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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03582</article-id><article-id pub-id-type="doi">10.7554/eLife.03582</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group><subj-group subj-group-type="heading"><subject>Microbiology and infectious disease</subject></subj-group></article-categories><title-group><article-title>A genetically attenuated malaria vaccine candidate based on <italic>P. falciparum b9/slarp</italic> gene-deficient sporozoites</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-15894" equal-contrib="yes"><name><surname>van Schaijk</surname><given-names>Ben C L</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15895" equal-contrib="yes"><name><surname>Ploemen</surname><given-names>Ivo H J</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="pa1">‡</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15896"><name><surname>Annoura</surname><given-names>Takeshi</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="pa2">§</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15897"><name><surname>Vos</surname><given-names>Martijn W</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-22033"><name><surname>Foquet</surname><given-names>Lander</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15899"><name><surname>van Gemert</surname><given-names>Geert-Jan</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15900"><name><surname>Chevalley-Maurel</surname><given-names>Severine</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15901"><name><surname>van de Vegte-Bolmer</surname><given-names>Marga</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15902"><name><surname>Sajid</surname><given-names>Mohammed</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15903"><name><surname>Franetich</surname><given-names>Jean-Francois</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15904"><name><surname>Lorthiois</surname><given-names>Audrey</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15905"><name><surname>Leroux-Roels</surname><given-names>Geert</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15906"><name><surname>Meuleman</surname><given-names>Philip</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15907"><name><surname>Hermsen</surname><given-names>Cornelius C</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15908"><name><surname>Mazier</surname><given-names>Dominique</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="aff" rid="aff7">7</xref><xref ref-type="fn" rid="con15"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15909"><name><surname>Hoffman</surname><given-names>Stephen L</given-names></name><xref ref-type="aff" rid="aff6">6</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con18"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-15910"><name><surname>Janse</surname><given-names>Chris J</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con16"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-22034"><name><surname>Khan</surname><given-names>Shahid M</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="con17"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15912"><name><surname>Sauerwein</surname><given-names>Robert W</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con19"/><xref ref-type="fn" rid="conf2"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Department of Medical Microbiology</institution>, <institution>Radboud University Nijmegen Medical Center</institution>, <addr-line><named-content content-type="city">Nijmegen</named-content></addr-line>, <country>Netherlands</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">Leiden Malaria Research Group, Parasitology</institution>, <institution>Leiden University Medical Center</institution>, <addr-line><named-content content-type="city">Leiden</named-content></addr-line>, <country>Netherlands</country></aff><aff id="aff3"><label>3</label><institution content-type="dept">Center for Vaccinology</institution>, <institution>Ghent University and University Hospital</institution>, <addr-line><named-content content-type="city">Ghent</named-content></addr-line>, <country>Belgium</country></aff><aff id="aff4"><label>4</label><institution content-type="dept">Centre d'Immunologie et des Maladies Infectieuses</institution>, <institution>Université Pierre et Marie Curie-Paris 6</institution>, <addr-line><named-content content-type="city">Paris</named-content></addr-line>, <country>France</country></aff><aff id="aff5"><label>5</label><institution content-type="dept">Centre d'Immunologie et des Maladies Infectieuses</institution>, <institution>INSERM, U1135, Paris</institution>, <addr-line><named-content content-type="city">Paris</named-content></addr-line>, <country>France</country></aff><aff id="aff6"><label>6</label><institution>Sanaria Inc.</institution>, <addr-line><named-content content-type="city">Rockville</named-content></addr-line>, <country>United States</country></aff><aff id="aff7"><label>7</label><institution content-type="dept">Service Parasitologie-Mycologie</institution>, <institution>Assistance Publique—Hôpitaux de Paris, Groupe hospitalier Pitié-Salpêtrière</institution>, <addr-line><named-content content-type="city">Paris</named-content></addr-line>, <country>France</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>White</surname><given-names>Nicholas J</given-names></name><role>Reviewing editor</role><aff><institution>Mahidol University</institution>, <country>Thailand</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>Robert.Sauerwein@radboudumc.nl</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>‡</label><p>Institute for Translational Vaccinology, Bilthoven, Netherlands</p></fn><fn fn-type="present-address" id="pa2"><label>§</label><p>Department of Tropical Medicine, Jikei University School of Medicine, Tokyo, Japan</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>19</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03582</elocation-id><history><date date-type="received"><day>04</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>19</day><month>11</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, van Schaijk et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>van Schaijk et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03582.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03582.001</object-id><p>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 <italic>Plasmodium falciparum</italic> 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 <italic>b9</italic> and <italic>slarp</italic>, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔ<italic>b9</italic>Δ<italic>slarp</italic>GAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔ<italic>b9</italic>Δ<italic>slarp</italic>GAP 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 PfΔ<italic>b9</italic>Δ<italic>slarp</italic>SPZ vaccine.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.001">http://dx.doi.org/10.7554/eLife.03582.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03582.002</object-id><title>eLife digest</title><p>Vaccines commonly contain a weakened or dead version of a disease-causing microorganism, or its toxins, or surface proteins. These prime the immune system to rapidly recognize, respond to, and eliminate the actual infectious pathogen if later encountered.</p><p>While vaccines are currently available to help prevent a large number of diseases, vaccines for many deadly diseases, including malaria, do not yet exist. Malaria is caused by a group of parasites called <italic>Plasmodium</italic>, which are transferred to humans by mosquitoes. While measures to control mosquito populations and prevent mosquito bites have helped to reduce the incidence of malaria in some countries, the number of people—and especially children—that die of malaria every year remains very high.</p><p>When a mosquito carrying <italic>Plasmodium</italic> in its salivary glands bites a human, the parasite is injected into the human's bloodstream and travels to the liver. The parasite reproduces in the liver cells until there are so many of them that the cells rupture, and the parasites are released back into the bloodstream. Any mosquito that then feeds on the blood of the infected individual may also suck up the parasite. The parasite then goes through a further stage of development in the mosquito, eventually migrating to the salivary glands, from where the parasite can be transmitted into a new human host.</p><p>Recent work in rodents suggests that genetically altered or weakened <italic>Plasmodium falciparum</italic> sporozoites—the form of the parasite found in mosquito saliva—could be used to vaccinate humans against malaria caused by this parasite species. Now, van Schaijk, Ploemen et al. evaluate whether a safe and effective vaccine could be made from sporozoites that lack two genes, called b9 and slarp, which are critical for the parasites to develop inside liver cells. When mice were injected with the modified sporozoites, their immune cells were able to detect the parasites and respond against them. The mice subsequently did not develop malaria when they were infected with normal, unmodified parasites. Furthermore, none of the mice contracted malaria from the modified sporozoites.</p><p>The modified sporozoites behaved similarly in human liver cells: after invading these cells, the parasites were unable to develop. Clinical testing and further development are now needed to see if a successful malaria vaccine can be made from these sporozoites.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.002">http://dx.doi.org/10.7554/eLife.03582.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd><italic>Plasmodium</italic></kwd><kwd>sporozoite</kwd><kwd>genetically attenuated parasite</kwd><kwd>malaria</kwd><kwd>vaccine</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>human</kwd><kwd>mouse</kwd><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004522</institution-id><institution>Top Institute Pharma (TI Pharma)</institution></institution-wrap></funding-source><award-id>T4-102</award-id><principal-award-recipient><name><surname>van Schaijk</surname><given-names>Ben C L</given-names></name><name><surname>Ploemen</surname><given-names>Ivo H J</given-names></name><name><surname>Annoura</surname><given-names>Takeshi</given-names></name><name><surname>Vos</surname><given-names>Martijn W</given-names></name><name><surname>van Gemert</surname><given-names>Geert-Jan</given-names></name><name><surname>Chevalley-Maurel</surname><given-names>Severine</given-names></name><name><surname>van de Vegte-Bolmer</surname><given-names>Marga</given-names></name><name><surname>Sajid</surname><given-names>Mohammed</given-names></name><name><surname>Hermsen</surname><given-names>Cornelius C</given-names></name><name><surname>Hoffman</surname><given-names>Stephen L</given-names></name><name><surname>Janse</surname><given-names>Chris J</given-names></name><name><surname>Sauerwein</surname><given-names>Robert W</given-names></name></principal-award-recipient></award-group><funding-statement>The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The preclinical evaluation of a malaria genetically attenuated parasite vaccine candidate suggests that it is both safe and effective.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>A vaccine that induces high-level (>90%) sterile protection by inducing immunity that attacks the non-pathologic, asymptomatic pre-erythrocytic stages of <italic>Plasmodium falciparum</italic> (Pf) will prevent infection, disease, and transmission and could be a powerful instrument to eliminate Pf malaria from geographically defined areas (<xref ref-type="bibr" rid="bib36">Plowe et al., 2009</xref>; <xref ref-type="bibr" rid="bib27">malERA Consultative Group on Vaccines, 2011</xref>). In rodent models, sterile protection can be induced by immunization with live <italic>Plasmodium</italic> sporozoites attenuated by either irradiation, genetic modification (GAP), or by concomitant anti-parasitic drug treatment (For reviews see <xref ref-type="bibr" rid="bib17">Hoffman et al., 2010</xref>; <xref ref-type="bibr" rid="bib6">Butler et al., 2012</xref>; <xref ref-type="bibr" rid="bib21">Khan et al., 2012</xref>; <xref ref-type="bibr" rid="bib31">Nganou-Makamdop and Sauerwein, 2013</xref>). In humans, induction of complete sustained protective immunity against a challenge infection has been achieved by previous exposure to the bites of mosquitoes infected with i) live radiation-attenuated <italic>Plasmodium</italic> sporozoites that invade but then completely arrest in the liver (<xref ref-type="bibr" rid="bib7">Clyde et al., 1973</xref>; <xref ref-type="bibr" rid="bib16">Hoffman et al., 2002</xref>) and ii) live sporozoites in volunteers taking chloroquine chemoprophylaxis (CPS) with full parasite liver-stage development; once released into the circulation asexual blood stages are killed by chloroquine (<xref ref-type="bibr" rid="bib41">Roestenberg et al., 2009</xref>, <xref ref-type="bibr" rid="bib42">2011</xref>). More recently it has been demonstrated for the first time that sterile immunity can be achieved by intravenous immunization with radiation-attenuated aseptic, purified, cryopreserved Pf sporozoites (SPZ) called PfSPZ Vaccine (<xref ref-type="bibr" rid="bib45">Seder et al., 2013</xref>).</p><p>From a product manufacturing perspective, GAPs have the clear advantage of representing a homogeneous parasite population with a defined genetic identity. The genetic attenuation is an irreversible, intrinsic characteristic of the parasite that does not require additional manufacturing steps like irradiation. Furthermore, in the manufacturing process of GAP-infected mosquitoes, operators are never exposed to Pf parasites that can cause disease. However, clinical development of GAPs has suffered from safety problems related to breakthrough infections during immunization leading to pathological blood stage infections responsible for clinical symptoms and complications. Strains of mice showed differential susceptibility to breakthrough infections after injection of sporozoites of rodent malaria GAPs, demonstrating the need for extensive preclinical rodent screening (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). The <italic>P. falciparum</italic> GAP PfΔ<italic>p52</italic>Δ<italic>p36</italic> is the only GAP so far that has been assessed in humans but the trial in which the Pf sporozoites were administered by mosquito bite had to be terminated, because of breakthrough infections in one volunteer during immunization (<xref ref-type="bibr" rid="bib47">Spring et al., 2013</xref>). Our in vitro experiments with PfΔ<italic>p52</italic>Δ<italic>p36</italic> confirm that this double gene deletion GAP (i.e. two genes removed from the genome) is not fully attenuated similar to the equivalent rodent GAP PbΔ<italic>p52</italic>Δ<italic>p36</italic> in the <italic>Plasmodium berghei/</italic>C57BL/6 model (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). Therefore, identification of additional genes critical and uniquely selective for liver-stage development has become a major challenge for GAP vaccine development (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>; <xref ref-type="bibr" rid="bib21">Khan et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Ploemen et al., 2012</xref>). Furthermore, single gene deletion GAPs will most likely not be adequate (<xref ref-type="bibr" rid="bib34">Ploemen et al., 2012</xref>).</p><p>This prompted us to generate and test a GAP with deletions of two independent genes critical for liver-stage development. We recently identified a novel <italic>P. berghei</italic> (Pb) gene deletion mutant, PbΔ<italic>b9</italic>, lacking the expression of the B9 protein (Pf ortholog: PFC_0750w; PF3D7_0317100) (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). This protein is a newly identified member of the <italic>Plasmodium</italic> 6-Cys family of proteins. Initial safety evaluation in rodents demonstrated that PbΔ<italic>b9</italic> mutants have a stronger attenuation phenotype than mutants lacking the 6-Cys proteins P52 and P36 (<xref ref-type="bibr" rid="bib51">van Dijk et al., 2005</xref>; <xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>; <xref ref-type="bibr" rid="bib56">VanBuskirk et al., 2009</xref>; <xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). As second target gene for liver-stage attenuation, we selected the <italic>slarp</italic> and <italic>sap1</italic> orthologs reported in Pb and <italic>Plasmodium yoelii</italic> (Py), respectively (Pf ortholog: PF11_0480; PF3D7_1147000; hereafter termed <italic>slarp</italic>). These <italic>slarp</italic> mutants show an excellent safety profile by full arrest in the liver in mice (<xref ref-type="bibr" rid="bib1">Aly et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>). The SLARP protein is expressed in sporozoites and in early liver-stages and is involved in the regulation of transcription (<xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>; <xref ref-type="bibr" rid="bib2">Aly et al., 2011</xref>).</p><p>In this study, we report the generation and evaluation of a rodent GAP lacking the genes encoding for B9 and SLARP (PbΔ<italic>b9</italic>Δ<italic>slarp</italic>) and the generation and evaluation of the equivalent human Pf GAP lacking the Pf ortholog genes. PfΔ<italic>b9</italic>Δ<italic>slarp</italic> was generated using constructs that allowed for the removal of the drug selectable marker from the genome by FRT/FLPe recombinase methodology (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>). The safety and efficacy of PbΔ<italic>b9</italic>Δ<italic>slarp</italic> and the lack of development of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> in human hepatocytes, in vitro, and, in vivo, in chimeric mice provide strong support for clinical development of a PfΔ<italic>b9</italic>Δ<italic>slarp</italic> PfSPZ vaccine.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>Arrest of liver-stage development and induced protection after <italic>P. berghei</italic> Δ<italic>b9</italic>Δ<italic>slarp</italic> GAP</title><p>Previously, we generated a Pb mutant with disruption of the <italic>b9</italic> locus (PbΔ<italic>b9</italic>) by standard genetic modification using a double cross-over integration event, followed by removal of the drug-selectable marker cassette by negative selection (<xref ref-type="bibr" rid="bib24">Lin et al., 2011</xref>). Characterization of the PbΔ<italic>b9</italic> phenotype showed that liver-stage development was fully abrogated in BALB/c mice and severely compromised in the more stringent C57BL/6 murine model for <italic>P. berghei</italic> (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). Immunization of a single dose of 10k (i.e. 10,000 sporozoites) or 5k PbΔ<italic>b9</italic> protected BALB/c mice against a 10k WT-sporozoite challenge, while 80% of mice were still protected after a single 1k immunizing dose (<xref ref-type="table" rid="tbl1">Table 1</xref>). In C57BL/6 mice, immunization with 50K/20K/20K of PbΔ<italic>b9</italic> resulted in complete protection lasting up to 180 days, reducing to 45% protection when challenged at 1 year post-immunization. However, sporozoite administration occasionally resulted in blood stage infections after administration of high doses, thereby compromising the safety profile (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03582.003</object-id><label>Table 1.</label><caption><p>Protection of mice after immunization with <italic>P. berghei</italic> PbΔ<italic>b9</italic> or PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.003">http://dx.doi.org/10.7554/eLife.03582.003</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center">Mouse strain</th><th align="center">Pb mutant</th><th align="center">Day of challenge<xref ref-type="table-fn" rid="tblfn1">*</xref></th><th colspan="3" align="center">Immunization regimes no. protected/no challenged</th></tr></thead><tbody><tr><td><bold>BALB/c</bold></td><td/><td/><td align="center"><bold>10k</bold><xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="center"><bold>5k</bold></td><td align="center"><bold>1k</bold></td></tr><tr><td/><td>PbΔ<italic>b9</italic></td><td align="center">10</td><td align="center">10/10<xref ref-type="table-fn" rid="tblfn3">‡</xref></td><td align="center">18/20</td><td align="center">8/10</td></tr><tr><td/><td>PbΔb9Δslarp</td><td align="center">10</td><td align="center">20/20</td><td align="center">10/10</td><td align="center">20/20</td></tr><tr><td><bold>C57Bl6</bold></td><td/><td/><td align="center"><bold>50/20/20k</bold><xref ref-type="table-fn" rid="tblfn4">§</xref></td><td align="center"><bold>10/10/10k</bold></td><td align="center"><bold>1/1/1k</bold></td></tr><tr><td rowspan="4"/><td rowspan="4">PbΔ<italic>b9</italic></td><td align="center">10</td><td align="center">4/4</td><td rowspan="4" align="center">nd</td><td rowspan="4" align="center">nd</td></tr><tr><td align="center">90</td><td align="center">5/5</td></tr><tr><td align="center">180</td><td align="center">9/9<xref ref-type="table-fn" rid="tblfn5">#</xref></td></tr><tr><td align="center">365</td><td align="center">5/11</td></tr><tr><td rowspan="2"/><td rowspan="2">PbΔb9Δslarp</td><td align="center">10</td><td align="center">Nd</td><td align="center">10/10</td><td align="center">6/10</td></tr><tr><td align="center">180</td><td align="center">6/6</td><td align="center">nd</td><td align="center">nd</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>Number of days post last immunization; 10<sup>4</sup> wild-type sporozoites were injected by IV route.</p></fn><fn id="tblfn2"><label>†</label><p>Immunization dose: number of sporozoites x1000.</p></fn><fn id="tblfn3"><label>‡</label><p>Protected/total # of immunized mice (%); protection was 0/15 in naive control BALB/c and 0/10 in C57BL/6 mice.</p></fn><fn id="tblfn4"><label>§</label><p>Immunization dose with 7 day intervals between immunizations.</p></fn><fn id="tblfn5"><label>#</label><p>Immunization dose 50/10/20k with 7 day intervals between immunizations. nd = not done.</p></fn></table-wrap-foot></table-wrap></p><p>Previously, it has been shown by others that PbΔ<italic>slarp</italic> parasites are completely arrested in liver-stage development with a complete lack of breakthrough blood-stage infections (<xref ref-type="bibr" rid="bib1">Aly et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>). Therefore, we generated a new single gene deletion mutant PbΔ<italic>slarp</italic> in a parasite line that constitutively expressed a fusion of the reporter proteins GFP and luciferase, using a <italic>slarp</italic>-targeting DNA-construct for deletion by double cross-over homologous integration (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). The PbΔ<italic>slarp</italic> mutant showed blood stage growth and mosquito infections with functional sporozoites similar to wild-type (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). However, intravenous injection of up to 500k PbΔ<italic>slarp</italic> sporozoites never led to full development of parasites in the liver as assayed by in vivo imaging (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>) or analysis of blood stage infection (<xref ref-type="table" rid="tbl2">Table 2</xref>). PbΔ<italic>slarp</italic> sporozoites arrested very soon after invasion of cultured Huh7 hepatocytes corroborating the excellent safety findings by <xref ref-type="bibr" rid="bib46">Silvie et al. (2008)</xref>.<table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03582.004</object-id><label>Table 2.</label><caption><p>Breakthrough blood-stage infections after intravenous injection of PbΔ<italic>slarp</italic> and PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.004">http://dx.doi.org/10.7554/eLife.03582.004</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Mouse strain</th><th>Mutant</th><th align="center">Infection<xref ref-type="table-fn" rid="tblfn6">*</xref> Spz x 10<sup>3</sup></th><th align="center">Breakthrough blood infection/total # mice</th><th align="center">Pre-patent period<xref ref-type="table-fn" rid="tblfn8">‡</xref> (days)</th></tr></thead><tbody><tr><td><bold>BALB/c</bold></td><td>WT<xref ref-type="table-fn" rid="tblfn7">†</xref></td><td align="center">10</td><td align="center">5/5</td><td align="center">4–5</td></tr><tr><td/><td>PbΔ<italic>slarp</italic></td><td align="center">50</td><td align="center">0/5</td><td/></tr><tr><td/><td>PbΔ<italic>slarp</italic></td><td align="center">25</td><td align="center">0/10</td><td/></tr><tr><td/><td>PbΔ<italic>b9</italic>Δ<italic>slarp</italic></td><td align="center">25</td><td align="center">0/10</td><td/></tr><tr><td><bold>C57BL/6</bold></td><td>WT<xref ref-type="table-fn" rid="tblfn7">†</xref></td><td align="center">10</td><td align="center">5/5</td><td align="center">4–5</td></tr><tr><td/><td>PbΔ<italic>slarp</italic></td><td align="center">500</td><td align="center">0/5</td><td/></tr><tr><td/><td>PbΔ<italic>slarp</italic></td><td align="center">200</td><td align="center">0/10</td><td/></tr><tr><td/><td>PbΔ<italic>b9</italic>Δ<italic>slarp</italic></td><td align="center">200</td><td align="center">0/10</td><td/></tr><tr><td/><td>PbΔ<italic>b9</italic>Δ<italic>slarp</italic></td><td align="center">150</td><td align="center">0/5</td><td/></tr></tbody></table><table-wrap-foot><fn id="tblfn6"><label>*</label><p>Inoculation dose of sporozoites administered IV.</p></fn><fn id="tblfn7"><label>†</label><p><italic>P. berghei</italic> ANKA strain: line cl15cy1.</p></fn><fn id="tblfn8"><label>‡</label><p>Day with parasitemia of 0.5–2%.</p></fn></table-wrap-foot></table-wrap></p><p>Therefore, in order to create a completely attenuated and safe rodent GAP, we additionally disrupted the <italic>slarp</italic> gene in the PbΔ<italic>b9</italic> genome by double cross-over integration (<xref ref-type="fig" rid="fig1">Figure 1</xref>). Asexual growth and sporogonic development/function equaled wild-type (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). However, PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites arrested soon after invasion of cultured Huh7 hepatocytes (<xref ref-type="fig" rid="fig1">Figure 1</xref>) and intravenous injection of 150–200K PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites never resulted in breakthrough blood-stage infections in mice (<xref ref-type="table" rid="tbl2">Table 2</xref>). Finally, protective efficacy induced by PbΔ<italic>b9</italic>Δ<italic>slarp</italic> was studied in both BALB/c and C57BL/6 mice. A single immunization dose of 10K, 5K, or even 1K of PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites in BALB/c mice induced full protection against a 10K wild-type sporozoite challenge (<xref ref-type="table" rid="tbl1">Table 1</xref>). C57BL/6 mice were 100% protected after 3 × 10K immunization with PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites, and the protective efficacy reduced to 60% after a 3 × 1K immunization dose. A challenge at day 180 post-immunization of a 50/20/20K dose still resulted in complete protection. The combined data showed that PbΔ<italic>b9</italic>Δ<italic>slarp</italic> completely arrest during liver-stage development and induce a highly efficient protective immunity in two different strains of mice.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03582.005</object-id><label>Figure 1.</label><caption><title>Generation and genotype analyses of <italic>P. berghei</italic> mutant PbΔ<italic>b9</italic>Δ<italic>slarp.</italic></title><p>(<bold>A</bold>) Generation of mutant PbΔ<italic>b9</italic>Δ<italic>slarp</italic>. For PbΔ<italic>b9</italic>Δ<italic>slarp</italic>, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette <italic>hdhfr/yfcy</italic>. This construct was subsequently used to generate the mutant PbΔ<italic>b9</italic>Δ<italic>slarp</italic> in the PbΔ<italic>b9</italic>Δ<italic>sm</italic> mutant. See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref> for the sequence of the primers. (<bold>B</bold>) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant PbΔ<italic>b9</italic>Δ<italic>slarp</italic> confirming correct disruption of the <italic>slarp</italic> and the <italic>b9</italic> locus. See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref> for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the <italic>slarp</italic> and the <italic>b9</italic> ORF. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR <italic>pbdhfr</italic> probe that recognizes the construct integrated into <italic>P. berghei slarp</italic> locus on chromosome 9, the endogenous locus of <italic>dhfr/ts</italic> on chromosome 7, and a 3′UTR <italic>pbdhfr</italic> probe that recognizes the construct integrated into <italic>P. berghei b9</italic> locus on chromosome 8. In addition, the chromosomes were hybridized with the <italic>hdhfr</italic> probe recognizing the integrated construct into the <italic>slarp</italic> locus on chromosome 9. (<bold>C</bold>) Development of liver-stages in cultured hepatocytes as visualized by staining with antibodies recognizing the parasitophorous vacuole membrane (anti-EXP1; green) and the parasite cytoplasm (anti-HSP70; red). Nuclei are stained with Hoechst-33342. Hpi: hours post-infection. Scale bar represents 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.005">http://dx.doi.org/10.7554/eLife.03582.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03582.006</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Generation and genotype analyses of <italic>P. berghei</italic> mutant PbΔ<italic>slarp-</italic>a<italic>.</italic></title><p>(<bold>A</bold>) Generation of mutant PbΔ<italic>slarp</italic>-a. For PbΔ<italic>slarp</italic>-a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette <italic>hdhfr</italic>/<italic>yfcy</italic>. This construct was subsequently used to generate the mutant PbΔ<italic>slarp</italic>-a (1839cl3) in the <italic>Pb</italic>GFP-Luc<sub>con</sub> reference line. See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref> for the sequence of the primers. (<bold>B</bold>) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ<italic>slarp</italic>-a confirming correct disruption of the <italic>slarp</italic>-locus. See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref> for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the <italic>slarp</italic> ORF. Mutant PbΔ<italic>slarp</italic>-a has been generated in the reference <italic>P. berghei</italic> ANKA line <italic>Pb</italic>GFP-Luc<sub>con</sub> which has a <italic>gfp-luciferase</italic> gene integrated into the silent <italic>230p</italic> locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR <italic>pbdhfr</italic> probe that recognizes the construct integrated into <italic>P. berghei slarp</italic> locus on chromosome 9, the endogenous locus of <italic>dhfr/ts</italic> on chromosome 7, and the <italic>gfp-luciferase</italic> gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the <italic>hdhfr</italic> probe recognizing the integrated construct into the <italic>slarp</italic> locus on chromosome 9. (<bold>C</bold>) Real time in vivo imaging of <italic>Δslarp</italic> luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10<sup>4</sup> <italic>Pb</italic>-GFPLuc<sub>con</sub> sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10<sup>5</sup> Pb<italic>Δslarp-a</italic> sporozoites (n = 5) (lower panel: representative image of Pb<italic>Δslarp-luc</italic> infected mice).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.006">http://dx.doi.org/10.7554/eLife.03582.006</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582fs001"/></fig></fig-group></p></sec><sec id="s2-2"><title>Generation of a <italic>P. falciparum</italic> Δ<italic>b9</italic>Δ<italic>slarp</italic> GAP</title><p>Considering the desired phenotype as observed in <italic>P. berghei</italic>, we generated a Pf mutant lacking expression of both B9 (PF3D7_0317100) and SLARP (PF3D7_1147000; sporozoite asparagine-rich protein). These genes are conserved between rodent and human species, both at the level of syntenic location in their respective genomes on chromosomes 3 and 11 respectively, and at the sequence level. Pf<italic>b9</italic> shows 37% amino acid sequence identity and 54% sequence similarity with Pb<italic>b9</italic> ((<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>)); Pf<italic>slarp</italic> shows 28% amino acid sequence identity and 46% sequence similarity with Pb<italic>slarp.</italic></p><p>First, we generated two independent Pf mutants lacking <italic>slarp</italic> by standard double cross-over integration of a DNA construct and analyzed their phenotype throughout the parasite life cycle (<xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplement 1,2</xref>). Blood-stage development of two independently derived PfΔ<italic>slarp</italic> (i.e. PfΔ<italic>slarp-a</italic> and<italic>–b</italic>) parasites was comparable to WT parasites. PfΔ<italic>slarp</italic> mutants produced WT numbers of gametocytes, oocysts and sporozoites (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The intra-cellular PfΔ<italic>slarp</italic>-<italic>a</italic> and -<italic>b</italic> parasite development in primary human hepatocytes was not significantly different in number and morphologically identical to WT parasites at 3 and 24 hours post-infection (hpi) (<xref ref-type="fig" rid="fig3">Figure 3</xref>). However, their number was more than 10-fold reduced at 48 hpi and not detectable from day 3 onwards to day 7 post-infection. Parasites lacking <italic>b9</italic> in <italic>P. falciparum</italic> arrested before day 2 post-infection of primary human hepatocytes with the exception of one observed liver schizont at a later timepoint (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). PfΔ<italic>slarp</italic>-<italic>a</italic> and -<italic>b</italic> parasites still showed positive HSP70 staining and morphologically normal parasites at 48 hpi in primary human hepatocytes, indicating time point of arrest later compared to PfΔ<italic>b9</italic> parasites.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03582.007</object-id><label>Figure 2.</label><caption><title>Phenotypes of <italic>P. falciparum</italic> PfΔ<italic>slarp</italic> and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites<italic>.</italic></title><p>(<bold>A</bold>) Gametocyte, oocyst, and sporozoite production. Gametocyte numbers (stage II and IV–V) per 1000 erythrocytes at day 8 and day 14 after the start of gametocyte cultures. Exflagellation (Exfl) of male gametocytes in stimulated samples from day 14 cultures (++ score = >10 exflagellation centers per microscope field at 400× magnification). Median number of oocysts at day 7, IQR is the inter quartile range and sporozoite (day 21) production (×1000) in <italic>A. stephensi</italic> mosquitoes. (<bold>B</bold>) Gliding motility of <italic>P. falciparum</italic> WT (cytochalasin D treated and untreated), PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 parasites. Gliding motility was quantified by determining the mean percentage ± standard deviation of parasites that exhibited gliding motility by producing characteristic CSP trails (≥1 circles) or parasites that did not produce CSP trails (0 circles). (<bold>C</bold>) Cell traversal ability of <italic>P. falciparum</italic> NF54, PfΔ<italic>slarp-b</italic> and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7 sporozoites as determined by FACS counting of Dextran positive Huh7 cells. Shown is the mean percentage ±standard deviation of FITC positive cells. Dextran control (control): hepatocytes cultured in the presence of Dextran but without the addition of sporozoites.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.007">http://dx.doi.org/10.7554/eLife.03582.007</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03582.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Consecutive gene deletion of <italic>slarp</italic> and <italic>b9</italic> in <italic>P. falciparum.</italic></title><p>Schematic representation of the genomic loci of (<bold>A</bold>) <italic>slarp</italic> (PF11_0480; PF3D7_1147000) on chromosome 11 (Chr. 11) and (<bold>B</bold>) <italic>b9</italic> (PFC_0750w; PF3D7_0317100) on chromosome 3 (Chr. 3) of wild-type (wt; NF54wcb), PfΔ<italic>slarp</italic> and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> gene deletion mutants before (PfΔ<italic>slarp</italic> a and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>) and after the FLPe mediated removal of the <italic>hdhfr::gfp</italic> resistance marker (PfΔ<italic>slarp</italic> b and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> clones F7/G9), respectively. The constructs for the targeted deletion of <italic>slarp</italic> (pHHT-FRT-GFP <italic>slarp</italic>) and <italic>b9</italic> (pHHT-FRT-GFP-B9) contain two FRT sequences (red triangles) that are recognized by FLPe. P1, P2 and P3, P4 primer pairs for LR-PCR analysis of <italic>slarp</italic> and <italic>b9</italic> loci respectively; T (<italic>Taq</italic>I) and R (<italic>Rca</italic>I): restriction sites used for Southern blot analysis and sizes of restriction fragment are indicated; <italic>cam:</italic> calmodulin<italic>; hrp:</italic> histidine rich protein<italic>; hsp:</italic> heatshock protein<italic>; fcu:</italic> cytosine deaminase/uracil phosphoribosyltransferase; <italic>hdhfr::gfp:</italic> human dihydrofolate reductase fusion with green fluorescent protein; <italic>pbdt: P. berghei dhfr</italic> terminator.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.008">http://dx.doi.org/10.7554/eLife.03582.008</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03582.009</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Genotype analysis of the generated PfΔ<italic>slarp</italic> and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites<italic>.</italic></title><p>(<bold>A</bold>) Long range PCR analysis of genomic DNA from WT, <italic>Pf</italic>Δ<italic>slarp</italic> and <italic>Pf</italic>Δ<italic>b9</italic>Δ<italic>slarp</italic> asexual parasites confirms the <italic>slarp</italic> gene deletion and consecutive gene deletions of both <italic>slarp</italic> and <italic>b9</italic> respectively and subsequent removal of the <italic>hdhfr::gfp</italic> 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 <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref>) and PCR products are also digested with restriction enzymes <italic>x</italic> (<italic>Xma</italic>I) and <italic>kx</italic> (<italic>Kpn</italic>I/<italic>Xcm</italic>I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ<italic>slarp-a</italic>, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ<italic>slarp-b</italic>, 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; Δ<italic>b9-b</italic>, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (<bold>B</bold>) Southern analysis of restricted genomic DNA from WT, PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 asexual parasites. DNA was digested with restriction enzyme (E: <italic>Taq</italic>I) and probed with the 5′ <italic>slarp</italic> targeting region (P: 5′ <italic>slarp</italic>-T; see <bold>A</bold>) on the left side of the <italic>slarp</italic> Southern or probed with the 3′<italic>slarp</italic> targeting region (P: 3′ <italic>slarp</italic>-T; see <bold>A</bold>) on the right side of the <italic>slarp</italic> panel. For analysis of the <italic>b9</italic>, integration DNA was digested with restriction enzymes (E: <italic>Rca</italic>I) and probed with the 5′ <italic>b9</italic> targeting region (P: 5′ <italic>b9</italic>-T; see <bold>A</bold>) on the right panel. The expected fragment sizes are indicated in panel (<bold>A</bold>). (<bold>C</bold>) RT-PCR analysis showing the absence of <italic>b9</italic> and <italic>slarp</italic> transcripts in <italic>P. falciparum</italic> PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-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 <italic>slarp</italic> and <italic>b9</italic> respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref>) and generated the expected 130 bp fragment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.009">http://dx.doi.org/10.7554/eLife.03582.009</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582fs003"/></fig></fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03582.010</object-id><label>Figure 3.</label><caption><title>Development of <italic>P. falciparum</italic> PfΔ<italic>slarp</italic> and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites in human primary hepatocytes.</title><p>(<bold>A</bold>) In vitro invasion of <italic>P. falciparum</italic> wt, PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 sporozoites in primary human hepatocytes. Invasion is represented as the mean ratio ± standard deviation of extra- and intra-cellular sporozoites by double staining at 3 and 24 hr post-infection, determined after three wash steps to remove sporozoites in suspension. (<bold>B</bold>) Immunofluorescence assay of PfΔ<italic>slarp-</italic>b parasites in human primary hepatocytes at 3 and 24 hr post-infection. Parasites are visualized by staining with anti-PfCSP antibodies (green; Alexa-488) and parasite, and hepatocyte nuclei are stained with DAPI (blue). Images were photographed on an Olympus FV1000 confocal microscope. Scale bar represents 5 µm. (<bold>C</bold>) Development of <italic>P. falciparum</italic> wt, PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b (top panel), PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 (bottom panel) liver-stages in primary human hepatocytes following inoculation with 40,000 sporozoites. From day 2 to 7, the mean number ± standard deviation of parasites per 96-well was determined by counting parasites stained with anti-<italic>P. falciparum</italic> HSP70 antibodies. The bottom panel represents experiments performed in primary human hepatocytes from 2 different donors. No parasites present (NP). (<bold>D</bold>) Development of liver-stages of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAP in chimeric mice engrafted with human hepatocytes. Mice were infected with 10<sup>6</sup> wt or PfΔ<italic>b9</italic>Δ<italic>slarp-G9</italic> sporozoites by intravenous inoculation. At 24 hr or at 5 days after sporozoite infection, livers were collected from the mice and the presence of parasites determined by qPCR of the parasite-specific 18S DNA. uPA HuHEP; chimeric homozygous uPA<sup>+/+</sup>-SCID mice engrafted with human hepatocytes. As controls, uPA mice; heterozygous uPA<sup>+/−</sup>-SCID mice not engrafted with human hepatocytes were used.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.010">http://dx.doi.org/10.7554/eLife.03582.010</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03582f003"/></fig></p><p>Next, we generated double gene-deletion PfΔ<italic>b9</italic>Δ<italic>slarp</italic> mutants using the FRT/FLPe recombinase methodology (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>). 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 PfΔ<italic>slarp-</italic>b parasites in <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplement 1,2</xref>. After cloning, this ‘marker-free’ line was subsequently transfected with the Pf<italic>b9</italic> gene-targeting construct pHHT-FRT-GFP-<italic>b9</italic> (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>) to delete the <italic>b9</italic> locus from the PfΔ<italic>slarp-</italic>b genome (<xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplement 1,2</xref>). Subsequently two ‘marker-free’ clones, PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>F7 and PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>G9, were obtained containing the correct genotype that is removal of the <italic>slarp</italic> and <italic>b9</italic> gene loci as well as both respective drug selection cassettes (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). In addition, we confirmed the loss of expression of both <italic>slarp</italic> and <italic>b9</italic> by RT-PCR analysis by demonstrating the absence of transcripts in mRNA collected from PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>F7 and PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>G9 salivary gland sporozoites (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). We then examined the phenotype of PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>F7 and PfΔ<italic>b9</italic>Δ<italic>slarp-</italic>G9 mutants during blood stage and mosquito development. Asexual blood stage growth of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites was normal as both clones reached an asexual parasitemia between 0.5 and 5% during cloning within 21 days and PfΔ<italic>b9</italic>Δ<italic>slarp</italic> clones produced WT-like numbers of gametocytes, oocysts, and sporozoites (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p></sec><sec id="s2-3"><title>Developmental arrest of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAPs in human hepatocytes</title><p>We next analyzed the development of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> in human hepatocytes using cultured primary hepatocytes and uPA<sup>+/+</sup>-SCID mice engrafted with human hepatocytes (human liver-uPA-SCID mice) (<xref ref-type="bibr" rid="bib28">Meuleman et al., 2005</xref>). PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites showed normal gliding motility, hepatic cell traversal (<xref ref-type="fig" rid="fig2">Figure 2</xref>), as well as invasion of primary human hepatocytes, but parasites were completely absent in two independent experiments at day 2 up to day 7 post-infection, following inoculation of primary human hepatocytes with 40,000 PfΔ<italic>b9</italic>Δ<italic>slarp</italic> F7 or G9 sporozoites (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Detailed analyses of 80 individual wells at day 4 post-infection did not result in identification of a single developing parasite. The combined day 2 and day 4 data of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> indicated that the timing of arrest is similar to PfΔ<italic>b9</italic> (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>) and there had been complete arrest of liver-stage development, similar to PfΔ<italic>slarp</italic> parasites.</p><p>In addition, human liver-uPA-SCID mice were intravenously inoculated with 1 × 10<sup>6</sup> WT or PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites. Two heterozygous uPA<sup>+/−</sup>-SCID mice, not engrafted with human hepatocytes, served as controls and were also challenged with <italic>P. falciparum</italic> sporozoites. Livers were collected either at 24 hpi or 5 days post-infection for detection of <italic>P. falciparum</italic> 18S DNA by quantitative real-time PCR (<xref ref-type="bibr" rid="bib11">Foquet et al., 2013</xref>). Both mice infected with WT Pf and 1 of the 2 mice infected with PfΔ<italic>b9</italic>Δ<italic>slarp</italic> were positive for Pf 18S DNA at 24 hr post-infection, demonstrating successful sporozoite infection in human hepatocytes (<xref ref-type="fig" rid="fig3">Figure 3</xref>). A lower signal was observed in PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-infected mice at day 1 after infection compared to WT parasites, likely reflecting the early time point of arrest of this GAP. All mice infected with Pf WT (3/3) showed a strong increase in parasite 18S DNA at day 5 post-infection, representing successful liver-stage development. In contrast, none of the human liver-uPA-SCID mice infected with PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites showed 18S DNA higher than heterozygous uPA<sup>+/−</sup>-SCID mice, not engrafted with human hepatocytes that had been infected with PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Although these studies were performed with a limited number of mice, these findings indicate that PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites can invade but do not develop in livers of humanized mice. Our combined results demonstrate abrogation of development of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> inside human hepatocytes.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>The Pf GAP PfΔ<italic>b9</italic>Δ<italic>slarp</italic> containing two gene deletions is proposed as a whole-parasite malaria vaccine candidate. Rationale and arguments are based on in vitro and in vivo experiments and supported by safety and protection data with rodent Pb GAP with deletions of the orthologous genes. The rodent GAP PbΔ<italic>b9</italic>Δ<italic>slarp</italic> completely arrested early in liver-stage development in two different mouse strains after injection of very high number of sporozoites. In addition, immunizations with PbΔ<italic>b9</italic>Δ<italic>slarp</italic> efficiently induced sterile and long-lasting protective immunity in both BALB/c and C57BL/6 mice. Similarly, the Pf GAP PfΔ<italic>slarp</italic>Δ<italic>b9</italic> completely aborted development in human hepatocytes 1 day after invasion, while sporozoites were fully motile and invasive with infectivity comparable to Pf WT sporozoites. Importantly, asexual parasite growth and production of salivary gland sporozoites in the mosquito were unaffected ensuring normal GAP production. PbΔ<italic>b9</italic>Δ<italic>slarp</italic> is to our knowledge the first completely attenuated rodent mutant in which multiple genes have been deleted that are critical for two independent biological processes during liver-stage development, that is regulation of parasite genes/transcripts that play a role in early liver-stage development stages (<xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>; <xref ref-type="bibr" rid="bib2">Aly et al., 2011</xref>) and the establishment of the PV within the infected hepatocyte (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>).</p><p>A number of Pb and Py GAPs have previously been reported to arrest at different time points during development in the liver (<xref ref-type="bibr" rid="bib21">Khan et al., 2012</xref>; <xref ref-type="bibr" rid="bib31">Nganou-Makamdop and Sauerwein, 2013</xref>). These include GAPs based on genes essential for i) the formation and maintenance of a parasitophorous vacuole (PV) (<italic>b9</italic>, <italic>p52, p36, uis3,</italic> and <italic>uis4</italic>; (<xref ref-type="bibr" rid="bib51">van Dijk et al., 2005</xref>; <xref ref-type="bibr" rid="bib22">Kumar et al., 2009</xref>; <xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>) and ii) type II fatty acid synthesis (i.e. <italic>fabb/f</italic>, <italic>fabz</italic>, <italic>pdh e1α</italic>; (<xref ref-type="bibr" rid="bib57">Vaughan et al., 2009</xref>; <xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>)), and iii) the regulation of gene expression in the liver-stages (<italic>sap1/slarp</italic> (<xref ref-type="bibr" rid="bib1">Aly et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>; <xref ref-type="bibr" rid="bib2">Aly et al., 2011</xref>)). A critical safety requirement for GAPs in order to qualify as vaccine candidate is the total absence of blood infections during immunization and therefore the complete abrogation of liver-stage development. Unfortunately many of the above mentioned target genes including p52, p36, and those involved in type II fatty acid synthesis show a leaky phenotype, resulting in blood stage infections after administration of high number of sporozoites. Incomplete liver-stage arrest obviously disqualifies GAPs for further clinical development for safety reasons.</p><p>In <italic>P. falciparum,</italic> GAPs have been generated that lack both the <italic>p52</italic> and <italic>p36</italic> genes (<xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>; <xref ref-type="bibr" rid="bib56">VanBuskirk et al., 2009</xref>). In the Pb rodent model, this GAP was not completely attenuated (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). Similarly, this Pf GAP while severely attenuated by the lack of both genes, a low percentage of parasites of this GAP are able to develop into mature liver-stage (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). These observations indicate a partially redundant function for these proteins; indeed, a breakthrough blood infection was observed in one out of the six volunteers after exposure to the bite of mosquitoes infected with sporozoites of a PfΔ<italic>p52</italic>Δ<italic>p36</italic> GAP (<xref ref-type="bibr" rid="bib47">Spring et al., 2013</xref>).</p><p>Since functional redundancy of related genes has been reported more often in <italic>Plasmodium</italic> (<xref ref-type="bibr" rid="bib26">Liu et al., 2006</xref>; <xref ref-type="bibr" rid="bib15">Heiss et al., 2008</xref>; <xref ref-type="bibr" rid="bib52">van Dijk et al., 2010</xref>; <xref ref-type="bibr" rid="bib25">Lin et al., 2013</xref>), we pursued the generation of GAPs from which multiple genes were removed from the genome, each governing a critical yet independent cellular process. The selection of those target genes excluded type II fatty acid synthesis (FAS II) because <italic>P. falciparum</italic> mutants lacking FAS II enzymes fail to generate sporozoites inside the oocyst, indicating that the FAS II pathway is essential for sporogony (<xref ref-type="bibr" rid="bib55">van Schaijk et al., 2013</xref>). The gene encoding liver-stage antigen 1 (LSA-1) may be an attractive candidate, but no orthologues are present in rodent or non-human primate <italic>Plasmodium</italic> species precluding sufficient pre-clinical testing (<xref ref-type="bibr" rid="bib29">Mikolajczak et al., 2011</xref>). The reverse is true for two published rodent GAPs with deletions of the genes <italic>uis3</italic> or <italic>uis4</italic> of which unequivocal orthologues are absent in the <italic>P. falciparum</italic> genome. Alternatively, genes encoding proteins with a role in the late stage parasite liver development could be an attractive target, since induction of protection by late arresting GAPs may be superior to early arresting GAPs (<xref ref-type="bibr" rid="bib5">Butler et al., 2011</xref>; <xref ref-type="bibr" rid="bib31">Nganou-Makamdop and Sauerwein, 2013</xref>) However, late arresting GAPs are likely more risky and prone to breakthrough infection as shown for GAPs lacking the genes <italic>palm</italic> or <italic>lisp</italic> (<xref ref-type="bibr" rid="bib21">Khan et al., 2012</xref>).</p><p>Therefore, we decided to focus on early liver-stage arrest and selected the newly identified <italic>b9</italic> as a prime candidate. PbΔ<italic>b9</italic> elicits long-lived protective immune responses in mice and only few breakthrough blood infections occur in mice, albeit less than were observed with PbΔ<italic>p52</italic>Δ<italic>p36</italic> GAP sporozoites (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). The genes <italic>p52, p36,</italic> and <italic>b9</italic>, all belong to the recently expanded 6-Cys family of <italic>Plasmodium</italic> proteins and may share a similar function in formation or maintenance of the PV membrane at the interface of parasite and host cell. Indeed, a triple gene-deletion mutant lacking <italic>p52, p36,</italic> and <italic>b9</italic> is no more attenuated than a mutant lacking <italic>b9,</italic> suggesting that these genes do not drive independent biological pathways (<xref ref-type="bibr" rid="bib51">van Dijk et al., 2005</xref>; <xref ref-type="bibr" rid="bib34">Ploemen et al., 2012</xref>; <xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). To date, the early arresting <italic>slarp</italic> mutant is the only rodent GAP with a Pf ortholog without a record of breakthrough blood infections in mice. Indeed, our data confirm that rodent sporozoites lacking <italic>slarp</italic> are fully capable of hepatocyte invasion and formation of a PV but completely abort development soon after invasion as previously reported (<xref ref-type="bibr" rid="bib1">Aly et al., 2008</xref>; <xref ref-type="bibr" rid="bib46">Silvie et al., 2008</xref>; <xref ref-type="bibr" rid="bib2">Aly et al., 2011</xref>). In this study, we report for the first time that <italic>P. falciparum</italic> mutants lacking <italic>slarp,</italic> that is PfΔ<italic>slarp,</italic> completely arrest at day 3 post-infection of primary human hepatocytes, while morphologically normal liver-stage parasites are still observed at 48 hpi. PfΔ<italic>b9</italic> parasites arrest at a point in time before day 2 after hepatocyte invasion, with the exception of a single liver schizont observed at a later time point (<xref ref-type="bibr" rid="bib4">Annoura et al., 2014</xref>). The multiple attenuated Pb<italic>Δb9Δslarp</italic> indeed passed our stringent pre-clinical safety screen and no breakthrough blood infections were observed in all conditions tested. In addition, we showed that immunization with PbΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites induced strong and sustained protective immunity in BALB/c and C57BL/6 mice, with similar efficacy as reported for mutant sporozoites lacking P52 (or P52 and P36) or γ-radiated sporozoites (<xref ref-type="bibr" rid="bib32">Nussenzweig et al., 1967</xref>; <xref ref-type="bibr" rid="bib51">van Dijk et al., 2005</xref>; <xref ref-type="bibr" rid="bib8">Douradinha et al., 2007</xref>; <xref ref-type="bibr" rid="bib23">Labaied et al., 2007</xref>).</p><p>Live vaccine strains (attenuated by natural selection or genetic engineering) may be potentially released into the environment. Therefore, safety issues concerning the medical as well as environmental aspects must be considered including the absence of heterologous DNA sequences (in particular drug resistance genes) from the genome of GAPs (<xref ref-type="bibr" rid="bib9">Committee for Medical Products for Human Use, 2006</xref>; <xref ref-type="bibr" rid="bib13">Frey, 2007</xref>). Thus, a PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAP was generated free of a drug resistance marker using FRT/FLPe-recombinase methodology. This approach permits the removal of drug resistance markers that were introduced to generate the mutant and results in an altered genome that retains only two 34 nucleotide FRT sequences. The removal of the drug resistance marker has the additional advantage that these parasites are easily amenable to further genetic modification (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>).</p><p>The PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAP aborted early development in cultured primary human hepatocytes, with a phenotype and timing similar to PfΔ<italic>b9,</italic> and studies performed in a limited number of chimeric mice engrafted with human hepatocytes confirm this arrest phenotype. From the combined Pb and Pf data, one can conclude that Δ<italic>b9</italic> attenuation phenotype induces highly effective protection, although it may at a low frequency produce a breakthrough blood infection. Therefore, the additional deletion of <italic>slarp</italic> in these mutants provides these parasites with complete attenuation that is essential in order to proceed with human trials.</p><p>An important prerequisite for further downstream clinical development and manufacturing (<xref ref-type="bibr" rid="bib45">Seder et al., 2013</xref>) is to show that production of PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites is unabated and similar to WT parasites. We have shown that the PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAP produces WT numbers of sporozoites that are fully capable of infecting hepatocytes. In addition, we have produced aseptic, purified, cryopreserved PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites (data not shown). Preliminary data from a 6-day attenuation assay in HC-04 cells showed that like irradiated PfSPZ (<xref ref-type="bibr" rid="bib17">Hoffman et al., 2010</xref>; <xref ref-type="bibr" rid="bib10">Epstein et al., 2011</xref>), none of the PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites developed to mature liver-stage parasites expressing PfMSP-1 (data not shown), as aseptic, purified, cryopreserved WT sporozoites (<xref ref-type="bibr" rid="bib43">Roestenberg et al., 2013</xref>).</p><p>In conclusion, we have generated a multiply attenuated PfΔ<italic>b9</italic>Δ<italic>slarp</italic> GAP, free of any drug resistance gene, and demonstrated that PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites invade hepatocytes comparably to WT sporozoites and are completely attenuated. These findings provide a solid foundation for clinical development and testing of a PfSPZΔ<italic>b9</italic>Δ<italic>slarp</italic> vaccine.</p><sec id="s3-1"><title>Note added at proof</title><p>While this manuscript was in preparation an article was published that also describes a multiple-gene deletion <italic>P. falciparum</italic> parasite that has undergone pre-clinical evaluation (<xref ref-type="bibr" rid="bib29a">Mikolajczak et al., 2014</xref>). In that study, the authors describe a <italic>P. falciparum</italic> mutant that, like our work, also lacks the gene <italic>slarp (sap1)</italic> as well as the paralogous pair of genes, p52 and p36.</p></sec></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4-1"><title><italic>P. berghei</italic> reference parasite lines</title><p>The following reference lines of the ANKA strain of <italic>P. berghei</italic> were used: line cl15cy1 (<xref ref-type="bibr" rid="bib1a">Janse et al., 2006a</xref>, <xref ref-type="bibr" rid="bib2a">2006b</xref>) and line 676m1cl1 (<italic>Pb</italic>GFP-Luc<sub>con</sub>; see RMgm-29 in <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.pberghei.eu/">www.pberghei.eu</ext-link>). <italic>Pb</italic>GFP-Luc<sub>con</sub> expresses a fusion protein of GFP and luciferase from the <italic>eef1a</italic> promoter (<xref ref-type="bibr" rid="bib12">Franke-Fayard et al., 2004</xref>; <xref ref-type="bibr" rid="bib1a">Janse et al., 2006a</xref>).</p></sec><sec id="s4-2"><title><italic>P. falciparum</italic> parasites and culture</title><p>For transfections, the parasite used was directly from a characterized good manufacturing process (GMP) and produced working cell bank (WCB) of the <italic>P. falciparum</italic> NF54 wild-type strain (<xref ref-type="bibr" rid="bib37">Ponnudurai et al., 1981</xref>), produced by Sanaria Inc, identical to that described previously (<xref ref-type="bibr" rid="bib17">Hoffman et al., 2010</xref>; <xref ref-type="bibr" rid="bib10">Epstein et al., 2011</xref>; <xref ref-type="bibr" rid="bib43">Roestenberg et al., 2013</xref>). Blood stages of wt, PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 were cultured in a semi-automated culture system using standard in vitro culture conditions for <italic>P. falciparum</italic> and induction of gametocyte production in these cultures was performed as previously described (<xref ref-type="bibr" rid="bib18">Ifediba and Vanderberg, 1981</xref>; <xref ref-type="bibr" rid="bib38">Ponnudurai et al., 1982</xref>, <xref ref-type="bibr" rid="bib39">1989</xref>). Fresh human red blood cells and serum were obtained from Dutch National blood bank (Sanquin Nijmegen, NL; permission granted from donors for the use of blood products for malaria research). Cloning of transgenic parasites was performed by the method of limiting dilution in 96-well plates as described (<xref ref-type="bibr" rid="bib50">Thaithong, 1985</xref>). Parasites of the positive wells were transferred to the semi-automated culture system and cultured for further phenotype and genotype analyses (See below).</p></sec><sec id="s4-3"><title>Experimental animals</title><p>For <italic>P. berghei</italic> infections, female C57BL/6J and BALB/c (12-week old; Janvier France) and Swiss OF1 (8 weeks old Charles River) were used. All animal experiments with rodent parasites performed at the LUMC (Netherlands) were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 07171; DEC 10099) and at the RUNMC (Netherlands) by the Radboud University Experimental Animal Ethical Committee (RUDEC 2008-123, RUDEC 2008-148, RUDEC 2010-250, RUDEC 2011-022, RUDEC 2011-208). The Dutch Experiments on Animal Act is established under European guidelines (EU directive 86/609/CEE) regarding the Protection of Animals used for Experimental and Other Scientific Purposes.</p><p>Human liver-uPA-SCID mice (chimeric mice) were produced as described before (<xref ref-type="bibr" rid="bib28">Meuleman et al., 2005</xref>). The study protocol for infecting these mice with <italic>P. falciparum</italic> sporozoites was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University.</p></sec><sec id="s4-4"><title>Generation and genotyping of <italic>P. berghei</italic> mutants</title><p>To disrupt the <italic>P. berghei slarp</italic> gene (PBANKA_090210), a construct was generated using the adapted ‘Anchor-tagging’ PCR-based method as described (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>) (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). The two targeting fragments (1195 bp and 823 bp) of <italic>slarp</italic> were amplified using genomic DNA (parasite line cl15cy1) as template with the primer pairs 5960/5961 (5′target sequence) and 5962/5963 (3′target sequence). See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2A</xref> for the sequence of the primers. Using this PCR-based targeting construct (pL1740), the mutant PbΔ<italic>slarp-a</italic> (1839cl3) was generated in the <italic>Pb</italic>GFP-Luc<sub>con</sub> reference line using standard methods of transfection and positive selection with pyrimethamine (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). The generation of the drug-selectable marker-free mutant PbΔ<italic>b9</italic>Δ<italic>sm</italic> (1309cl1m0cl2; RMgmDB no. 934) has been described by <xref ref-type="bibr" rid="bib4">Annoura et al. (2014</xref>). This mutant, which contains a disrupted <italic>b9</italic> gene and is drug-selectable marker free, was used for deleting the <italic>slarp</italic> gene (PBANKA_090210). To delete the <italic>slarp</italic> gene, the gene-deletion construct pL1740 was used as described above. Using this construct the mutant PbΔ<italic>b9</italic>Δ<italic>slarp</italic> (line 1844cl1) was generated in the PbΔ<italic>b9</italic>Δ<italic>sm</italic> line using standard methods of transfection and positive selection with pyrimethamine (<xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><p>Correct integration of the constructs into the genome of mutant parasites was analyzed by diagnostic PCR-analysis and Southern analysis of PFG-separated chromosomes as shown in <xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>. PFG-separated chromosomes were hybridized with a probe recognizing <italic>hdhfr</italic> or the 3′-UTR <italic>dhfr/ts</italic> of <italic>P. berghei</italic> (<xref ref-type="bibr" rid="bib2a">Janse et al., 2006b</xref>)<italic>.</italic></p></sec><sec id="s4-5"><title>Generation and genotyping of <italic>P. falciparum</italic> mutants</title><p>The <italic>slarp</italic> gene (PF3D7_1147000) in <italic>P. falciparum</italic> WT parasites (NF54wcb) was deleted using a modified construct based on plasmid pHHT-FRT-(GFP)-Pf52 (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>) (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Targeting regions were generated by PCR using primers BVS179 and BVS180 for the 5′ target region and primers BVS182 and BVS184 for the 3′ target region (see <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref> for primer sequences). The 5′and 3′ target regions were cloned into pHHT-FRT-(GFP)-Pf52 digested with <italic>Bsi</italic>WI, <italic>Bss</italic>HII and <italic>Nco</italic>I, <italic>Xma</italic>I, respectively, resulting in the plasmid pHHT-FRT-GFP-<italic>slarp.</italic> The <italic>b9</italic> gene (PF3D7_0317100) of PfΔ<italic>slarp</italic>-b <italic>P. falciparum</italic> parasites was deleted using a modified construct based on plasmid pHHT-FRT-(GFP)-Pf52 (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>) (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Targeting regions were generated by PCR using primers BVS84 and BVS85 for the 5′ target region and primers BVS88 and BVS89 for the 3′ target region. The 5′and 3′ target regions were cloned into pHHT-FRT-(GFP)-Pf52 digested with <italic>Nco</italic>I, <italic>Xma</italic>I and <italic>Mlu</italic>I, <italic>Bss</italic>HII resulting in the plasmid pHHT-FRT-GFP-<italic>b9.</italic> All DNA fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic <italic>P. falciparum</italic> DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen, Leek, The Netherlands) sub-cloning. Transfection of WT (NF54wcb) parasites with the plasmid pHHT-FRT-GFP-<italic>slarp</italic> and selection of mutant parasites were performed, as described (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>), resulting in the selection of the parasite line PfΔ<italic>slarp-</italic>a. The second PfΔ<italic>slarp</italic> parasite line<italic>,</italic> originating from an independent transfection, was subsequently transfected with pMV-FLPe to remove the drug-selectable marker cassette using FLPe as described (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>) and cloned resulting in the parasite clone PfΔ<italic>slarp-</italic>b. Subsequent transfection of PfΔ<italic>slarp-</italic>b parasites with the plasmid pHHT-FRT-GFP-<italic>b9</italic> and selection were performed, as described above, resulting in the parasite line PfΔ<italic>b9</italic>Δ<italic>slarp</italic>. The parasite line PfΔ<italic>b9</italic>Δ<italic>slarp</italic> was subsequently transfected with pMV-FLPe to remove the drug-selectable marker cassette using FLPe and cloned, as described above, resulting in the cloned parasite lines PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7 and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 that are free of drug resistance markers.</p><p>Genotype analysis of PfΔ<italic>slarp and</italic> PfΔ<italic>b9</italic>Δ<italic>slarp</italic> parasites was performed by Expand Long range dNTPack (Roche) diagnostic, long-range, PCR (LR-PCR) and Southern blot analysis (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Genomic DNA of blood stages of WT or mutant parasites was isolated and analyzed by LR-PCR using primer pair p1, p2 (<italic>slarp</italic>) and p3, p4 (<italic>b9</italic>) (See <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2B</xref> for primer sequences) for correct integration of the constructs in the respective <italic>slarp</italic> and <italic>b9</italic> loci by double cross-over homologous recombination. The LR-PCR program has an annealing step of 48°C for 30 s and an elongation step of 62°C for 10–15 min. All other PCR settings were according to manufacturer's instructions. PCR products were directly analyzed by standard agarose gel electrophoresis or first digested with restriction enzymes for further confirmation of the genotype and removal of resistance markers was confirmed by sequencing. For Southern blot analysis, genomic DNA was digested with <italic>Taq</italic>I or <italic>Rca</italic>I restriction enzymes for analysis of integration into the <italic>slarp</italic> and <italic>b9</italic> loci, respectively. Southern blot was generated by capillary transfer as described (<xref ref-type="bibr" rid="bib44">Sambrook and Russel, 2001</xref>) and DNA was hybridized to radioactive probes specific for the targeting regions used for the generation of the mutants and generated by PCR (See above).</p><p>The presence or absence of <italic>slarp</italic> and <italic>b9</italic> transcripts in WT and mutant sporozoites was analyzed by reverse transcriptase-PCR (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Total RNA was isolated using the RNeasy mini Kit (Qiagen) from 10<sup>6</sup> salivary gland sporozoites collected by dissection of mosquitoes 16 days after feeding with WT, PfΔ<italic>slarp-</italic>a, PfΔ<italic>slarp-</italic>b, PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7, and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 parasites. Remaining DNA was degraded using DNAseI (Invitrogen). cDNA was synthesized using the First Strand cDNA synthesis Kit for RT-PCR AMV (Roche). As a negative control for the presence of genomic DNA, reactions were performed without reverse transcriptase (RT−). PCR amplification was performed for regions of <italic>slarp</italic> using primers BVS290, BVS292 and for regions of <italic>b9</italic> using primers BVS286 and BVS288. Positive control was performed by PCR of 18S rRNA using primers 18Sf and 18Sr.</p></sec><sec id="s4-6"><title>Phenotype analyses of blood stages of <italic>P. berghei</italic> and <italic>P. falciparum</italic> mutants</title><p>Asexual multiplication rate and gametocyte production of <italic>P. berghei</italic> blood stages were determined as described (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). The <italic>P. berghei</italic> mutants were maintained in Swiss mice. The multiplication rate of blood stages and gametocyte production were determined during the cloning procedure (<xref ref-type="bibr" rid="bib2a">Janse et al., 2006b</xref>) and were not different from parasites of the reference ANKA lines. <italic>P. falciparum</italic> blood stage development and gametocyte production were analyzed as described (<xref ref-type="bibr" rid="bib54">van Schaijk et al., 2010</xref>).</p></sec><sec id="s4-7"><title>Analysis of <italic>P. berghei</italic> and <italic>P. falciparum</italic> sporozoite production and in vitro motility, hepatocyte traversal, and infectivity of sporozoites</title><p>Feeding of <italic>A. stephensi</italic> mosquitoes with <italic>P. berghei</italic> and <italic>P. falciparum,</italic> determination of oocyst production and sporozoite collection, as well as <italic>P. berghei</italic> gliding motility were performed as described (<xref ref-type="bibr" rid="bib3">Annoura et al., 2012</xref>). <italic>P. falciparum</italic> gliding motility of sporozoites was determined as described (<xref ref-type="bibr" rid="bib48">Stewart and Vanderberg, 1988</xref>; <xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>). <italic>P. falciparum</italic> cell traversal and invasion of hepatocytes were determined in Huh7 cells and primary human hepatocytes respectively as described (<xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>). Infectivity of <italic>P. berghei</italic> sporozoites and development was determined in cultures of Huh7 cells as described (<xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>). For analysis of liver-stage development by immunofluorescence, parasites were stained with the following primary antibodies: anti-PbEXP1 (PBANKA_092670; raised in chicken (<xref ref-type="bibr" rid="bib49">Sturm and Heussler, 2007</xref>)) and anti-PbHSP70 (PBANKA_081890; raised in mouse (<xref ref-type="bibr" rid="bib30">Mueller et al., 2005</xref>)). Infectivity of <italic>P. falciparum</italic> sporozoites and development was analyzed in primary human hepatocytes as described (<xref ref-type="bibr" rid="bib53">van Schaijk et al., 2008</xref>). Briefly for analysis of development by immunofluorescence, parasites were stained with the following primary antibodies: anti-HSP70 (PF3D7_0930300 (<xref ref-type="bibr" rid="bib40">Renia et al., 1990</xref>)) and anti-CSP (PF3D7_0304600; 3SP2) using double labeling. Anti-mouse secondary antibodies, conjugated to Alexa-488 or Alexa-594 (Invitrogen), were used for visualization. Primary human hepatocytes were isolated from healthy parts of human liver fragments, which were collected during unrelated surgery in agreement with French national ethical regulations (<xref ref-type="bibr" rid="bib14">Gouagna et al., 2007</xref>) and after oral informed consent from adult patients undergoing partial hepatectomy as part of their medical treatment (Service de Chirurgie Digestive, Hépato-Bilio-Pancréatique et Transplantation Hépatique, Hôpital Pitié-Salpêtrière, Paris, France). The collection and use of this material for the purposes of the study presented here were undertaken in accordance with French national ethical guidelines under Article L. 1121-1 of the ‘Code de la Santé Publique’. Given that the tissue samples are classed as surgical waste, that they were used anonymously (the patient's identity is inaccessible to the researchers), and that they were not in any way genetically manipulated, article L. 1211-2 stipulates that their use for research purposes is allowed provided that the patient does not express any opposition to the surgeon prior to surgery and after being informed of the nature of the research in which they might be potentially employed. Within this framework, the collection and use of this material was furthermore approved by the Institutional Review Board (Comité de Protection des Personnes) of the Centre Hospitalo-Universitaire Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, France.</p></sec><sec id="s4-8"><title>Analysis of <italic>P. berghei</italic> sporozoite infectivity in mice and in vivo imaging of liver-stage development</title><p>C57BL/6 or BALB/c mice were inoculated with sporozoites by intravenous injection of different sporozoite numbers, ranging from 1 × 10<sup>4</sup> to 5 × 10<sup>5</sup>. Blood stage infections were monitored by analysis of Giemsa-stained thin smears of tail blood collected on day 4–14 after inoculation of sporozoites. The prepatent period (measured in days post sporozoite infection) is defined as the day when a blood stage infection with a parasitemia of 0.5–2% is observed. Liver-stage development in live mice was monitored by real time in vivo imaging of liver-stages as described (<xref ref-type="bibr" rid="bib35">Ploemen et al., 2012</xref>). Liver-stages were visualized by measuring luciferase activity of parasites (expressing luciferase under the <italic>eef1a</italic> promoter) in whole bodies of mice (<xref ref-type="bibr" rid="bib33">Ploemen et al., 2009</xref>).</p></sec><sec id="s4-9"><title>Immunizations of mice with <italic>P. berghei</italic> sporozoites</title><p>Prior to immunization, <italic>P. berghei</italic> sporozoites were collected at day 21–27 after mosquito infection by hand-dissection. Salivary glands were collected in DMEM (Dulbecco's Modified Eagle Medium from GIBCO) and homogenized in a homemade glass grinder. The number of sporozoites was determined by counting in triplicate in a Bürker-Türk counting chamber using phase-contrast microscopy. BALB/c and C57BL/6 mice were immunized by intravenous injection using different numbers of mutant sporozoites. BALB/c mice received one immunization and C57BL/6 mice received three immunizations with two 7 day intervals. Immunized mice were monitored for blood infections by analysis of Giemsa stained films of tail blood at day 4–16 after immunization. Immunized mice were challenged at different time points after immunization by intravenous injection of 1 × 10<sup>4</sup> sporozoites from the <italic>P. berghei</italic> ANKA reference line cl15cy1. In each experiment, age matched naive mice were included to verify infectivity of the sporozoites used for challenge. After challenge, mice were monitored for blood infections by analysis of Giemsa stained films of tail blood at day 4–21.</p></sec><sec id="s4-10"><title>Development of Pf Δ<italic>b9</italic>Δ<italic>slarp</italic> GAP in chimeric mice engrafted with human hepatocytes</title><p>Human liver-uPA-SCID mice were produced as described before (<xref ref-type="bibr" rid="bib28">Meuleman et al., 2005</xref>). Briefly, within two weeks after birth homozygous uPA<sup>+/+</sup>-SCID mice (<xref ref-type="bibr" rid="bib11">Foquet et al., 2013</xref>) were transplanted with approximately 10<sup>6</sup> cryopreserved primary human hepatocytes obtained from a single donor (BD Biosciences, Erembodegem, Belgium). To evaluate successful engraftment, human albumin was quantified in mouse plasma with an in-house ELISA (Bethyl Laboratories Inc., Montgomery, TX). The study protocol was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University. Human liver-uPA-SCID mice (n = 10) and non-chimeric heterozygous uPA<sup>+/−</sup>-SCID mice (control, n = 2) were intravenously injected with 10<sup>6</sup> fresh isolated PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 or as a control WT sporozoites. One and 5 days post-infection livers were removed and each liver was cut into 12 standardized sections and stored in RNAlater (Sigma) at 4°C until analysis as described (<xref ref-type="bibr" rid="bib11">Foquet et al., 2013</xref>). From each part DNA was extracted to assess the parasite load by Pf18S qPCR and to assess the number of human and mouse hepatocytes by Multiplex qPCR PTGER2 analysis (<xref ref-type="bibr" rid="bib11">Foquet et al., 2013</xref>).</p><p>While this manuscript was in preparation an article was published that also describes a multiple-gene deletion <italic>P. falciparum</italic> parasite that has undergone pre-clinical evaluation (<xref ref-type="bibr" rid="bib29a">Mikolajczak et al, 2014</xref>). In that study, the authors describe a <italic>P. falciparum</italic> mutant that, like our work, also lacks the gene <italic>slarp</italic> (<italic>sap1</italic>) as well as the paralogous pair of genes, p52 and p36.</p></sec><sec id="s4-11"><title>Data and Materials availability</title><p>The materials described in this study must be acquired through a material transfer agreement.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We would like to thank the following people from RUMC (Nijmegen) for technical support: Claudia Lagarde, Alex Ignacio, Daniëlle Janssen, Rianne Siebelink-Stoter, Wouter Graumans, Jolanda Klaassen, Laura Pelser-Posthumus, Astrid Pouwelsen, and Jacqueline Kuhnen; and the following people from LUMC (Leiden) for technical support: Jai Ramesar, Jing-wen Lin, and Hans Kroeze. We acknowledge the Sanaria Manufacturing Team for the GMP produced working cell bank of PfNF54. Sanaria’s development efforts supported in part by NIAID Small Business Innovation Research grants, 5R44AI069631 and 5R44AI058375.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>SLH: CEO of Sanaria Inc, biotechnology company focused on whole sporozoite malaria vaccines.</p></fn><fn fn-type="conflict" id="conf2"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>BCLS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>IHJP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>TA, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>MWV, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>LF, Conception and design, Acquisition of data</p></fn><fn fn-type="con" id="con6"><p>G-JG, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>SC-M, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con8"><p>MV-B, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>MS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>J-FF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con11"><p>AL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con12"><p>GL-R, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con13"><p>PM, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con14"><p>CCH, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con15"><p>DM, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con16"><p>CJJ, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con17"><p>SMK, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con18"><p>SLH, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con19"><p>RWS, Conception and design, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Human subjects: Primary human hepatocytes were isolated from healthy parts of human liver fragments which were collected during unrelated surgery in agreement with French national ethical regulations and after oral informed consent from adult patients undergoing partial hepatectomy as part of their medical treatment (Service de Chirurgie Digestive, Hépato-Bilio-Pancréatique et Transplantation Hépatique, Hôpital Pitié-Salpêtrière, Paris, France). The collection and use of this material for the purposes of the study presented here were undertaken in accordance with French national ethical guidelines under Article L. 1121-1 and article L. 1211-2.</p></fn><fn fn-type="other"><p>Animal experimentation: All animal experiments with rodent parasites performed at the LUMC (Netherlands) were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 07171; DEC 10099) and at the RUNMC (Netherlands) by the Radboud University Experimental Animal Ethical Committee (RUDEC 2008-123, RUDEC 2008-148, RUDEC 2010-250, RUDEC 2011-022, RUDEC 2011-208). The Dutch Experiments on Animal Act is established under European guidelines (EU directive 86/609/CEE) regarding the Protection of Animals used for Experimental and Other Scientific Purposes. Human liver-uPA-SCID mice (chimeric mice) were produced as described before. The study protocol for infecting these mice with <italic>P. falciparum</italic> sporozoites was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University. The study protocol was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of the Ghent University.</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03582.011</object-id><label>Supplementary file 1.</label><caption><p>Oocyst and sporozoite production and sporozoite characteristics (motility, traversal, hepatocyte invasion) of the <italic>P. berghei</italic> mutants PbΔ<italic>slarp</italic> and PbΔ<italic>b9</italic>Δ<italic>slarp</italic>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.011">http://dx.doi.org/10.7554/eLife.03582.011</ext-link></p></caption><media xlink:href="elife03582s001.docx" mimetype="application" mime-subtype="docx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03582.012</object-id><label>Supplementary file 2.</label><caption><p>Primer sequences.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03582.012">http://dx.doi.org/10.7554/eLife.03582.012</ext-link></p></caption><media xlink:href="elife03582s002.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “A genetically attenuated malaria vaccine candidate based on <italic>P. falciparum</italic> b9/slarp gene-deficient sporozoites” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Prabhat Jha (Senior editor), Nicholas White (Reviewing editor), and 3 reviewers.</p><p>The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The paper describes an important step on the pathway to the development of a genetically attenuated parasite (GAP) for use as a live-attenuated vaccine for human malaria.</p><p>1) Could you explain why you have focused on Δb9, given that it suffers from the same 'redundancy' issues affecting previous liver-stage genes examined in GAP mutants? Why did you not focus on slarp until another truly 'essential' liver-stage gene is found for combination? The b9 deletion seems to have limited value.</p><p>2) Are you sure that the Pb double KO parasites did not give rise to sub-patent infections at densities only detectable by PCR?</p><p>3) Please provide numbers of sporozoites added to the human hepatocyte line for the in vitro infectivity study (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><p>4) Only 5 humanized mice were studied for the <italic>in vivo</italic> infectivity study, with one becoming PCR positive above background at day 1 and 0 of 3 above background at day 5. These data are encouraging but don't exclude the possibility that a rare parasite might break through. The limitations of the small numbers should be acknowledged.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03582.014</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Could you explain why you have focused on Δb9, given that it suffers from the same 'redundancy' issues affecting previous liver-stage genes examined in GAP mutants? Why did you not focus on slarp until another truly 'essential' liver-stage gene is found for combination? The b9 deletion seems to have limited value</italic>.</p><p>First of all it was considered to be essential to delete two functionally independent genes to minimize the chances of breakthrough infections based on experiences in the past with single gene deletions. It is critical that GAPs are incapable of ‘reverting’ to a wild-type mode of (blood stage) development, and this is best achieved by removing multiple genes from the parasite’s genome. In that perspective, slarp was considered the best partner gene to ensure the safety profile as best and only the single gene deletion able to generate completely liver-stage arrested parasites in multiple <italic>Plasmodium</italic> species to date. Subsequently the b9 gene has been selected while comparing other gene candidates as best target for the following reasons:</p><p>(1) Δ<italic>b9</italic> GAP are strongly attenuated as only very high doses of Δ<italic>b9</italic> sporozoites (>50 000) can produce a blood stage infection, in 1 of 3 mouse strains examined and only a very low percentage of these mice become infected even at very high sporozoite inocula (i.e. 500 000); the Δ<italic>b9</italic> GAP has the strongest attenuation of all reported single gene GAPs, after Δ<italic>slarp</italic>-GAP, and is considerably more attenuated than parasites that lack BOTH <italic>p52</italic> and <italic>p36</italic> (the next best GAP candidates)</p><p>(2) <italic>b9</italic> encodes for a protein that is involved in a completely different biological process from SLARP, thereby any ‘reversion’ to wild type phenotype would require a very unlikely functional compensation of 2 independent cellular process;</p><p>(3) Δ<italic>b9</italic> GAP are most potent in induction of protective immunity in several murine models.</p><p>Taken together the combination of Δ<italic>slarp</italic> with Δ<italic>b9</italic> should result in a GAP that is both safe and more potent than any other combination currently available.</p><p><italic>2) Are you sure that the Pb double KO parasites did not give rise to sub-patent infections at densities only detectable by PCR?</italic></p><p>Indeed we have not analysed the blood of immunized and challenged mice by PCR or blood transfer to naive mice. Notwithstanding, we do not believe that sub-patent infections may occur or persist.</p><p>Δ<italic>b9</italic>Δ<italic>slarp</italic> blood stage parasite produces a normal course of infection reaching a parasitemia of 0.2-5% parasitemia on the same day similar to wild-type parasites. Therefore any Δ<italic>b9</italic>Δ<italic>slarp</italic> parasite that would emerge in the blood after liver stage development, in an unimmunised mouse, will become patent.</p><p>Extensive blood smear examinations in a large number of mice after single dose infections, has never provided any evidence for a sub-patent infection. Indeed many GAP immunized mice were housed for longer than 1 year after immunisation and some establish a normal blood stage infection after wild-type challenge.</p><p>We therefore believe it is unlikely that a sub-patent Δ<italic>slarp</italic>Δ<italic>b9</italic> blood stage infection is possible or present.</p><p><italic>3) Please provide numbers of sporozoites added to the human hepatocyte line for the in vitro infectivity study (</italic><xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref><italic>).</italic></p><p>The number of 40.000 sporozoites is now indicated in the results section and reads:</p><p>“PfΔ<italic>b9</italic>Δ<italic>slarp</italic> sporozoites showed normal gliding motility, hepatic cell traversal (<xref ref-type="fig" rid="fig1">Fig. 1</xref>) as well as invasion of primary human hepatocytes, but parasites were completely absent in two independent experiments at day 2 up to day 7 post-infection following inoculation of primary human hepatocytes with 40.000 PfΔ<italic>b9</italic>Δ<italic>slarp</italic> F7 or G9 sporozoites (<xref ref-type="fig" rid="fig2">Figure 2</xref>)”</p><p>Additionally the amount of sporozoites is indicated in the figure legend of <xref ref-type="fig" rid="fig2">Figure 2c</xref> and now reads:</p><p>“C. Development of <italic>P. falciparum</italic> wt, PfΔ<italic>slarp-</italic>a PfΔ<italic>slarp-</italic>b (top panel), PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-F7 and PfΔ<italic>b9</italic>Δ<italic>slarp</italic>-G9 (bottom panel) liver-stages in primary human hepatocytes following inoculation with 40.000 sporozoites. From day 2 to 7 the mean number ± standard deviation of parasites per 96-well was determined by counting parasites stained with anti-<italic>P. falciparum</italic> HSP70 antibodies. The bottom panel represents experiments performed in primary human hepatocytes from 2 different donors. No parasites present (NP).”</p><p><italic>4) Only 5 humanized mice were studied for the in vivo infectivity study, with one becoming PCR positive above background at day 1 and 0 of 3 above background at day 5. These data are encouraging but don't exclude the possibility that a rare parasite might break through. The limitations of the small numbers should be acknowledged</italic>.</p><p>We agree with the reviewers that based on the limited number of mice that we used we cannot exclude the possibility of a breakthrough infections in humanised mice. However, in all our studies performed with both the single slarp deletion and the double slarp/b9 deletion mutants we were unable to detect developing parasites in cultured primary human hepatocytes. In order to address the reviewers concern we have now modified sentences in the Results section as follows:</p><p>“Although these studies were performed with a limited number of mice, these findings indicate that PfΔb9Δslarp parasites can invade but do not develop in livers of humanized mice. Our combined results demonstrate abrogation of development of PfΔb9Δslarp inside human hepatocytes.”</p><p>And in the Discussion section:</p><p>“The PfΔb9Δslarp GAP completely aborted early development in cultured primary human hepatocytes with a phenotype and timing similar to PfΔb9 and studies performed in a limited number of chimeric mice engrafted with human hepatocytes also confirm this arrest phenotype.”</p></body></sub-article></article> |