated oocyst without sporozoite formation (scale bar, 10 µm). Upper panel, right, Percentage of degenerated oocyst in An. stephensi mosquitoes (n = 5) at day 14 after feeding (***P =

Additional file 4. Growth of asexual blood stages and mCherry expression in blood stages. Growth of asexual blood stages of two chimeric P. falciparum parasite lines (pf-pvcsp(vk210) and pf-pvcsp(vk247), a P. falciparum line lacking expression of CSP (PfΔcsp) and P. falciparum wild type (WT) parasites. Parasites of the different cloned lines were cultured in semi-automated culture system for a period of 7 days. Cultures were initiated with a parasitaemia of 0.5%. Arrows indicate the dilution of the cultures with fresh red blood cells to have a final parasitaemia of 1%. B. mCherry-expressing blood stages of PfΔcsp parasites where the csp gene has been disrupted by insertion of an mCherry expression cassette (see Additional file 5 for details of the generation of PfΔcsp). Scale bar, 7 µm.

Additional file 3. Sequence analysis of the P. vivax csp genes introduced into P. falciparum. Long-range PCR fragments (see Fig. 1) were cloned into pET TOPO TA (Invitrogen) and sequenced. See Additional file 2 for primer sequences used for sequencing the complete fragment. A. LR-PCR fragment of parasites of pf-pvcsp(vk210). B. LR-PCR fragment of parasites of pf-pvcsp(vk247).

Additional file 4. Growth of asexual blood stages and mCherry expression in blood stages. Growth of asexual blood stages of two chimeric P. falciparum parasite lines (pf-pvcsp(vk210) and pf-pvcsp(vk247), a P. falciparum line lacking expression of CSP (PfΔcsp) and P. falciparum wild type (WT) parasites. Parasites of the different cloned lines were cultured in semi-automated culture system for a period of 7 days. Cultures were initiated with a parasitaemia of 0.5%. Arrows indicate the dilution of the cultures with fresh red blood cells to have a final parasitaemia of 1%. B. mCherry-expressing blood stages of PfΔcsp parasites where the csp gene has been disrupted by insertion of an mCherry expression cassette (see Additional file 5 for details of the generation of PfΔcsp). Scale bar, 7 µm.

Additional file 5. Degenerated oocysts of two chimeric P. falciparum parasite lines (pf-pvcsp) and a PfCSP knockout line (PfΔcsp). Light microscope pictures of degenerated oocysts at day 10 after feeding gametocytes to Anopheles stephensi mosquitoes. These oocysts are classified as degenerate based on the absence of sporozoite formation and vacuolated cytoplasm. See Fig. 2 for pf-pvcsp and wild type P. falciparum (WT) oocyst in which sporozoite formation occurred. No sporozoite formation was observed in PfΔcsp oocysts. Scale bar, 20 µm.

Additional file 1. Vector maps of the P. vivax csp genes introduced into P. falciparum. A. Vector maps of the different plasmids used to generate two chimeric P. falciparum parasite lines (pf-pvcsp) expressing P. vivax CSP(VK210) of CSP(VK247) and a P. falciparum line lacking expression of CSP (PfÎ csp). See â Methodsâ section for description and details of the generation of these plasmids.

Additional file 3. Sequence analysis of the P. vivax csp genes introduced into P. falciparum. Long-range PCR fragments (see Fig. 1) were cloned into pET TOPO TA (Invitrogen) and sequenced. See Additional file 2 for primer sequences used for sequencing the complete fragment. A. LR-PCR fragment of parasites of pf-pvcsp(vk210). B. LR-PCR fragment of parasites of pf-pvcsp(vk247).