Compositions Incorporating Genetically Attenuated Plasmodium Having Modified Liver Stage Nuclear Protein (LINUP) and Related Methods

Abstract

The present disclosure is directed to malaria-causing parasites, specifically Plasmodium species parasites, and more specifically Plasmodium species parasites that have been genetically modified to develop normally only to the late liver stage, but are completely inhibited from entering the blood stage or infection of red blood cells. Specifically, the inventors have identified a genetic modification resulting from disruption of the genetic function of the liver stage nuclear protein (LINUP) gene that results in a late liver stage arrest of the development of these parasites. This complete late liver stage arrest allows enhanced expression of the parasite antigen array during liver stage development, but prevents entry into the blood stage, red blood cell infection, and associated signs, symptoms, and pathology of malarial disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 234,872, filed August 19, 2021.

[0002] Description of sequence listing The sequence listing XML accompanying this application is provided in XML format and is hereby incorporated by reference. The name of the XML file containing the sequence listing is 3399-P33WO.xml. The XML file is 300KB, was created on August 19, 2022, and has been submitted through the Patent Center with the application herein.

[0003] Government License Rights Statement This invention was made with Government support under R01 AI125706 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] background The present invention relates generally to malaria and to the identification, production and use of genetically altered Plasmodium parasites. More specifically, the present invention relates to genetically attenuated Plasmodium parasites that are disrupted at single and / or multiple loci and are nevertheless capable of progressing to the late liver stage of development, but attenuated at this stage and unable to progress to the blood stage.

[0005] Malaria has a significant impact on human health, causing hundreds of thousands of deaths each year and severely impeding social and economic development in areas where malaria is endemic, particularly in sub-Saharan African countries [1]. The causative agent of malaria, a parasite of the genus Plasmodium, is transmitted to vertebrate hosts in the saliva of infected Anopheles mosquitoes via an infectious bite. After transmission, the Plasmodium parasite, specifically the sporozoite stage of its life cycle, exits the bite site and enters the host's bloodstream, from which it quickly migrates to the liver. The sporozoites infect hepatocytes, then grow and replicate within the infected hepatocytes as liver-stage parasites, maturing from early to late liver stages and producing tens of thousands of blood-stage infectious merozoites, which are released into the bloodstream and infect red blood cells. Here, they undergo further development and replication, after which they cause the red blood cell to burst, releasing a wave of new merozoites into the blood. The majority of the new merozoites continue the repeated replication cycle by invading additional red blood cells. This circulation of red blood cell infection and rupture potentially causes the severe symptoms associated with malaria, such as fever, chills, weakness, malaise, enlarged spleen, and death. A small minority of the circulating merozoites, after invading the red blood cell, develop into male or female gametocytes, which continue to circulate within the body until they are taken up in a blood meal through the bite of another mosquito. Assuming that this mosquito is compatible for Plasmodium transmission (i.e., another Anopheles mosquito), the gametocytes proceed to develop into gametes and fuse to form a diploid zygote. The zygote develops into a motile ookinete form, which penetrates the wall of the mosquito's midgut and forms an oocyst. Each oocyst undergoes multiple divisions, eventually producing an infective sporozoite, which accumulates in the mosquito's salivary glands and can then be injected into the next host, thus continuing the life cycle.

[0006] The liver stage of Plasmodium infection is a promising target for preventive interventions in malaria, as it is asymptomatic for the disease and progresses to symptomatic blood stage infection. Indeed, vaccines targeting the liver stage of the life cycle have shown great promise in numerous clinical trials. To achieve this, vaccines have been attenuated at the liver stage of development by three distinct processes outlined below:

[0007] Radiation-attenuated vaccines: Decades ago, it was discovered that irradiated Plasmodium species sporozoites ("radiation-attenuated sporozoites" or RAS) could confer sterile protective immunity in host-specific rodents and humans when used as an experimental vaccine [2, 3]. This was surprising because natural malaria infection does not induce sterile protective immunity in endemic regions of the world. This approach has been used to produce a radiation-attenuated P. falciparum (Pf) whole-parasite vaccine that has proven highly effective in multiple clinical trials. The Sanaria® PfSPZ vaccine has been evaluated in 19 clinical trials in nine countries to date, and has been found to be extremely safe and well tolerated, with adverse event rates that are no different from saline controls [4-12]. The PfSPZ vaccine has induced vaccine efficacy (VE) of over 90% at 3–11 weeks post-immunization against a homologous challenge (same Pf strain in vaccine and challenge) in a controlled human malaria infection (CHMI), 80% VE at 9.5 weeks post-immunization (unpublished) against a heterologous challenge (different Pf strains in vaccine and challenge), and 54% heterologous VE at 8 months against a heterologous challenge [5, 7, 8]. In four field trials in Africa, axenic VE was sustained for at least 18 months against strong naturally transmitted Pf malaria, and VE against strong transmitted Pf infection was 52%, 51%, and 47% at 6 months and 58% at 2 years by survival analysis [9, 13]. Radiation-attenuated PfSPZ induces DNA damage and impairs DNA replication during liver stage development [2, 14, 15]. PfSPZ develops into an early liver stage parasite on approximately days 2-3, but does not replicate. Plasmodium-specific immunogens produced during this liver stage elicit immunity primarily due to CD8+ T cells, however, relatively large doses (9 × 10 5 ~1.8×10 6 A total of 10 PfSPZ (PfSPZ) are required [4-9, 16, 17].

[0008] Chemically attenuated vaccines: Vaccines based on live non-attenuated whole parasites targeting (i) the liver stage of the Plasmodium life cycle or (ii) first wave parasite-infected erythrocytes, administered under the cover of an antimalarial drug (chemically attenuated), have shown good efficacy in clinical trials. In a recent trial

[18] , three doses of 2 × 10 5 The PfSPZ vaccine Sanaria® PfSPZ-CVac (CQ) provided 100% protection against heterologous CHMI at 12 weeks after the last vaccine dose. However, when chemical attenuation relies on the blood-stage antimalarial chloroquine (CQ), as in Sanaria® PfSPZ CVac vaccine-CQ, there may be a brief period of transient parasitemia 7-8 days after the first injection of PfSPZ non-attenuated whole parasites, which may result in up to grade 3 malaria symptoms if ibuprofen is not taken. In addition, there is a risk of developing severe malaria if the partner drug is not taken (for whatever reason).

[0009] Genetically attenuated vaccines: Genetically attenuated parasites (GAPs) are created by deletion of specific genes that prevent complete liver stage development. GAPs have been extensively studied in rodent malaria models. The first GAPs were produced by deleting genes that are upregulated in infective sporozoites (UIS) compared to oocyst sporozoites [19-22]. Such deletions did not affect the viability of the GAPs when they were at the sporozoite stage, but led to growth arrest early in the liver stage of development, before active liver stage replication can occur. Such GAPs are termed "early-arrested replication-deficient" (EARD). These GAPs not only exhibited favorable immunogenic properties, but also, at times, showed incomplete attenuation, allowing progression of the life cycle from the liver stage to the blood stage (also termed "breakthrough") and causing active infection. One early-stop GAP, Sanaria® PfSPZ-GA1, was manufactured in compliance with GMP and evaluated in clinical trials. It was shown to be well tolerated, safe and fully attenuated as it did not show any breakthrough blood-stage infections, but the efficacy obtained against CHIM was low

[23] .

[0010] More recently, in an effort to generate parasitic vaccine strains, next-generation GAPs have been developed that undergo growth, development, and replication in the liver but stop development and die just prior to progression to the blood stage [see, for example, 24-27]. These GAPs, termed LARC (late-arrested replication-adapted), are more potent immunogens than EARD GAPs or RAS because they result in significant vaccine biomass expansion in the liver and further progress to the hepatic life cycle, thus presenting a larger and more extensive parasite antigen to the immune system for a more effective subsequent immune response. Indeed, in a rodent malaria model, Plasmodium yoelii LARC GAPs have been shown to provide superior protection from sporozoite challenge compared to EARD GAPs and RAS.

[28] Furthermore, P. yoelii LARC GAPs have been shown to also provide stage-transfer protection from direct blood-stage challenge, demonstrating the presence of antigens in late liver-stage GAPs, which are also characteristic of blood-stage morphology

[29] . However, the published P. yoelii GAPs do not provide information on gene deletions in the human parasitic homolog, P. falciparum, and / or do not show complete attenuation [26, 30]. Thus, there is a need to identify additional targets for gene deletion to ensure complete attenuation. Ultimately, the goal is to generate human malaria LARC GAPs that are fully attenuated in malaria clinical trials and can be used as safe and highly effective vaccines to prevent malaria. However, identifying additional target genes for deletion that result in complete attenuation in parasites that cause human malaria, such as P. falciparum, remains a continuing challenge.

[0011] Despite advances in the art in generating attenuated Plasmodium parasites useful for priming the vertebrate immune system against future infection, there remains a need to identify novel specific gene-based modifications or novel combinations of gene-based modifications that provide complete attenuation, i.e., complete arrest of life cycle progression at late liver stage development, without breakthrough to blood stage infection. This would allow for the safe and long-term development of liver stage parasite vaccine immunogens that provide a greater variety of antigens for more complete immunity against subsequent parasite infection. The present disclosure addresses these and related needs. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] World Health O. World malaria report 2020: 20 years of global progress and challenges. Geneva: World Health Organization2020. [Non-Patent Document 2] Nussenzweig RS, Vanderberg J, Most H, and Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei. Nature 216: 160-162, 1967. [Non-Patent Document 3] Clyde DF. Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971-75. Bull World Health Organ 68(Suppl): 9-12.

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Summary of the Invention

Means for Solving the Problems

[0013] Summary The present disclosure is directed to malaria-causing parasites, specifically Plasmodium species parasites, more specifically Plasmodium species parasites that have been genetically altered to develop normally into the late liver stage of development, but are blocked from transitioning from the liver stage to the blood stage and subsequent red blood cell infection. Specifically, the inventors have identified genetic alterations that attenuate these parasites in the late liver stage of development, causing arrest of this late liver stage, through deletion of the Liver Stage Nuclear Protein (LINUP) gene, which leads to the disruption of the LINUP gene product. The arrested LINUP mutants allow expression of a broad array of parasite-specific antigenicity during liver stage development, but are severely attenuated with respect to blood stage entry, subsequent red blood cell infection, and associated signs, symptoms, and pathology of malaria disease. A family of genetically attenuated Plasmodium species parasites containing LINUP disruptions, including double knockout LARC2 (where both LINUP and PlasMei2 gene functions are deleted), are ideal as live immunogens in malaria vaccines and related therapeutics. [Brief description of the drawings]

[0014] [Figure 1A] 1A-1C. P. yoelii Liver Stage Nuclear Protein (LINUP) (PY17X_1465200) localizes to the nucleus of liver stage parasites and will be referred to as Liver Stage Nuclear Protein (LINUP). Amino acid alignment between APfalciparum LINUP (PF3D7_1249700) (SEQ ID NO:1) and its syntenic orthologs P. yoelii LINUP (PY17X_1465200) (SEQ ID NO:2) and P. vivax LINUP (PVP01_1466800) (SEQ ID NO:3) shows 40% amino acid sequence identity and 60% sequence similarity. See consensus sequence (SEQ ID NO:4), sequence identity / similarity is shown in grey. The predicted and conserved nuclear localization sequence (NLS) is underlined and spans amino acids 132-156. [Figure 1B] Figure 1A-1C. P. yoelii liver stage nuclear protein (LINUP) (PY17X_1465200) localizes to the nucleus of liver stage parasites and will be referred to as liver stage nuclear protein (LINUP). To visualize the localization of P. yoelii LINUP, an mCherry epitope tagged parasite strain was generated by fusing an mCherry tag to the C-terminus of LINUP. The tagged transgenic parasite replaces endogenous LINUP with the tagged copy. Immunofluorescence assay (IFA) on mouse liver infected with P. yoelii LINUPmCherry using mCherry antibody, an antibody against the endoplasmic reticulum marker BiP, and DNA strain DAPI, shows that the protein is expressed 24 h post liver stage infection and localizes to the nucleus (schizont) of late liver stages at both 36 h and 48 h time points. Scale bar size is 5 μm. [Figure 1C] Figure 1A-1C. P. yoelii liver stage nuclear protein (LINUP) (PY17X_1465200) localizes to the nucleus of liver stage parasites and will be referred to as liver stage nuclear protein (LINUP). C. At 48 h, LINUP shows partial colocalization with the histone H3 markers acetylated lysine 9 and DAPI, scale bar is 5 µm, which is further shown in the enlarged image on the right where the scale bar size is 2 µm.

[0015] [Figure 2A]Figures 2A-2E. Generation of P. yoelii LINNUP gene deleted parasites (linup-) and analysis of mosquito stage development. A. Schematic showing generation of P. yoelii linup- parasites using CRISPR / Cas9 mediated gene editing. B. Blood stage growth was compared for P. yoelii XNL wild type and P. yoelii linup- clones c3 and c5 in groups of 5 Swiss Webster mice. One million infected erythrocytes were injected intravenously into each mouse and parasitemia was measured every other day for 10 days. Growth rates were comparable, suggesting that LINUP does not have a significant role in blood stage replication. Data are presented as mean ± SEM, n = 5 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. P. yoelii linup- clones c3 and c5 had no deficits in mosquito infection, as C. oocysts / midgut, D. oocyst abundance, and E; salivary gland sporozoites / mosquito counts were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. E; salivary gland sporozoites / mosquito were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, with P>0.05 interpreted as no significant difference. [Figure 2B]Figures 2A-2E. Generation of P. yoelii LINNUP gene deleted parasites (linup-) and analysis of mosquito stage development. A. Schematic showing generation of P. yoelii linup- parasites using CRISPR / Cas9 mediated gene editing. B. Blood stage growth was compared for P. yoelii XNL wild type and P. yoelii linup- clones c3 and c5 in groups of 5 Swiss Webster mice. One million infected erythrocytes were injected intravenously into each mouse and parasitemia was measured every other day for 10 days. Growth rates were comparable, suggesting that LINUP does not have a significant role in blood stage replication. Data are presented as mean ± SEM, n = 5 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. P. yoelii linup- clones c3 and c5 had no deficits in mosquito infection, as C. oocysts / midgut, D. oocyst abundance, and E; salivary gland sporozoites / mosquito counts were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. E; salivary gland sporozoites / mosquito were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, with P>0.05 interpreted as no significant difference. [Figure 2C]Figures 2A-2E. Generation of P. yoelii LINNUP gene deleted parasites (linup-) and analysis of mosquito stage development. A. Schematic showing generation of P. yoelii linup- parasites using CRISPR / Cas9 mediated gene editing. B. Blood stage growth was compared for P. yoelii XNL wild type and P. yoelii linup- clones c3 and c5 in groups of 5 Swiss Webster mice. One million infected erythrocytes were injected intravenously into each mouse and parasitemia was measured every other day for 10 days. Growth rates were comparable, suggesting that LINUP does not have a significant role in blood stage replication. Data are presented as mean ± SEM, n = 5 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. P. yoelii linup- clones c3 and c5 had no deficits in mosquito infection, as C. oocysts / midgut, D. oocyst abundance, and E; salivary gland sporozoites / mosquito counts were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. E; salivary gland sporozoites / mosquito were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, with P>0.05 interpreted as no significant difference. [Figure 2D]Figures 2A-2E. Generation of P. yoelii LINNUP gene deleted parasites (linup-) and analysis of mosquito stage development. A. Schematic showing generation of P. yoelii linup- parasites using CRISPR / Cas9 mediated gene editing. B. Blood stage growth was compared for P. yoelii XNL wild type and P. yoelii linup- clones c3 and c5 in groups of 5 Swiss Webster mice. One million infected erythrocytes were injected intravenously into each mouse and parasitemia was measured every other day for 10 days. Growth rates were comparable, suggesting that LINUP does not have a significant role in blood stage replication. Data are presented as mean ± SEM, n = 5 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. P. yoelii linup- clones c3 and c5 had no deficits in mosquito infection, as C. oocysts / midgut, D. oocyst abundance, and E; salivary gland sporozoites / mosquito counts were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. E; salivary gland sporozoites / mosquito were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, with P>0.05 interpreted as no significant difference. [Figure 2E]Figures 2A-2E. Generation of P. yoelii LINNUP gene deleted parasites (linup-) and analysis of mosquito stage development. A. Schematic showing generation of P. yoelii linup- parasites using CRISPR / Cas9 mediated gene editing. B. Blood stage growth was compared for P. yoelii XNL wild type and P. yoelii linup- clones c3 and c5 in groups of 5 Swiss Webster mice. One million infected erythrocytes were injected intravenously into each mouse and parasitemia was measured every other day for 10 days. Growth rates were comparable, suggesting that LINUP does not have a significant role in blood stage replication. Data are presented as mean ± SEM, n = 5 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. P. yoelii linup- clones c3 and c5 had no deficits in mosquito infection, as C. oocysts / midgut, D. oocyst abundance, and E; salivary gland sporozoites / mosquito counts were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. E; salivary gland sporozoites / mosquito were comparable between P. yoelii linup- c3 (dark grey) and c5 (light grey) and P. yoelii wild type (black). Data are presented as mean ± SD, n = 3 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, with P>0.05 interpreted as no significant difference.

[0016] [Figure 3A]Figure 3A-3D. Analysis of P. yoelii linup- liver stage development. Tissue sections were prepared from BALB / cJ mice infected with 250,000 sporozoites of either P. yoelii wild-type or P. yoelii linup- at 24, 36, and 48 h postinfection and analyzed by IFA. (A). Comparison of liver stage parasite size (based on the area at the largest perimeter of the parasite) between P. yoelii wild-type and P. yoelii linup- at the 24, 36, and 48 h time points shows that P. yoelii wild-type liver stage schizonts are significantly larger than P. yoelii linup- at the 36 and 48 h time points. Data are expressed as mean ± SD. Each data point refers to the average size of at least 20 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *=P.05, ***P<0.001, P>0.05 interpreted as no significant difference. Liver stage development was compared at 48 h using antibodies against (B) parasite mitochondria (mHSP70) and apicoplast (ACP), (C) parasite plasma membrane and mature extraerythrocytic merozoite marker MSP1, and (D) inner membrane complex protein and mature extraerythrocytic merozoite marker mTIP. DNA was stained with DAPI. Scale bar: 10 μm. P. yoelii linup- liver stage parasites are smaller and show less branching of mitochondrial and apicoplast organelles compared to P. yoelii wild type. P. yoelii linup- expresses late liver stage proteins MSP1 and mTIP, however there is abnormal separation of cytomeres and incomplete formation of mature extraerythrocytic merozoites. [Figure 3B]Figure 3A-3D. Analysis of P. yoelii linup- liver stage development. Tissue sections were prepared from BALB / cJ mice infected with 250,000 sporozoites of either P. yoelii wild-type or P. yoelii linup- at 24, 36, and 48 h postinfection and analyzed by IFA. (A). Comparison of liver stage parasite size (based on the area at the largest perimeter of the parasite) between P. yoelii wild-type and P. yoelii linup- at the 24, 36, and 48 h time points shows that P. yoelii wild-type liver stage schizonts are significantly larger than P. yoelii linup- at the 36 and 48 h time points. Data are expressed as mean ± SD. Each data point refers to the average size of at least 20 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *=P.05, ***P<0.001, P>0.05 interpreted as no significant difference. Liver stage development was compared at 48 h using antibodies against (B) parasite mitochondria (mHSP70) and apicoplast (ACP), (C) parasite plasma membrane and mature extraerythrocytic merozoite marker MSP1, and (D) inner membrane complex protein and mature extraerythrocytic merozoite marker mTIP. DNA was stained with DAPI. Scale bar: 10 μm. P. yoelii linup- liver stage parasites are smaller and show less branching of mitochondrial and apicoplast organelles compared to P. yoelii wild type. P. yoelii linup- expresses late liver stage proteins MSP1 and mTIP, however there is abnormal separation of cytomeres and incomplete formation of mature extraerythrocytic merozoites. [Figure 3C]Figure 3A-3D. Analysis of P. yoelii linup- liver stage development. Tissue sections were prepared from BALB / cJ mice infected with 250,000 sporozoites of either P. yoelii wild-type or P. yoelii linup- at 24, 36, and 48 h postinfection and analyzed by IFA. (A). Comparison of liver stage parasite size (based on the area at the largest perimeter of the parasite) between P. yoelii wild-type and P. yoelii linup- at the 24, 36, and 48 h time points shows that P. yoelii wild-type liver stage schizonts are significantly larger than P. yoelii linup- at the 36 and 48 h time points. Data are expressed as mean ± SD. Each data point refers to the average size of at least 20 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *=P.05, ***P<0.001, P>0.05 interpreted as no significant difference. Liver stage development was compared at 48 h using antibodies against (B) parasite mitochondria (mHSP70) and apicoplast (ACP), (C) parasite plasma membrane and mature extraerythrocytic merozoite marker MSP1, and (D) inner membrane complex protein and mature extraerythrocytic merozoite marker mTIP. DNA was stained with DAPI. Scale bar: 10 μm. P. yoelii linup- liver stage parasites are smaller and show less branching of mitochondrial and apicoplast organelles compared to P. yoelii wild type. P. yoelii linup- expresses late liver stage proteins MSP1 and mTIP, however there is abnormal separation of cytomeres and incomplete formation of mature extraerythrocytic merozoites. [Figure 3D]Figure 3A-3D. Analysis of P. yoelii linup- liver stage development. Tissue sections were prepared from BALB / cJ mice infected with 250,000 sporozoites of either P. yoelii wild-type or P. yoelii linup- at 24, 36, and 48 h postinfection and analyzed by IFA. (A). Comparison of liver stage parasite size (based on the area at the largest perimeter of the parasite) between P. yoelii wild-type and P. yoelii linup- at the 24, 36, and 48 h time points shows that P. yoelii wild-type liver stage schizonts are significantly larger than P. yoelii linup- at the 36 and 48 h time points. Data are expressed as mean ± SD. Each data point refers to the average size of at least 20 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *=P.05, ***P<0.001, P>0.05 interpreted as no significant difference. Liver stage development was compared at 48 h using antibodies against (B) parasite mitochondria (mHSP70) and apicoplast (ACP), (C) parasite plasma membrane and mature extraerythrocytic merozoite marker MSP1, and (D) inner membrane complex protein and mature extraerythrocytic merozoite marker mTIP. DNA was stained with DAPI. Scale bar: 10 μm. P. yoelii linup- liver stage parasites are smaller and show less branching of mitochondrial and apicoplast organelles compared to P. yoelii wild type. P. yoelii linup- expresses late liver stage proteins MSP1 and mTIP, however there is abnormal separation of cytomeres and incomplete formation of mature extraerythrocytic merozoites.

[0017] [Figure 4A]Figures 4A-4E. Generation of P. falciparum linup- and analysis of mosquito stage development. A. Schematic showing generation of P. falciparum linup- knockout parasites using CRISPR / Cas9-mediated gene editing. Primers used to verify gene deletion are indicated, and PCR amplicon sizes are shown in kilobases. B. Agarose gel electrophoresis shows PCR products corresponding to gene deletion of P. falciparum LINUP in clones B1 and B4. P. falciparum linup- did not have a defect in mosquito infectivity, as C. oocysts / midgut, D. oocyst abundance, and E. salivary gland sporozoite / mosquito counts were comparable between P. falciparum linup- clones B1 (dark grey) and B4 (light grey) and P. falciparum NF54 (black). Data are presented as mean ± SD, n = 2 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. [Figure 4B]Figures 4A-4E. Generation of P. falciparum linup- and analysis of mosquito stage development. A. Schematic showing generation of P. falciparum linup- knockout parasites using CRISPR / Cas9-mediated gene editing. Primers used to verify gene deletion are indicated, and PCR amplicon sizes are shown in kilobases. B. Agarose gel electrophoresis shows PCR products corresponding to gene deletion of P. falciparum LINUP in clones B1 and B4. P. falciparum linup- did not have a defect in mosquito infectivity, as C. oocysts / midgut, D. oocyst abundance, and E. salivary gland sporozoite / mosquito counts were comparable between P. falciparum linup- clones B1 (dark grey) and B4 (light grey) and P. falciparum NF54 (black). Data are presented as mean ± SD, n = 2 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference. [Figure 4C-E]Figures 4A-4E. Generation of P. falciparum linup- and analysis of mosquito stage development. A. Schematic showing generation of P. falciparum linup- knockout parasites using CRISPR / Cas9-mediated gene editing. Primers used to verify gene deletion are indicated, and PCR amplicon sizes are shown in kilobases. B. Agarose gel electrophoresis shows PCR products corresponding to gene deletion of P. falciparum LINUP in clones B1 and B4. P. falciparum linup- did not have a defect in mosquito infectivity, as C. oocysts / midgut, D. oocyst abundance, and E. salivary gland sporozoite / mosquito counts were comparable between P. falciparum linup- clones B1 (dark grey) and B4 (light grey) and P. falciparum NF54 (black). Data are presented as mean ± SD, n = 2 biological replicates. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. P > 0.05 is interpreted as no significant difference.

[0018] [Figure 5A]Figures 5A-5B. P. falciparum linup- liver stages show abnormal growth and abnormal late liver stage development. Tissue sections were prepared from FRG NOD huHep mice infected with 1 million sporozoites of P. falciparum NF54 and P. falciparum linup- at days 5 and 7 post-sporozoite infection and analyzed for size and protein expression. A. Comparison of liver stage parasite size (based on area at largest parasite perimeter) between P. falciparum NF54 and P. falciparum linup- at days 5 and 7 post-sporozoite infection. There was no growth defect between P. falciparum NF54 and P. falciparum linup- liver stage schizonts at day 5, but there was a statistically significant difference in size of late liver stage schizonts at day 7. Data are presented as mean ± SD. Each data point refers to the average size of at least 30-50 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. ***P<0.001, P>0.05 interpreted as no significant difference. B. IFA shows that at 7 days post-infection, wild-type NF54 parasite vacuolar membrane protein EXP1 has the circumferential staining pattern in the wild type, as expected. In comparison, EXP1 expression in mature P. falciparum linup- at day 7 of the liver stage is uneven and aberrant. [Figure 5B]Figures 5A-5B. P. falciparum linup- liver stages show abnormal growth and abnormal late liver stage development. Tissue sections were prepared from FRG NOD huHep mice infected with 1 million sporozoites of P. falciparum NF54 and P. falciparum linup- at days 5 and 7 post-sporozoite infection and analyzed for size and protein expression. A. Comparison of liver stage parasite size (based on area at largest parasite perimeter) between P. falciparum NF54 and P. falciparum linup- at days 5 and 7 post-sporozoite infection. There was no growth defect between P. falciparum NF54 and P. falciparum linup- liver stage schizonts at day 5, but there was a statistically significant difference in size of late liver stage schizonts at day 7. Data are presented as mean ± SD. Each data point refers to the average size of at least 30-50 parasites for each time point. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. ***P<0.001, P>0.05 interpreted as no significant difference. B. IFA shows that at 7 days post-infection, wild-type NF54 parasite vacuolar membrane protein EXP1 has the circumferential staining pattern in the wild type, as expected. In comparison, EXP1 expression in mature P. falciparum linup- at day 7 of the liver stage is uneven and aberrant.

[0019] [Figure 6A]Figures 6A-6C. P. falciparum linup- shows severe attenuation of liver stage development and blood stage transition in infected FRG NOD huHep humanized mice. A. Schematic showing the experimental design of the liver stage to blood stage transition experiment in FRG NOD huHep mice. One million sporozoites derived from P. falciparum NF54 and P. falciparum linup- are injected into one and three FRG NOD huHep mice, respectively. Mice were repopulated with human erythrocytes as indicated. All mice were euthanized on day 7 and 50 μl blood samples were taken from all mice for parasite burden based on 18S rRNA qRT-PCR sampling. The remaining blood was transferred to in vitro culture and grown in culture for 7 days. An additional 50 μl blood sample was taken from the in vitro cultured samples 7 days after transfer to in vitro culture for analysis of parasite 18S rRNA qRT-PCR samples (day 14 of sporozoite infection). B. Analysis of parasite burden by 18S rRNA qRT-PCR was performed on RNA extracted from blood of mice infected with P. falciparum NF54 and P. falciparum linup- sporozoites 7 days after completion of expected liver stage development and liver to blood stage transition (day 7, black bars). One of three mice infected with P. falciparum linup- sporozoites showed no blood stages in the blood, whereas the other two infected mice had a 10,000-fold reduction in parasite RNA load compared to wild type, indicating a severe defect in the transition to blood stages of P. falciparum linup-. Blood was transferred to in vitro culture and parasite load was measured after an additional 7 days (day 14, grey bar). Wild type NF54 blood stages replicated during the 7 days in culture, but no significant replication was seen for P. falciparum linup- blood stages.This indicates that the liver-to-blood stage transition of P. falciparum linup- did not release viable blood stage parasites capable of initiating blood stage replication. C. Analysis of parasite liver burden by 18S rRNA qRT-PCR was performed on RNA extracted from livers infected with P. falciparum NF54 and P. falciparum linup- at day 7 post-infection and is shown as copy number of P. falciparum 18S rRNA per μg of extracted liver RNA. Parasite liver burden is comparable between P. falciparum NF54 and P. falciparum linup-. [Figure 6B-C]Figures 6A-6C. P. falciparum linup- shows severe attenuation of liver stage development and blood stage transition in infected FRG NOD huHep humanized mice. A. Schematic showing the experimental design of the liver stage to blood stage transition experiment in FRG NOD huHep mice. One million sporozoites derived from P. falciparum NF54 and P. falciparum linup- are injected into one and three FRG NOD huHep mice, respectively. Mice were repopulated with human erythrocytes as indicated. All mice were euthanized on day 7 and 50 μl blood samples were taken from all mice for parasite burden based on 18S rRNA qRT-PCR sampling. The remaining blood was transferred to in vitro culture and grown in culture for 7 days. An additional 50 μl blood sample was taken from the in vitro cultured samples 7 days after transfer to in vitro culture for analysis of parasite 18S rRNA qRT-PCR samples (day 14 of sporozoite infection). B. Analysis of parasite burden by 18S rRNA qRT-PCR was performed on RNA extracted from blood of mice infected with P. falciparum NF54 and P. falciparum linup- sporozoites 7 days after completion of expected liver stage development and liver to blood stage transition (day 7, black bars). One of three mice infected with P. falciparum linup- sporozoites showed no blood stages in the blood, whereas the other two infected mice had a 10,000-fold reduction in parasite RNA load compared to wild type, indicating a severe defect in the transition to blood stages of P. falciparum linup-. Blood was transferred to in vitro culture and parasite load was measured after an additional 7 days (day 14, grey bar). Wild type NF54 blood stages replicated during the 7 days in culture, but no significant replication was seen for P. falciparum linup- blood stages.This indicates that the liver-to-blood stage transition of P. falciparum linup- did not release viable blood stage parasites capable of initiating blood stage replication. C. Analysis of parasite liver burden by 18S rRNA qRT-PCR was performed on RNA extracted from livers infected with P. falciparum NF54 and P. falciparum linup- at day 7 post-infection and is shown as copy number of P. falciparum 18S rRNA per μg of extracted liver RNA. Parasite liver burden is comparable between P. falciparum NF54 and P. falciparum linup-.

[0020] [Figure 7A-1] Figures 7A-7D. Generation of P. falciparum plasmei2 / linup (P. falciparum LARC2). A. The diagram shows the generation of P. falciparum plasmei2 parasites using CRISPR / Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. B. The diagram shows the generation of P. falciparum linup parasites using CRISPR / Cas9-mediated gene editing. The marker-free P. falciparum PlasMei2 clone F3 from (A) was used for transfection of the P. falciparum LINUP KO plasmid. Recombinant parasites were cloned by limiting dilution to generate P. falciparum plasmei2 / linup double knockout (LARC2) clones. The primers used to verify the gene deletion are shown. The primer combinations used and the size of the PCR products are shown in Table 2. Agarose gel electrophoresis of the absence of P. falciparum PlasMei2, the absence of BPfalciparum LINUP C., and the absence of plasmid DNA D. in four P. falciparum LARC2 clones - C12, F7, B7, and B5. [Figure 7A-2]Figures 7A-7D. Generation of P. falciparum plasmei2 / linup (P. falciparum LARC2). A. The diagram shows the generation of P. falciparum plasmei2 parasites using CRISPR / Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. B. The diagram shows the generation of P. falciparum linup parasites using CRISPR / Cas9-mediated gene editing. The marker-free P. falciparum PlasMei2 clone F3 from (A) was used for transfection of the P. falciparum LINUP KO plasmid. Recombinant parasites were cloned by limiting dilution to generate P. falciparum plasmei2 / linup double knockout (LARC2) clones. The primers used to verify the gene deletion are shown. The primer combinations used and the size of the PCR products are shown in Table 2. Agarose gel electrophoresis of the absence of P. falciparum PlasMei2, the absence of BPfalciparum LINUP C., and the absence of plasmid DNA D. in four P. falciparum LARC2 clones - C12, F7, B7, and B5. [Figure 7B]Figures 7A-7D. Generation of P. falciparum plasmei2 / linup (P. falciparum LARC2). A. The diagram shows the generation of P. falciparum plasmei2 parasites using CRISPR / Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. B. The diagram shows the generation of P. falciparum linup parasites using CRISPR / Cas9-mediated gene editing. The marker-free P. falciparum PlasMei2 clone F3 from (A) was used for transfection of the P. falciparum LINUP KO plasmid. Recombinant parasites were cloned by limiting dilution to generate P. falciparum plasmei2 / linup double knockout (LARC2) clones. The primers used to verify the gene deletion are shown. The primer combinations used and the size of the PCR products are shown in Table 2. Agarose gel electrophoresis of the absence of P. falciparum PlasMei2, the absence of BPfalciparum LINUP C., and the absence of plasmid DNA D. in four P. falciparum LARC2 clones - C12, F7, B7, and B5. [Figure 7C]Figures 7A-7D. Generation of P. falciparum plasmei2 / linup (P. falciparum LARC2). A. The diagram shows the generation of P. falciparum plasmei2 parasites using CRISPR / Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. B. The diagram shows the generation of P. falciparum linup parasites using CRISPR / Cas9-mediated gene editing. The marker-free P. falciparum PlasMei2 clone F3 from (A) was used for transfection of the P. falciparum LINUP KO plasmid. Recombinant parasites were cloned by limiting dilution to generate P. falciparum plasmei2 / linup double knockout (LARC2) clones. The primers used to verify the gene deletion are shown. The primer combinations used and the size of the PCR products are shown in Table 2. Agarose gel electrophoresis of the absence of P. falciparum PlasMei2, the absence of BPfalciparum LINUP C., and the absence of plasmid DNA D. in four P. falciparum LARC2 clones - C12, F7, B7, and B5. [Figure 7D]Figures 7A-7D. Generation of P. falciparum plasmei2 / linup (P. falciparum LARC2). A. The diagram shows the generation of P. falciparum plasmei2 parasites using CRISPR / Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. B. The diagram shows the generation of P. falciparum linup parasites using CRISPR / Cas9-mediated gene editing. The marker-free P. falciparum PlasMei2 clone F3 from (A) was used for transfection of the P. falciparum LINUP KO plasmid. Recombinant parasites were cloned by limiting dilution to generate P. falciparum plasmei2 / linup double knockout (LARC2) clones. The primers used to verify the gene deletion are shown. The primer combinations used and the size of the PCR products are shown in Table 2. Agarose gel electrophoresis of the absence of P. falciparum PlasMei2, the absence of BPfalciparum LINUP C., and the absence of plasmid DNA D. in four P. falciparum LARC2 clones - C12, F7, B7, and B5.

[0021] [Figure 8A] Figures 8A and 8B. Absence of P. falciparum PlasMei2 and P. falciparum LINUP in P. falciparum LARC2 clones. Eight P. falciparum LARC2 clones were analyzed by whole genome sequencing to confirm A. deletion of P. falciparum PlasMei2 on chromosome 6, and B. deletion of P. falciparum LINUP on chromosome 12. P. falciparum LARC2 clones B7, C12, D5, and F7 will be used for further phenotypic analysis. [Figure 8B]Figures 8A and 8B. Absence of P. falciparum PlasMei2 and P. falciparum LINUP in P. falciparum LARC2 clones. Eight P. falciparum LARC2 clones were analyzed by whole genome sequencing to confirm A. deletion of P. falciparum PlasMei2 on chromosome 6, and B. deletion of P. falciparum LINUP on chromosome 12. P. falciparum LARC2 clones B7, C12, D5, and F7 will be used for further phenotypic analysis.

[0022] [Figure 9A-B] Figure 9A-9D. A. 15 days after establishment of the four P. falciparum LARC2KO clones B7, C12, D5, and F7, flagellar extrusion centers were counted in mature gametocyte cultures of P. falciparum LARC2 and found to be comparable to P. falciparum NF54. These gametocyte cultures were fed by female Anopheles stephensi mosquitoes in a standard membrane blood-feeding assay, and 8 days after blood-feeding, midguts were dissected. B. Oocyst abundance, C. oocyst / mosquito counts were comparable to wild type for P. falciparum LARC2KO clones F7 and B7. These two clones will be used for further phenotypic analysis. D. sporozoite / mosquito counts were comparable to wild type for P. falciparum LARC2 clones F7 and B7. Clone F7 was used for further phenotypic analysis. [Figure 9C]Figure 9A-9D. A. 15 days after establishment of the four P. falciparum LARC2KO clones B7, C12, D5, and F7, flagellar extrusion centers were counted in mature gametocyte cultures of P. falciparum LARC2 and found to be comparable to P. falciparum NF54. These gametocyte cultures were fed by female Anopheles stephensi mosquitoes in a standard membrane blood-feeding assay, and 8 days after blood-feeding, midguts were dissected. B. Oocyst abundance, C. oocyst / mosquito counts were comparable to wild type for P. falciparum LARC2KO clones F7 and B7. These two clones will be used for further phenotypic analysis. D. sporozoite / mosquito counts were comparable to wild type for P. falciparum LARC2 clones F7 and B7. Clone F7 was used for further phenotypic analysis. [Figure 9D] Figure 9A-9D. A. 15 days after establishment of the four P. falciparum LARC2KO clones B7, C12, D5, and F7, flagellar extrusion centers were counted in mature gametocyte cultures of P. falciparum LARC2 and found to be comparable to P. falciparum NF54. These gametocyte cultures were fed by female Anopheles stephensi mosquitoes in a standard membrane blood-feeding assay, and 8 days after blood-feeding, midguts were dissected. B. Oocyst abundance, C. oocyst / mosquito counts were comparable to wild type for P. falciparum LARC2KO clones F7 and B7. These two clones will be used for further phenotypic analysis. D. sporozoite / mosquito counts were comparable to wild type for P. falciparum LARC2 clones F7 and B7. Clone F7 was used for further phenotypic analysis.

[0023] [Figure 10A]Figures 10A-10C. P. falciparum LARC2 parasites are severely attenuated at liver stage development and are unable to generate infectious extra-erythrocytic merozoites. A. Schematic diagram showing the experimental design. P. falciparum NF54 and P. falciparum LARC2 sporozoites were isolated from the salivary glands of infected Anopheles stephensi mosquitoes. To evaluate the blood stage transition of P. falciparum LARC2 in FRG NOD huHep mice, 1 x 106 sterile cryopreserved P. falciparum NF54 (PFSPZ) and sterile cryopreserved P. falciparum LARC2 (PFSPZ LARC2) sporozoites were injected intravenously into 4 and 6 FRG NOD huHep mice per group, respectively. On days 6 and 7, 400 μl of 70% RBCs were injected intravenously to allow migration of liver-stage parasites into the blood. After 4 h of human RBC repopulation on day 7, mice were euthanized, blood was collected by cardiac puncture, and 50 μl of blood from each mouse was used for qRT-PCR analysis to detect parasite 18S RNA. Blood was washed three times in sex-free medium, a volume of human RBCs equivalent to the packed RBC volume was added, and blood was transferred to in vitro culture. Fresh medium was replaced daily and cultures were analyzed for the presence of parasites by overlay smears every 2-3 days for up to 6 weeks. [Figure 10B]Figures 10A-10C. P. falciparum LARC2 parasites are severely attenuated during liver stage development and are unable to generate infectious extraerythrocytic merozoites. B. On day 7, parasite densities in mice infected with PfSPZ ranged from 103 to 105 parasite equivalents / ml by P. falciparum 18S qRT-PCR, which further increased to 109 parasite equivalents / ml after 7 days of in vitro culture. In contrast, in mice infected with PFSPZ LARC2, 0 to 103 parasite equivalents / ml were detected by P. falciparum 18S qRT-PCR, and there was a dramatic drop in P. falciparum 18S rRNA signal after 7 days of culture, indicating that viable extraerythrocytic merozoites were not released from PFSPZ LARC2 liver stage schizonts. This was further confirmed by thin blood smears. Blood stage parasites were detected by Giemsa-stained thin blood smears within 1-3 days of transfer to in vitro culture in all four mice infected with PfSPZ (see Table 5). In contrast, no blood stage parasites were detected by Giemsa-stained thin blood smears in all six mice infected with PfSPZ LARC2. [Figure 10C] Figures 10A-10C. P. falciparum LARC2 parasites are severely attenuated in liver stage development and are unable to generate infectious extraerythrocytic merozoites. (C) Livers of infected mice were also harvested on day 7 to extract RNA and for analysis of parasite liver burden by P. falciparum 18S qRT-PCR. Parasite liver burden is shown as log10 copy number of P. falciparum 18S rRNA per µg of total liver RNA. Parasite liver burden was comparable between PfSPZ and PfSPZ LARC2, indicating that LARC2 persists in the liver until day 7 and undergoes significant biomass expansion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description definition "Additives," as used herein as a noun, are compounds or compositions added to a sporozoite preparation. Additives include diluents, carriers, excipients, cryoprotectants, and the like.

[0025] "Sterile" as used herein means free from the introduction of detectable contaminants such as other microorganisms, e.g., bacteria, fungi, pathogenic viruses, etc. A sterile sporozoite preparation results in a sterile preparation of sporozoites that is free of any other type of microorganism or infectious agent. Microbiological assays used to monitor sterility procedures evaluate the presence or absence of contaminants. These include the Microbial Limits Test, current USP <61> These include, but are not limited to, those disclosed herein which are incorporated by reference.

[0026] "Attenuated" as used herein means rendering a living organism unable to complete its life cycle without killing it. The organism may have a limited ability to replicate, express proteins, and develop through several life cycle stages, but stops development at a particular life cycle stage and is unable to progress beyond that stage. With respect to the attenuated Plasmodium parasites disclosed herein, they retain the ability to infect host liver cells and express stage-specific proteins, but are unable to develop beyond the liver stage, are unable to transition to a blood stage infection in the bloodstream of an infected host after liver stage development, and are unable to cause malarial disease pathology.

[0027] "Challenge", as used herein, refers to the presentation of an infectious pathogen to a subject who has previously been provided with a vaccine intended to confer protective immunity against infection / disease caused by the challenging pathogen. For malaria, challenge can be by inoculation with infectious Plasmodium sporozoites (CHMI) [31, 32], by exposure to Anopheles mosquitoes carrying infectious Plasmodium sporozoites, or by field trials monitoring vaccinated subjects in areas where the malaria parasite is naturally transmitted by the bite of infected mosquitoes.

[0028] "Conferring protective immunity," as used herein, refers to providing a population or host (i.e., an individual) with the ability to generate an immune response to protect against an infection / disease (e.g., malaria) caused by a pathogen (e.g., Plasmodium), such that clinical symptoms, pathology, or symptoms of the disease in the host are reduced compared to an untreated host, or such that the rate at which clinical symptoms, pathology, or symptoms of the infection or disease appear within the population is reduced compared to an untreated population.

[0029] As used herein, the term "disrupt" with respect to gene function means to interfere with the function of a gene, e.g., to inhibit, inactivate, attenuate, or prevent the function of a gene or an encoded gene product. Interference or disruption can be accomplished, for example, by altering (e.g., substituting, modifying, deleting, adding, knocking down, knocking out, or knocking in) the gene sequence in such a manner and / or to such an extent that the translated protein, if present, is unable to perform its wild-type function. Alternatively, the gene sequence may be deleted.

[0030] As used herein, the term "genetically modified" refers to a modification to the genome of a wild-type Plasmodium organism that results in a critical difference from the wild-type genome sequence. The genetic modification is brought about by human manipulation, for example, by genetic engineering. In the context of the inventions disclosed herein, the genetic modification can be one or more insertions or deletions of genes, knockouts of genes, or other modifications. In these inventions, the genetic modification results in the functional disruption of the LINUP gene, and in certain embodiments, the LINUP gene and the PlasMei2 gene.

[0031] "Immune response" as used herein means the recipient's response to the introduction of attenuated sporozoites, typically characterized by, but not limited to, the production of antibodies and / or T cells. In general, the immune response can be a cellular response specific for Plasmodium species epitopes, e.g., induction or activation of CD4+ or CD8+ T cells, a humoral response that is an increase in the production of Plasmodium-specific antibodies, or both a cellular and humoral response. In the context of malaria vaccines, the immune response built by a vaccine containing live sporozoites includes, but is not limited to, responses to proteins expressed by extracellular sporozoites, intracellular liver stages, or other stages of the parasite. Mononuclear cells, e.g., dendritic cells, are expected to obtain components of the parasite and present these antigens to relevant immune cells. In the present invention, upon subsequent challenge with an infectious organism, the immune response prevents the development of the pathogenic parasite to a disease-causing asexual erythrocytic stage.

[0032] As used herein, the term "live" refers to continued metabolic activity in the Plasmodium organism. In some embodiments, "live" refers to the ability of the Plasmodium organism to eventually establish at least a transient infection, for example, in hepatocytes (culture or in vivo). The Plasmodium organism may be at any relevant developmental stage, as practical given the genetic attenuation. Thus, for example, the Plasmodium organism may be at a developmental stage within a mosquito, at an infective sporozoite stage, or at an intrahepatocyte (liver) stage.

[0033] "In vitro" produced sporozoites, or "iSPZ", as used herein, refers to sporozoites that have developed externally to the mosquito, i.e., sporogony from the gametocyte stage to the mature infective sporozoite stage is external to the mosquito.

[33]

[0034] "Metabolically active" as used herein means alive and capable of carrying out persistence functions and some life cycle processes. With respect to attenuated sporozoites, this includes, but is not limited to, sporozoites that are capable of invading hepatocytes in culture and in vivo, potentially dividing, have a substantial ability to progress to several developmental stages, and can express stage-specific proteins de novo.

[0035] As used herein, the term "Plasmodium organism" or "Plasmodium species" refers to any parasitic organism belonging to the genus Plasmodium. In some embodiments, the Plasmodium organism has a human host range, e.g., P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. In some embodiments, the Plasmodium organism is P. falciparum.

[0036] A "promoter," as used herein, is a region of DNA upstream of a gene to which relevant proteins (e.g., RNA polymerase and transcription factors) bind and initiate and regulate transcription of that gene.

[0037] A "transgene" as used herein is a gene that is not normally present in the organism into which the transgene is introduced. In some embodiments of the present invention, the expression of the transgene or the encoded polypeptide functions to disrupt one or more gene functions of the organism. In other embodiments, the transgene encodes a foreign antigen, the expression of which induces an immune response.

[0038] A "vaccine" as used herein is a preparation that includes an immunogenic substance and pharma- ceutically acceptable additives, such as excipients, adjuvants, and / or additives or protectants. The immunogen may consist of the entire infectious agent, or a molecular subset of the infectious agent (synthetically or recombinantly produced by the infectious agent). When the vaccine is administered to a subject, the immunogen is expected to stimulate an immune response that will protect the subject from disease upon subsequent challenge with the infectious agent, or reduce the pathology, symptoms, or clinical symptoms caused by the agent. Therapeutic (treatment) vaccines are provided after infection and are intended to reduce or halt the progression of the disease. Preventive (prophylactic) vaccines are intended to prevent initial infection or reduce the infection rate or dose.

[0039] The present disclosure is directed to genetically attenuated malaria parasites that develop into late liver stages but are blocked from transitioning to blood stages of red blood cell infection. Specifically, the attenuated malaria parasites contain a genetic modification that disrupts the functionality of the liver stage nucleoprotein (LINUP) gene. In some embodiments, the malaria parasites include human host-range Plasmodium species, such as P.falciparum, P.vivax, P.malariae, P.ovale, P.knowlesi. In some embodiments, the malaria parasites contain additional genetic modifications that block other genes that are critical for the transition from liver to blood stages. As a non-limiting example, the malaria parasites of certain embodiments additionally contain genetic modifications that disrupt both LINUP and PlasMei2 gene functions (this double knockout is variously referred to as "linup / plasmei2 double knockout (KO)", "LARC2", "LARC2 KO", and the like, and these terms are equivalent). The disclosed attenuated malaria parasites are useful in additives, vaccines for the prevention and mitigation of malaria, methods for inducing and / or stimulating the human immune system against Plasmodium specific antigens, methods for inducing an immune response to one or more Plasmodium antigens in a human subject, or methods for conferring protective immunity against malaria caused by Plasmodium species parasites in a human subject, which are also encompassed by the present disclosure. In some embodiments, the disclosed attenuated malaria parasites are useful in compositions and methods for mitigating, reducing, preventing, treating, and / or protecting against malaria infection.

[0040] As described in more detail below, the inventors have discovered a novel protein (LINUP) described as a liver stage nuclear protein or liver stage specific protein that localizes in the parasite nucleus. Knocking out the gene or gene function that codes for this protein results in life cycle arrest at a late stage of liver stage development. In addition, the inventors have developed a double knockout called LARC2 (Late Arrested Replication Competent Double KO) that contains functional deletion of both the PlasMei2

[27] and LINUP genes. This double knockout is a superior immunogen compared to previous vaccines based on replication-deficient whole parasites because it undergoes almost complete development to late liver stage in the liver, resulting in an expansion of the biomass and diversity of parasite antigens. In addition, LARC2 is more robustly attenuated due to genetic engineering of two genes required for blood-stage transition, ensuring batch-to-batch uniformity and immunizing with a live non-attenuated sporozoite vaccine under drug cover, overcoming the need for antimalarial drug cover (another late liver-stage approach mentioned above), a major limitation associated with chemically attenuated vaccination. Genetically attenuated Plasmodium species (e.g., P. falciparum) having a gene knockout of LINUP, and in another embodiment, another liver stage specific gene, such as PlasMei2, or in yet another embodiment, another gene encoding a protein required for the transition from the liver stage to the blood stage, can be used to induce an immune response to one or more Plasmodium species antigens in a subject, particularly a human subject, to confer protective immunity to malaria caused by Plasmodium species parasites in a subject, particularly a human subject, to immunize a subject, particularly a human subject, as a pre-erythrocytic stage vaccine to provide or boost immunity against reinfection, and / or to treat, prevent, or alleviate malaria infection. Complete blocking of the Plasmodium species parasite life cycle at the liver stage also results in the prevention of the transmission of the parasite to other individuals.This is vital in the eradication campaign to eliminate malaria.

[0041] In one embodiment, a live Plasmodium species organism has been genetically modified to disrupt the LINUP gene, which encodes a liver stage nuclear protein, thereby preventing the biological function of the protein encoded by the wild-type LINUP gene.

[0042] In one embodiment, live Plasmodium species linup - The parasite arrests its life cycle development at the late liver stage within the intermediate mammalian host.

[0043] In another embodiment, Plasmodium species linup - Or LARC2 parasites contain at least one transgene, and in yet other embodiments, the transgene is under the control of a promoter that provides transcription of the transgene during the sporozoite or liver stage of development. In certain embodiments, the one or more transgenes encode blood stage or gametocyte associated antigens. By providing expression of one or more blood stage or gametocyte associated antigens, GAP has additional immunogenicity and provides protection against blood stage parasites (asexual and sexual). Thus, even if GAP is completely arrested before development to the blood stage, it is expected that it will still be able to stimulate immune cells against antigens characteristic of blood stage parasites. This provides additional protection against blood stage parasites, reducing the risk of clinical symptoms as well as the spread of infection in gametocyte antigens. In other embodiments, the one or more transgenes encode antigens derived from other pathogens, thus providing an immune response against these additional pathogens.

[0044] In certain embodiments, the Plasmodium species organism is P. falciparum, P. vivax, P. malariae, P. ovale, or P. knowlesi.

[0045] In certain embodiments, the Plasmodium species organism is in the sporozoite stage of development.

[0046] In one embodiment, the live Plasmodium species linup before genetic modification. - A functional LINUP gene has at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% nucleotide sequence identity to SEQ ID NO:35.

[0047] In one embodiment, live Plasmodium species linup - In the parasite, a functional LINUP gene prior to genetic modification encodes a LINUP polypeptide having at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to the amino acid sequence set forth in PF3D7_1249700, SEQ ID NO:1.

[0048] In one embodiment, the vaccine composition comprises Plasmodium species linup in the sporozoite stage. - The vaccine composition comprises a parasite and an excipient, wherein the vaccine composition is prepared sterile.

[0049] In one embodiment, Plasmodium species linup - The parasite is genetically modified to disrupt one or more additional gene functions, each of which is required for the transition from liver stage to blood stage, and in another embodiment, the additional gene function is PlasMei2. In certain embodiments, the functional PlasMei2 gene before genetic modification comprises a nucleic acid sequence that is at least about 70%, 80%, 90%, or 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the amino acid sequence of the functional PlasMei2 polypeptide encoded by the PlasMei2 gene comprises is at least about 70%, 80%, 90%, or 95% identical to the sequence set forth in SEQ ID NO: 36.

[0050] In certain embodiments, the vaccine composition comprises Plasmodium species LARC2 (linup- / plasmei2-) and an excipient. In certain embodiments, the vaccine composition is prepared sterile.

[0051] Another embodiment is a method for inducing an immune response to one or more Plasmodium antigens in a human subject, comprising administering one or more doses of Plasmodium species linup - Yet another embodiment relates to a method for inducing an immune response to one or more Plasmodium antigens in a human subject, comprising administering to the subject one or more doses of Plasmodium species LARC2. - Still other embodiments relate to methods comprising administering a vaccine composition to a human subject, wherein the immune response generated by administration of these vaccines mitigates or protects against infection by a subsequent wild-type Plasmodium challenge. Still other embodiments relate to methods of conferring protective immunity against malaria caused by Plasmodium species parasites in a human subject by administration of these vaccines.

[0052] In one embodiment, the Plasmodium species LINUP - Sporozoites are produced in vitro and, in one embodiment, Plasmodium species LARC2 - Sporozoites are produced in vitro. In vitro produced Plasmodium species sporozoites have been disclosed

[34] , and in particular, in vitro produced genetically attenuated Plasmodium species sporozoites have been disclosed

[35] , both of which references are incorporated herein in their entireties.

[0053] Purification method Methods for purifying live organisms of about 10 microns in size, such as Plasmodium species sporozoites, e.g., P. falciparum SPZ, particularly genetically attenuated Plasmodium species SPZ

[37] , are known in the art, as exemplified by

[36] . These methods utilize a series of different types of size-exclusion filters with different pore sizes assembled in a non-intuitive manner. This approach excludes accompanying material from preparations of live, motile parasites. A unique aspect of this method is that the pore size of adjacent size-exclusion filters is not necessarily smaller than the pore size of the size-exclusion filter preceding it. Another inventive aspect is that some filters provide a matrix of nominal pore sizes, with at least one filter providing a track-etched filter with a precise pore size. At least one filter has a pore size close to or slightly smaller than the diameter of the parasite.

[0054] In preparations for purification, salivary glands from 150-400 mosquitoes are typically dissected. Sporozoites are released from the glands by passing them in and out of a needle and syringe (trituration), and the sporozoites from these glands are purified collectively. However, in scaled-up preparations, several times more mosquitoes may be dissected, in some embodiments up to 1,000 mosquitoes, in other embodiments up to 5,000 mosquitoes, and in other embodiments up to 10,000 mosquitoes. Sporozoites are released from the glands by trituration, and the triturated salivary gland preparation (pre-purification preparation) is purified by a size-exclusion filtration process as disclosed herein. Sporozoites are maintained in an excipient, typically 1 percent human serum albumin (HSA) in Earle's salts-containing 199 medium (E-199), during the purification process.

[0055] A - Preparation of material for purification The crushed dissection products (pre-purification preparation) are placed into a single tube all at once. This is the mosquito salivary gland material (SGM) pre-purification preparation. It represents approximately 100,000 to 1 billion sporozoites, preferably at least 1 million sporozoites, more preferably at least 25 million sporozoites. The amount of SGM measured in the pre-purification preparation is usually 300 ng to 12,000 ng per 25,000 sporozoites, more typically 400 ng to 1,100 ng per 25,000 sporozoites. The pre-purification preparation was then diluted to 10 ml with excipient. The solutions and samples were kept at 15 to 30° C. for the duration of the purification.

[0056] B - Purification Procedure A peristaltic pump is used to pump the diluted pre-purified preparation through a series of size exclusion filters at a flow rate of at least 1 ml / min but not more than 1000 ml / min, preferably at least 2 ml / min but not more than 500 ml / min, more preferably at least 3 ml / min and not more than 200 ml / min. The corresponding flow rate through each filter is at least 1 L / hr / m. 2 However, it is 2000L / hour / m 2 Not to exceed 3 L / h / m 2 ~1500L / hour / m 2 , most preferably at least 125 L / hr / m 2 but 250L / hour / m 2The filters are usually connected in series with medical grade silicone tubing. Preferably, the first filter (filter number 1) or the first two filters (filter numbers 1 and 2) are matrix filters and are made of polypropylene, however, nylon, mixed cellulose esters, and borosilicate glass, or other materials known to those skilled in the art, may be used. Preferably, the penultimate filter (filter number 3) is a membrane filter, most preferably a track-etched polycarbonate filter, although other filters with similar properties known to those skilled in the art may be used. For aseptic procedures, the filters are sterile. In an embodiment, three filters (filter number 1, filter number 2, and filter number 3) are connected in series, and sporozoites are captured by filter number 4 by dead-end filtration. Additional filters may be used. In an embodiment, filter number 1 is a membrane matrix with a nominal pore size of at least about 2.5 microns but not more than about 30 microns, preferably at least about 5 microns but not more than 20 microns. In one embodiment, the filter used has a nominal pore size of about 10 microns and is 17.5 cm 2 (Polygard®-CN Optiscale - Millipore Catalog No. SN1HA47HH3). In a scaled-up embodiment, the filtration area is 1800 cm 2 Filter number 2, which is also a membrane matrix, has a nominal pore size of at least about 0.3 microns but not greater than about 1.2 microns. In one embodiment, the nominal pore size is about 0.6 microns and the nominal pore size is 17.5 cm 2 (Polygard®-CN Optiscale filter - Millipore Catalog No. SN06A47HH3), which is smaller than the diameter of a Plasmodium sporozoite. In a scaled-up embodiment, the filtration area is 1800 cm 2In one embodiment, filter number 3 is a track etched membrane filter with precise pore size and constant pore size, with a pore size of at least 1.2 microns but not more than 3 microns, larger than the nominal pore size of the preceding filter. In one embodiment, the filter used has a pore size of 1.2 microns and a diameter of 11.3 cm. 2 (Isopore membrane, 47 mm diameter - Millipore catalog number RTTP04700) held in a Swin-Loc filter holder (Whatman catalog number 420400). In a scaled-up embodiment, the filtration area is 127 cm 2 The filtered material is 28.7 cm 2 The filtration area is 162 cm2 and the track etched pore size is 0.8 microns, preferably 0.6 microns, preferably 0.2 microns. In one embodiment, the pore size is 0.4 microns (Millipore catalog number HTTP09030). In a scaled up embodiment, the filtration area is 162 cm2. 2 In another scaled-up embodiment, the filtration area is 63 cm 2The system is washed multiple times with medium. When the retentate volume reaches about 40 ml in the stirred cell, the outlet of the stirred cell vessel is opened and the remaining retentate is allowed to drain by gravity leaving about 5-10 ml; the volume of the retentate can be reduced by other methods, for example by applying pressure with compressed gas such as nitrogen, or by mechanical devices such as pistons, but gravity is the preferred method. This remaining retentate is collected along with three washes with purification medium and transferred to a total of about 35 ml, typically to a sterile 35 ml Oak Ridge or similar centrifuge tube (the size of the tube will vary depending on the volume of the preparation). The purified sporozoites in the medium of the 35 ml Oak Ridge tube are centrifuged at 5,000 g to 25,000 g, preferably 16,300 g, for 2 minutes to 12 minutes, preferably 5 minutes to pellet the sporozoites. The supernatant medium is decanted. This step further purifies the sporozoite preparation by removing smaller, more buoyant and soluble material remaining in the supernatant.

[0057] Using the three size exclusion filter approach, this procedure provides a substantial reduction of the associated material in the purified sporozoite preparations by more than 200-10,000 times (reduction factor) compared to the associated material in the pre-purified preparations. The amount of residual SGM in the purified preparations of sterile purified sporozoites is routinely less than 25 ng of associated material per 25,000 sporozoites (a reduction of more than 97% compared to the initial amount of SGM), preferably less than 15 ng per 25,000 sporozoites (a reduction of 98%), more preferably less than 1 ng per 25,000 sporozoites (99.9%). The contaminating SGM in each of the purified preparations described herein is usually reduced by several thousand times compared to the SGM in the initial ground pre-purified salivary gland material from which each of the purified preparations is derived. Preferably, the purification reduction factor is at least 15 times, more preferably, the purification factor is at least 1,500 times, and most preferably, at least 3,500 times. The geometric mean of the reduction factors in the 10 campaigns described in Example 1 is 1625, which is 99.93% in SGM during the purification process.

[0058] This method is effective in reducing contaminants and associated mosquito salivary gland material in crude sporozoite preparations. The sporozoites can then be stored frozen, as known in the art.

[0059] Vaccine Compositions and Methods of Use In some embodiments, Plasmodium sporozoites, and in particular purified Plasmodium sporozoites, are prepared sterile, a technique known in the art as exemplified by

[38] and further exemplified in the description of GMP production of sterile, purified Pf-LARC2 in Example 7 below.

[0060] Genetically attenuated Plasmodium species sporozoites, including vaccines in which the immunogen is Plasmodium species P. falciparum linup- and Plasmodium species P. falciparum-LARC2, are generally administered in a one to three dose regimen, usually parenterally, including subcutaneously, intradermally, and intravenously, preferably by intravenous direct intravenous inoculation (DVI) techniques known in the art. A suitable dose of genetically attenuated Plasmodium sporozoites, e.g., genetically attenuated sporozoites of P. falciparum, per inoculation can be from about 10,000 to about 10 million sporozoites, preferably from 100,000 to 1,000,000 sporozoites. In some embodiments of the invention, the vaccine is administered with an adjuvant, such as 7DW8-5 as described in

[39] , and it is expected that such administration in combination with an adjuvant is likely to reduce the number of sporozoites required per dose.

[0061] Such vaccines are useful for preventing or reducing the severity of malaria, its clinical symptoms, symptoms, or pathology. In certain embodiments, compositions and vaccines comprising sterilely prepared genetically attenuated purified sporozoites provide partial, enhanced, or complete protection in human and other mammalian subjects that have not been previously exposed to the pathogen that causes malaria, or have been exposed but not fully protected. These compositions and vaccines are similarly useful for reducing the likelihood of developing disease-causing infections with malaria-causing parasites, including species of Plasmodium, such as P. falciparum or P. vivax, reducing the likelihood of becoming ill if infected, reducing the severity of illness, e.g., fever, if infected, reducing the concentration of the parasite in an infected person, or reducing malaria mortality in a population exposed to the malaria parasite. In many cases, even partial protection or a delay in the time it takes for an immunized individual to become infected with the parasite or become ill from infection, compared to a non-immunized individual, is beneficial. Similarly, a vaccine treatment strategy that provides any of these benefits in about 30% of a population could have a large impact on the health of a community and the individuals within that community. In general, it is contemplated that inoculating a subject with genetically attenuated Plasmodium sporozoites of one Plasmodium species according to the methods of the present invention would be expected to induce protective immunity against challenge with wild-type Plasmodium parasites of the same species. However, it has been shown that immunization with sporozoites of one Plasmodium species can protect against challenge with sporozoites of another Plasmodium species, and therefore it is also within the scope of the present invention to induce cross-species protection in this manner.

[0062] Also provided is a method for preventing malaria in a subject, comprising administering to the subject a genetically attenuated Plasmodium species vaccine lacking LINUP gene function, the vaccine being prepared sterilely and comprising substantially purified live genetically attenuated Plasmodium sporozoites in an amount effective to prevent malaria.

[0063] Prevention and / or treatment of malaria can be readily ascertained by one of skill in the art by assessment of clinical or pathological symptoms associated with malaria infection, such as elevated body temperature, headache, fatigue, lethargy, or percent parasitized red blood cells. Thus, according to the methods of the present invention, a subject exhibits improvement or absence of clinical signs, symptoms, or pathological symptoms of malaria following administration of a vaccine comprising purified live attenuated Plasmodium sporozoites.

[0064] In some embodiments, the administering step results in infection of the subject's hepatocytes, development into a liver stage of genetically attenuated Plasmodium parasites, providing an array of Plasmodium-specific antigens, and an immune response is generated to mitigate or prevent infection from a subsequent wild-type Plasmodium challenge. Thus, in some embodiments, the disclosed methods confer sufficient protective immunity to reduce or prevent symptoms of malaria in at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of subjects following exposure to wild-type Plasmodium.

[0065] Effective and optimal dosage ranges of vaccines and immunogens can be determined using methods known in the art. Guidance regarding the appropriate dosage to achieve antimalarial effect is provided from the illustrated assays disclosed herein. More specifically, the results obtained from the immunization patterns described herein and in the cited references can be extrapolated by those skilled in the art to obtain test vaccination schedules. Volunteer subjects are inoculated with various dosages at scheduled intervals, and test blood samples are evaluated for the level of protection against malaria when challenged with a subsequent infectious parasite. Such results can be used to refine the optimized immunization dose and dosing regimen (schedule) for effective immunization of mammalian subjects, particularly human subjects.

[0066] The immune response in a subject can be measured by standard tests, including, but not limited to, assessment of humoral and cellular immune responses, including, but not limited to, measurement of antigen-specific or parasite stage-specific antibody responses, direct measurement of peripheral blood lymphocytes by means known in the art, natural killer cell cytotoxicity assays

[40] , cell proliferation assays

[41] , immunoassays of immune cells and subsets [42, 43], and skin tests for cell-mediated immunity

[44] . Various methods and assays for measuring the strength of the immune system have been described, for example, in

[45] .

[0067] The vaccines provided include sterile and non-sterile compositions (preferably sterile) of purified live attenuated Plasmodium sporozoites substantially free of associated materials, as well as compositions having pharmaceutically acceptable diluents, excipients, or carriers. These vaccines are effective in preventing or ameliorating malaria upon subsequent challenge with an infectious parasite. Methods of formulating pharmaceutical compositions and vaccines are well known to those skilled in the art [see, e.g., 46].

[0068] Vaccine compositions, prepared aseptically or otherwise, containing purified live attenuated or non-attenuated Plasmodium sporozoites together with suitable diluents and buffers are encompassed by the present invention. The diluents, typically phosphate buffered saline (PBS) or normal saline (NS), are of various buffer contents pH and ionic strength. Such compositions may also contain excipients, such as serum albumin, particularly human serum albumin. Serum albumin may be purified from naturally occurring sources, such as human blood, or produced by recombinant DNA or synthetic techniques. Such compositions may also contain additives, such as antioxidants, e.g., ascorbic acid, sodium metabisulfite, and / or preservatives or cryopreservatives. Particulate preparations of polymeric compounds, such as polylactic acid, polyglycolic acid, or incorporation of the material into liposomes may also be used. (See, e.g.,

[46] pp. 1435-1712, which are incorporated herein by reference).

[0069] To determine the effective amount of the vaccine, a person skilled in the art would be able to ascertain the appropriate dosage, taking into account the treatment status, age and general health of the recipient. The dosage selected will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. Experiments to determine dosage levels can be confirmed by a person skilled in the art through suitable human clinical trials in which various dosage regimens are evaluated for their ability to induce protection against malaria.

[0070] The disclosed vaccines and the disclosed methods of using these vaccines can be useful as one component in a vaccine regimen, each component comprising a different vaccine to be administered separately to a subject. The regimen can include sequential immunization with attenuated Plasmodium species sporozoites and other types of Plasmodium vaccines, the so-called prime-boost strategy. This can include attenuated sporozoites as a prime and Plasmodium-related recombinant protein(s) in adjuvant as a boost, or vice versa. This can also include Plasmodium-related DNA vaccines or recombinant viruses expressing Plasmodium-related proteins, such as adenoviruses, as a prime and purified attenuated sporozoite vaccines as a boost, or vice versa. This can also include sequential or mixed immunization with attenuated Plasmodium species sporozoites and any form of erythrocytic stage parasites, including killed and live attenuated ones. A vaccine complex that includes separate components may be referred to as a vaccine regimen, a prime / boost regimen, a component vaccine, a component vaccine kit, or a component vaccine package that includes separate vaccine components. For example, a vaccine complex may include a vaccine that includes purified, sterile, live, attenuated sporozoites as a component. The complex may additionally include one or more recombinant or synthetic subunit vaccine components, including but not limited to recombinant proteins, synthetic polypeptides, the DNA that encodes these elements itself, or those functionally incorporated into recombinant viruses, bacteria, or parasites. A vaccine component may also include sterile, attenuated, axenic sporozoites that can develop extracellularly to early liver stages.

[0071] Below is an experimental disclosure of the discovery and analysis of LINUP as a viable target for genetic attenuation, and the development of a double knockout attenuated Plasmodium.

[0072] It is generally noted that use of the term "or" in the claims means "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, but that the present disclosure supports definitions that refer to alternatives only and to "and / or."

[0073] In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in a claim or the specification, refer to one or more, unless otherwise indicated.

[0074] Unless otherwise clearly indicated by the context, throughout this specification and claims, words such as "comprise", "comprising" and the like shall be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, e.g., "including but not limited to". Words using the singular and plural forms also encompass the plural and singular, respectively. In addition, the words "herein", "above", and "hereunder", and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portion of this application. Words such as "about" and "approximately" refer to slight variations in the stated value, usually within a standard error range, e.g., 10% or 5% of the stated value.

[0075] Disclosed are materials, compositions, and components that can be used in, with, and in the preparation of the disclosed methods and compositions. When combinations, subsets, interactions, groups, etc. of these materials are disclosed, it should be understood that each of the various individual and collective combinations is specifically contemplated, even if specific reference to each of these compounds and each individual single combination and permutation is not explicitly disclosed. This concept applies to all aspects of the present disclosure, including but not limited to the steps in the described methods. Thus, specific elements of any of the above-mentioned embodiments may be combined with or substituted for elements in other embodiments. For example, when there are various additional steps that can be performed, it is understood that each of these additional steps may be performed with any specific method step or combination of method steps of the disclosed methods, and each such combination or subset of combinations should be considered to be specifically contemplated and disclosed. In addition, it is understood that the embodiments described herein can be implemented using any suitable materials, such as those described elsewhere herein or known in the art.

[0076] Publications cited herein and the subject matter for which they are cited are specifically incorporated herein by reference in their entireties. EXAMPLES

[0077] Example 1 P. yoelii LINUP is expressed exclusively during liver stage development. To identify novel genes for which attenuation occurs only during liver stage development, we utilized the late liver stage P. vivax transcriptome isolated from infected livers of human liver chimeric FRG NOD huHep mice. Mice were infected with 1 million sporozoites of P. vivax Thailand local strain isolated from Anopheles dirus mosquitoes. Mice were euthanized 8 days post-infection, late in liver stage development, and livers were processed for RNA extraction. Probes specific for the P. vivax exome were hybridized to purified RNA, converted to cDNA, and bulk sequenced. Reads were normalized and expressed as reads per kilobase of transcript per million mapped reads (RPKM). This resulted in the capture of 4397 unique gene transcripts, with an RPKM value greater than 2. To enrich for liver stage-specific reads, the RPKM reads of P. vivax were expressed as fold change relative to the maximum RPKM read count of a P. falciparum orthologous gene expressed during the blood stage of the life cycle. This allowed us to compare and contrast the liver stage development of asexual P. vivax with the blood stage development of asexual P. falciparum, and to specifically isolate genes that are highly expressed only during liver stage development. The top 10 genes resulting from this analysis (Table 1) included genes or metabolic pathways already known to be expressed during liver stage development but not during blood stage development.

[0078] [Table 1-1] [Table 1-2]

[0079] Known genes included LISP1 (30), LISP2 (31-33), PALM (34), and SIAP2 (35). Malonyl-CoA-acyl carrier protein transacylase, a precursor of type II fatty acid biosynthesis, was also detected, a pathway known to be expressed during liver stage development in P. falciparum and important for liver stage development in P. yoelii and P. berghei (36-39). This result gave us confidence that our approach to discovering liver stage-specific transcripts was sound. We also had the requirement that orthologs were present in the P. yoelii genome, allowing us to efficiently analyze spatial and temporal expression and essentiality using this rodent malaria model. Of the remaining five genes in the top ten, four had orthologs in the P. yoelii genome, including the putative protoporphyrinogen oxidase PY17X_0513300 and three hypothetical genes PY17X_1003700, PY17X_1465200, and PY17X_1369800. Analysis of both P. yoelii genes and their P. falciparum orthologues for protein expression (P. yoelii and P. falciparum) and essentiality in blood stages (P. falciparum) using PlasmoDB suggested that PY17X_051300 / PF3D7_102800 was deleteable in P. falciparum ansexual blood stages, but this protein was expressed in P. falciparum blood stage gametocytes and P. yoelii oocyst sporozoites, and therefore this gene was not studied further. Analysis of PY17X_1369800 / PF3D7_1351300 suggested that this gene was essential for P. falciparum ansexual blood stage replication, and therefore it was not studied further.Analysis of PY17X_1003700 / PF3D7_0404600 suggested that this gene was deleteable in P. falciparum asexual blood stages, but the protein was expressed in both merozoites and sporozoites of P. falciparum blood stages, and therefore this gene was not studied further. Notably, analysis of PY17X_1465200 / PF3D7_1249700 suggested that this gene was deleteable with respect to P. falciparum asexual blood stage replication. Furthermore, there was no evidence of protein expression in P. falciparum and expression in liver stages of P. yoelii at the 40-hour developmental time point. Therefore, PY17X_1465200 / PF3D7_1249700 was carried forward for further study.

[0080] PY17X_1465200 is a single-exon gene encoding a protein of 746 amino acids with no known protein properties and is conserved across Plasmodium species (Figure 1A). The overall amino acid identity between P. yoelii and the synthetic orthologues of P. falciparum and P. vivax was 40%, while the amino acid similarity was 60%. The identity in a stretch of 122 amino acids proximal to the N-terminus (amino acids 44 to 161) was 89%. In addition, this gene has no orthologues in other Apicomplexa or any other eukaryotes and is therefore unique to Plasmodium. To determine the spatial-temporal expression pattern of PY17X_1465200, we generated transgenic P. yoelii parasites in which an mCherry tag was attached to the C-terminus of endogenous PY17X_1465200 (Figure 1B, top panel). Standard transfection procedures allowed successful double crossover homologous recombination, transgenic parasites were cloned, and incorporation of the mCherry tag was confirmed by transgene-specific PCR. Comparison of PY17X_1465200 mCherry-tagged parasite clones with wild-type parasites revealed no statistically significant differences in life cycle progression. Specifically, intravenous injection of 10,000 salivary gland sporozoites from both wild-type and tagged parasites into groups of Swiss Webster mice revealed that all mice showed blood stage infection 3 days after sporozoite injection based on Giemsa-stained thin-layer blood smears, and thus the mosquito and liver stages of the life cycle were comparable. Since tagged parasites were found to have successfully completed the life cycle, expression of the mCherry tag was monitored throughout the complete life cycle by immunofluorescence assay (IFA) of infected tissues. Interestingly, mCherry expression was only seen during liver stage development, which for P. yoelii has a duration of approximately 52 h. Expression was not detected at the 24 h time point (Figure 1B), but was detected at both the 36 and 48 h time points, the time point at which extraerythrocytic merozoites are formed (Figure 1B).Expression was punctate and did not overlap with the punctate expression of the endoplasmic reticulum marker BiP (Figure 1B). However, expression partially overlapped with the nuclear stain DAPI, as well as the active histone marker histone 3 acetylated lysine 9 (Figure 1C). Thus, we concluded that PY17X_1465200 has a nuclear expression pattern exclusively during mid- to late liver stage development. We therefore named this gene and its protein product LINUP, liver stage nuclear protein, and tagged the parasite P. yoelii LINUP. mCherry We named this protein LINUP IL-1, which is a nuclear localization protein that is essential for the development of liver stage LINUP. Further analysis of the LINUP amino acid sequence revealed a conserved N-terminal 25 amino acid nuclear localization sequence (NLS) in P. yoelii, P. falciparum, and P. vivax, which was nearly identical across the three species (Figure 1A). The presence of the NLS explains the nuclear localization of P. yoelii LINUP. To further study the importance of LINUP in liver stage development, we used a gene knockout approach.

[0081] Example 2 P. yoelii LINUP is crucial for liver stage development. To study the essentiality of LINUP expression in P. yoelii liver stage development, we used CRISPR / Cas9 technology to delete the LINUP gene from the P. yoelii genome (Figure 2A). After transfection of schizonts, in vivo positive drug selection with pyrimethamine in mice, and isolation of drug-resistant parasites, gene knockout was confirmed by PCR on isolated genomic DNA using specific primers.

[0082] [Table 2]

[0083] P.yoelii linup -Parasites were cloned and two clones, c3 and c5, were initially used for downstream phenotypic analysis. The two clones were initially compared to the wild-type parent during blood stage growth (Figure 2B) and mosquito stages of development, and there were no statistical differences in overall oocyst numbers (Figure 2C), as well as oocyst abundance (Figure 2D), and salivary gland sporozoites per mosquito (Figure 2E) in biological replicate experiments, suggesting that deletion of LINUP did not affect the mosquito stage of the life cycle. This was expected as LINUP expression was not seen until the mid-to-late liver stage.

[0084] To determine whether liver stage development is affected by the deletion of LINUP, P. yoelii wild-type and linup - Sporozoites were isolated from the salivary glands of infected Anopheles stephensi mosquitoes and injected intravenously into groups of laboratory mice, including outbred Swiss Webster mice and the susceptible inbred strains BALB / cJ and BALB / cByJ (Table 3).

[0085] [Table 3]

[0086] The time to blood stage infection in days was then determined from Giemsa stained thin blood smears. All Swiss Webster mice infected with 50,000 wild type sporozoites all showed blood stage infection on day 3, whereas P. yoelii linup - When infected with sporozoites, 9 out of 10 mice showed blood-stage infection, and mice became infected on days 8 and 10 postinfection, with a significant delay in patency, suggesting that P. yoelii linup -The liver stage was suggested to be severely attenuated (Figure 3). Similarly, BALB / cByJ mice infected with 50,000 wild-type sporozoites all showed blood stage infection on day 3, whereas P. yoelii linup - Only 9 of 20 mice infected with sporozoites showed blood stage infection, with days to infection ranging from 8 to 10 days (Table 3). A dose escalation study in BALB / cJ mice provided further evidence of liver stage attenuation. Mice were infected with 1,000, 10,000, and 50,000 sporozoites. All mice infected with wild-type sporozoites showed blood stage infection on days 4 (1,000), 3-4 (10,000), and 3 (50,000), whereas P. yoelii linup - For sporozoite infection, only 1 of 10 mice showed blood-stage infection on day 12 (1,000), 7 of 20 mice showed blood-stage infection on days 7-12 (10,000), and 5 of 10 mice were symptomatic on days 7-9 (50,000) (Table 3). - The parasite is severely attenuated during liver stage development.

[0087] Example 3 P.yoelii linup - Liver stage growth is reduced and extraerythrocytic merozoite formation is attenuated. P.yoelii linup - To further study liver stage attenuation, BALB / cByJ mice were inoculated with 250,000 P. yoelii linup - Mice were euthanized at 24, 36, and 48 hours post-infection and livers were removed, perfused, fixed, sectioned, and subjected to IFA to determine both size and protein / DNA expression patterns compared to wild-type infection (Figure 3). At 24 hours, P. yoelii linup -Liver stages appeared to be comparable in size to wild-type (Fig. 3A). However, at 36 h, P. yoelii linup - Liver stages were statistically smaller than wild type (Figure 3A). The reduction in size was even more pronounced at 48 hours (Figure 3A), at which point clear differences in protein / DNA expression were also identified. At 48 hours, mitochondrial and apicoplast branching was observed in the P. yoelii linup. - In the liver stage, expression was reduced compared to the wild type (Figure 3B). In addition, the expression pattern of the parasite plasma membrane marker MSP1, which represents extraerythrocytic merozoites, was also abnormal (Figure 3C), indicating incomplete cytomere formation. Furthermore, expression of MTIP, which is part of the inner membrane complex of fully mature merozoites, was seen in 17% of wild-type liver stages, but was absent in P. yoelii linups. - P. yoelii linup showed MTIP expression in only 7% of liver-stage cases. - Its expression was abnormal in the liver stage (Fig. 3D). - There was also no evidence of extensive nuclear division during the formation of extraerythrocytic merozoites in the liver stage. - Merozoites are the P. yoelii linup, indicating liver stage infection after sporozoite injection. - They can be released from the liver stage, and therefore in a few cases, viable extra-erythrocytic merozoites can be formed.

[0088] Example 4 P.yoelii linup - Immunization of mice with sporozoites protects against wild-type challenge. Rodent malaria GAPs that are arrested during liver stage development are potent immunogens that can protect against wild-type sporozoite challenge [26-28]. -To determine whether immunization of susceptible BALC / cJ mice with sporozoites could protect against challenge, groups of mice were immunized with either 1,000 or 10,000 P. yoelii linups. - The mice were either intravenously immunized with sporozoites or mock immunized with an equivalent volume of salivary gland extract from uninfected mosquitoes (Table 4). - Only mice that did not develop liver stage infection after sporozoite infection were finally challenged with wild-type sporozoites. Mice were immunized twice, approximately 33 days apart, and then challenged an additional 34 days later with an intravenous injection of 10,000 wild-type sporozoites. All mice immunized with salivary gland extracts developed blood stage infection 3-4 days after challenge (Table 4).

[0089] [Table 4]

[0090] 1,000 P. yoelii linups - Six of nine mice immunized twice with sporozoites were protected from challenge, while the remaining three mice showed a significant delay in blood-stage infection, becoming infected 8-10 days after challenge (Table 4). - All mice (13) immunized twice with sporozoites were protected after challenge. - We show that immunization with GAP results in a highly effective immune response that protects mice from significant wild-type sporozoite challenge.

[0091] Example 5 P. falciparum linup in FRG NOD huHep mice - Parasite phenotypic breakthrough analysis: P. falciparum NF54 and P. falciparum linup -Sporozoites were isolated from the salivary glands of infected Anopheles stephensi mosquitoes. - For liver stage analysis, 1 x 10 6 1 × 10 P. falciparum NF54 and 1 × 10 6 P. falciparum linup - Sporozoites were injected intravenously (retro-orbitally) into FRG NOD huHep mice, 4 per group. Livers were harvested on days 5 and 7 and used for IFA (Figure 6A). - To assess the blood stage progression of 1 × 10 clones, 6 P. falciparum NF54 and P. falciparum linup - Sporozoites were injected intravenously into one and three FRG NOD huHep mice per group, respectively. On days 6 and 7, 400 μl of 70% RBCs were injected intravenously to allow migration of liver-stage parasites into the blood. On day 7, 4 h after human RBC repopulation, mice were euthanized, blood was collected by cardiac puncture, and 50 μl of blood from each mouse was used for qRT-PCR analysis to detect parasite 18S RNA. Blood was washed three times in sex-free medium, a volume of human RBCs equivalent to the packed RBC volume was added, and blood was transferred to in vitro culture. Fresh medium was replaced daily and cultures were analyzed for the presence of parasites by overlay smears every 2-3 days for up to 10 days. Samples from in vitro cultures were analyzed for the presence of 18S rRNA by qRT-PCR after 10 days.

[0092] P. falciparum linup - The values ​​for the three mice injected with sporozoites ranged from 0 to 1 × 10 4, indicating severe attenuation in migration (Figure 6B). Blood obtained from exsanguinated animals was placed in in vitro culture and sampled after 7 days, 14 days after sporozoite injection. P. falciparum NF54 wild-type parasite replication during this period was significant, reaching 7 × 10 per ml of blood. 8 18S rRNA copies, a more than 100-fold increase (Figure 6B), and parasites were demonstrated by evaluation of Giemsa-stained thin blood smears. In contrast, P. falciparum linup - Cultures of blood from mice injected with sporozoites showed no significant increase in parasite biomass, with 0–2 × 10 parasites per ml of blood. 4 The results showed that the linup 18S rRNA gene was 100 copies in total (Fig. 6B). - These results suggest that the initial quantification of parasite biomass in blood from mice injected with sporozoites was due to release of parasite rRNA from attenuated and non-viable liver-stage parasites into the mouse bloodstream, rather than viable extra-erythrocytic merozoites. - To further evaluate liver stage attenuation, parasite biomass, measured as 18S rRNA copies per ng of liver RNA, was determined in mice used for the transfer experiment (Figure 6C). - We suggested that liver stage biomass was reduced compared to wild type, likely due to abnormal maturation of extraerythrocytic merozoites seen by IFA (Figure 5B).

[0093] Example 6 Phenotypic breakthrough analysis of P. falciparum LARC2 parasites in FRG NOD huHep mice P. falciparum NF54 and P. falciparum LARC2 sporozoites were isolated from the salivary glands of infected Anopheles stephensi mosquitoes. To assess the transition of P. falciparum LARC2 to the blood stage in FRG NOD huHep mice, 1 × 10 6 Sporozoites of sterile cryopreserved P. falciparum NF54 (PFSPZ) and sterile cryopreserved P. falciparum LARC2 (PFSPZ LARC2) were injected intravenously into 4 and 6 FRG NOD huHep mice, respectively (Figure 10A). On days 6 and 7, 400 μl of 70% RBCs were injected intravenously to allow migration of liver stage parasites into the blood. After 4 h of human RBC repopulation on day 7, mice were euthanized, blood was collected by cardiac puncture, and 50 μl of blood from each mouse was used for qRT-PCR analysis to detect parasite 18S RNA. Blood was washed three times in sex-free medium, a volume of human RBCs equivalent to the packed RBC volume was added, and blood was transferred to in vitro culture. Fresh medium was replaced daily and cultures were analyzed for the presence of parasites by overlay smears every 2-3 days for up to 6 weeks.

[0094] On day 7, the parasite density in mice infected with PfSPZ was estimated to be 10 3 ~10 6 The results ranged from 10 parasite equivalents / ml to 10 parasite equivalents / ml (Figure 10B and Table 5).

[0095] [Table 5]

[0096] Blood stage parasites were detected by Giemsa-stained smears within 1 to 3 days of transfer to in vitro culture in all four mice infected with PfSPZ (Table 5). In contrast, no parasites were detected by Giemsa-stained thin smears in all six mice infected with PfSPZ LARC2. On day 7, parasite densities in mice infected with PfSPZ LARC2 ranged from 0 to 10 by P. falciparum 18S qRT-PCR. 3 parasite equivalents / ml, suggesting that parasite 18S rRNA is released into the bloodstream from non-viable and attenuated P. falciparum LARC2 liver stage parasites. However, viable extraerythrocytic merozoites are not formed in P. falciparum LARC2. Livers of infected mice were also harvested on day 7 for RNA extraction and analysis of parasite liver burden by P. falciparum 18S qRT-PCR. Parasite liver burden is shown as log10 copy number of P. falciparum 18S rRNA per μg of total liver RNA (Figure 10).

[0097] Example 7 Growth parameters were optimized to be compatible with GMP manufacturing and the production, purification, and cryopreservation of sterile PfSPZ-LARC2 vaccine are demonstrated. The production of live, sterile, purified and cryopreserved GMP quality PfSPZ-LARC2 vaccine has been achieved, which follows the life cycle steps of the genetically attenuated Pf parasite, where sporozoites are produced in a biological system, the salivary glands of intact germ-free mosquitoes. This requires the simultaneous production of Pf gametocytes from a master cell bank and germ-free mosquitoes from colonies of A. stephensi. Pf was cultured in vitro to produce gametocytes. P. falciparum infected RBCs were mixed with uninfected RBCs and supplemented RPMI. These were then placed in wells of a (6-well) culture plate containing supplemented RPMI, and the cultures were expanded. To initiate the production of gametocytes in the cultures, complete growth medium (RPMI1640 supplemented with 10% human O+ serum and 367 μM hypoxanthine) was added and changed daily. Giemsa-stained thin blood smears from induced culture samples were examined to monitor gametocyte growth. The number of stage V gametocytes was assessed from days 14 to 19 after the initiation of gametocyte production. Cultures were selected for preparation of infectious blood meals for mosquito feeding. Gametocytes in infectious blood meals were fed by sterile adult mosquitoes cultured from sterile eggs, larvae, and pupae.

[0098] Axenic mosquito production includes all stages of the mosquito life cycle, starting with sterilization of eggs and preparation of solutions, and ending when the mosquitoes are ready to feed (U.S. Patent No. 8,802,919). An artificial membrane was placed over the mouth of the container and autoclaved. The container was kept at approximately 35-37°C and adult mosquitoes were introduced. Pf gametocyte-infected blood was added to the membrane and the mosquitoes were allowed to feed on the gametocyte-infected blood meal for 30 min. After feeding, the mosquitoes were returned to the incubator and maintained at 26°C, 77% relative humidity, and a 12-h light:dark cycle.

[0099] Infected, germ-free adult mosquitoes were then dissected, salivary glands removed, and sporozoites were extracted and purified as described. The formulated, germ-free, purified PfSPF vaccine was then cryopreserved. Cryopreservation was initiated by adding 2xCPA at a 1:1 ratio. The preparation was dispensed in 20 μL aliquots into cryovials. Vials were prepared for cryopreservation by rate freezing, a multi-step process that cools the vials to a freezing temperature below -150°C. The cryovials were finally transferred to storage in liquid nitrogen vapor phase (LNVP), which is -150°C to -196°C.

[0100] Evaluation of two separate preparations of the PfSPZ-LARC2 vaccine is shown in Table 6.

[0101] [Table 6] 200 million sterile purified PfSPZ-LARC2 per vial, 10 6 PfSPZ-LARC2 was placed in a vial.

[0102] [Table 7-1] [Table 7-2] [Table 7-3]

[0103] [Table 8]

[0104] [Table 9]

[0105] [Table 10-1] [Table 10-2] [Table 10-3]

[0106] [Table 11]

[0107] While exemplary embodiments have been illustrated and described, it will be understood that various changes can be made therein without departing from the spirit and scope of the invention.

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Claims

1. A live Plasmodium species organism that has been genetically modified to disrupt the LINUP gene, which encodes a liver-stage nuclear protein, thereby preventing the biological function of the protein encoded by the wild-type LINUP gene.

2. 10. The live Plasmodium species organism of claim 1, which arrests life cycle development at a late liver stage in a mammalian intermediate host.

3. The Plasmodium species organism of claim 2, wherein the Plasmodium species comprises P. falciparum, P. vivax, P. malariae, P. ovale, or P. knowlesi.

4. 4. The Plasmodium species organism of claim 3, wherein the Plasmodium species comprises P. falciparum.

5. A Plasmodium species organism described in claim 3, wherein the organism is in the sporozoite stage of development.

6. 4. The Plasmodium species organism of claim 3, wherein the functional LINUP gene before genetic modification is a LINUP having at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO:

35.

7. 4. The Plasmodium species organism of claim 3, wherein the functional LINUP gene prior to genetic modification encodes a LINUP polypeptide having at least about 40%, 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to the amino acid sequence set forth in PF3D7_1249700, SEQ ID NO:

1.

8. 10. A vaccine composition comprising the Plasmodium species organism of claim 5 and an additive, wherein the vaccine composition is prepared sterile.

9. 3. The Plasmodium species organism of claim 2, wherein said Plasmodium species organism comprises at least one transgene.

10. 10. The Plasmodium species organism of claim 9, wherein said transgene is under the control of a promoter that provides for transcription of said transgene during the sporozoite or liver stage of development.

11. 3. The Plasmodium species organism of claim 2, wherein said Plasmodium species organism has been genetically modified to disrupt one or more additional gene functions, each additional gene function being necessary for the transition from the liver stage to the blood stage of the Plasmodium organism's life cycle.

12. 12. The Plasmodium species organism of claim 11, wherein the Plasmodium species organism has been genetically modified to disrupt PlasMei2 gene function.

13. 13. The Plasmodium species organism of claim 12, wherein the Plasmodium species organism is in the sporozoite stage of development.

14. 13. The Plasmodium species organism of claim 12, wherein the functional PlasMei2 gene prior to genetic modification comprises a nucleic acid sequence that is at least about 70%, 80%, 90%, or 95% identical to the nucleic acid sequence set forth in SEQ ID NO:

34.

15. A Plasmodium species organism as described in claim 12, comprising: an amino acid sequence of a functional PlasMei2 polypeptide encoded by the PlasMei2 gene that is at least about 70%, 80%, 90%, or 95% identical to the sequence set forth in SEQ ID NO:

36.

16. 14. A vaccine composition comprising the Plasmodium species organism of claim 13 and an additive, wherein the vaccine composition is prepared sterile.

17. 17. A vaccine composition according to claim 8 or claim 16 for inducing an immune response to one or more Plasmodium antigens in a human subject, wherein one or more doses of the vaccine composition are administered to the subject.

18. 18. The vaccine composition of claim 17, wherein the immune response mitigates or protects against infection from a subsequent wild-type Plasmodium challenge.

19. 14. A composition comprising the Plasmodium species organism of claim 5 or claim 13 for conferring protective immunity against malaria caused by a Plasmodium species parasite in a human subject, wherein one or more doses of the composition are administered to the human subject.

20. 6. The Plasmodium species organism of claim 5, produced in vitro.

21. 14. The Plasmodium species organism of claim 13 produced in vitro.

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