Patent Application
20240382573
The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3399-P33WO.xml. The XML file is 300 KB; was created on Aug. 19, 2022; and is being submitted via Patent Center with the filing of the specification.
This invention relates generally to malaria and the identification, creation and uses of genetically altered Plasmodium parasites. More particularly, the invention relates to genetically attenuated Plasmodium parasites, disrupted at single and/or multiple genetic loci, that can nevertheless proceed to late liver stage of development, but become attenuated at this stage and cannot proceed to blood stage.
Malaria has a tremendous impact on human health, killing hundreds of thousands annually and creating a major impediment for social and economic development of nations in malaria-endemic areas, particularly in sub-Saharan Africa [1]. Parasites of the genus Plasmodium, the causative agents of malaria, are transmitted to the vertebrate host via an infectious bite, in the saliva of an infected Anopheles mosquito. After transmission, Plasmodium parasites, specifically, the sporozoite stage of the life cycle, exit the bite site, enter the bloodstream of the host and migrate quickly through the blood stream to the liver. Sporozoites that infect hepatocytes and then grow and replicate within the infected hepatocyte as live stage parasites and mature from early liver stage through late liver stage, producing tens of thousands of blood stage-infectious merozoites, which are released into the blood stream and infect red blood cells. Here, they undergo further development and replication, after which they cause the rupture of the red blood cells, releasing a new wave of merozoites into the blood. Most of the new merozoites continue to repeat the replicative cycle by invading additional red blood cells. This cycling of infecting and rupturing of the red blood cells manifests in the potentially severe symptoms associated with malaria, such as fever, chills, weakness, malaise, an enlarged spleen, and death. A minority of the cycling merozoites, after invading red blood cells, develop into male or female gametocytes that remain in circulation within the body until being taken up in a blood meal through the bite of another mosquito. Assuming 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. Zygotes develop into motile ookinete forms, which penetrate the wall of the mosquito's midgut and form oocysts. Each oocyst undergoes numerous rounds of division to eventually produce infectious sporozoites, which accumulate in the mosquito's salivary glands and can then be injected into the next host, thus continuing the lifecycle.
The hepatic stage of Plasmodium infection is an attractive target for malaria prophylactic intervention as it is asymptomatic for this disease and precedes the symptomatic blood stage infection. Indeed, vaccines that target 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.
RADIATION-ATTENUTED VACCINE: Decades ago, it was found that irradiated Plasmodium-species sporozoites (“radiation attenuated sporozoites” or RAS) can confer sterile, protective immunity in host-specific rodents and humans when used as an experimental vaccine [2,3]. This was surprising, as a natural infection with malaria does not induce sterile protective immunity in endemic areas of the world. This approach has been used to produce radiation-attenuated P. falciparum (Pf) whole parasite vaccines that have proven to be highly efficacious in numerous clinical trials. Sanaria® PfSPZ Vaccine has been assessed to date in 19 clinical trials in 9 countries, is exceptionally safe and well tolerated, with rates of adverse events no different than in normal saline controls [4-12]. PfSPZ Vaccine has induced >90% vaccine efficacy (VE) against homologous challenge by controlled human malaria infection (CHMI) (same strain of Pf in vaccine and challenge) at 3-11 weeks after immunization: 80% VE against heterologous (different strains of Pf in vaccine and challenge) at 9.5 weeks (unpublished) after immunization; 54% heterologous VE against heterologous at 8 months [5, 7, 8]; and sterile VE durable for at least 18 months against intense naturally transmitted Pf malaria in 4 field trials in Africa, where VE against intensely transmitted Pf infection was 52%, 51%, and 47% for 6 months, and 58% in the second year by time to event analysis [9, 13]. PfSPZ attenuated by irradiation induces DNA damage and prevents DNA replication during liver stage development [2, 14, 15]. PfSPZ develop into approximately day 2 to 3 early liver stage parasites, but do not replicate. Plasmodium-specific immunogens produced during these liver stages elicit immunity primarily due to CD8+T cells, however, relatively large doses (9×105 to 1.8×106 PfSPZ) are required for engendering sterilizing immunity [4-9, 16, 17].
CHEMO-ATTENUATED VACCINE: Live, non-attenuated whole parasite-based vaccines administered under antimalarial drug cover (chemo-attenuation) targeting (i) the liver stage of the Plasmodium life cycle, or (ii) the first wave of parasite-infected red blood cells, have shown superior efficacy in clinical trials. In a recent trial [18], 3 doses of 2×105 PfSPZ of Sanaria® PfSPZ-CVac (CQ) gave 100% protection against a heterologous CHMI at 12 weeks after last dose of vaccine. However, if chemo-attenuation relies on the blood stage anti-malarial, chloroquine (CQ), as does Sanaria® PfSPZ CVac Vaccine-CQ, there can be can a brief period of transient parasitemia 7-8 days after the initial injection of PfSPZ non-attenuated whole parasites, resulting in up to Grade 3 malaria symptoms unless ibuprofen is taken. In addition, if (for whatever reason) the partner drug is not taken, there is a risk of developing severe malaria.
GENETICALLY-ATTENUATED VACCINE: Genetically attenuated parasites (GAPs), are created by the 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) as compared to oocyst sporozoites [19-22]. Such deletions did not affect viability of GAP when in the sporozoite stage, but resulted in growth arrest early in the liver stage of development before active liver stage replication takes place. Such GAPs are termed “early-arresting, replication deficient” (EARD) These GAPs exhibited favorable immunogenic properties, but have also, on occasion, exhibited incomplete attenuation, allowing for liver stage-to blood stage lifecycle progression (also called “breakthrough”), leading to an active infection. One early arresting GAPs, Sanaria® PfSPZ-GA1, was manufactured in compliance with GMP, and assessed in a clinical trial. It was shown to be well tolerated, safe, and completely attenuated as it did not show any breakthough blood stage infection, but conferred low efficacy against CHIM [23].
Next generation GAPs have been more recently developed in an attempt to create a parasite vaccine strain, which undergoes growth, development, and replication within the liver, only to arrest in development and die just before progressing to the blood stage [See, e.g. 24-27). These GAP are termed LARC (late-arresting, replication-competent) and these GAP are more powerful immunogens than the EARD GAP or RAS because they generate a significant vaccine biomass expansion in the liver and transit further through the liver life cycle, and thus present a larger and broader range of parasitic antigens to the immune system rendering the ensuing immune response more effective. Indeed, in the rodent malaria model, Plasmodium yoelii, LARC GAPs have been shown to provide superior protection from sporozoite challenge as compared to EARD GAPs and RAS. [28] Moreover, P. yoelii LARC GAPs have also been shown to provide stage-transcending protection from a direct blood stage challenge, indicating the presence of antigens in the late liver stage GAPs that are also characteristic of the blood stage forms [29]. However, published P. yoelii GAPs have either not successfully informed gene deletion in the human parasitic homologue P. falciparum and/or have shown in complete attenuation [26, 30]. Thus, there is a need to identify further targets for gene deletion to ensure complete attenuation. Ultimately, the aim is the creation of a human malaria LARC GAP that is completely attenuated in malaria clinical trials and can be used as a safe and highly efficacious vaccine to prevent malaria. However, the identification of additional target genes for deletion in human malaria-causing parasites, such as P. falciparum, which results in complete attenuation has been a continuing challenge.
Despite the advances in the art of creating attenuated Plasmodium parasites useful for stimulating the vertebrate immune system against a later infection, a need remains to identify novel specific genetic-based modifications, or novel combinations of multiple genetic-based modification, that provide complete attenuation, i.e., complete cessation of lifecycle progression in late liver stage development, without breakthrough to blood stage infection. This in turn will permit safe and prolonged development of the liver stage parasite vaccine immunogen to provide greater quantity and variety of antigens for a more complete immunity against subsequent parasitic infection. The present disclosure addresses these and related needs.
The present disclosure is directed to parasites that cause malaria, particularly Plasmodium-species parasites, and more particularly to Plasmodium-species parasites that have been genetically altered to develop normally to the late liver stage of development, but are blocked in the transition from liver stage to blood stage and subsequent infection of erythrocytes. Specifically, the inventors have identified genetic alternations that cause these parasites to be attenuated at the late liver stage of development by the deletion of the liver stage nuclear protein (LINUP) gene that result in the disruption of the LIMUP gene product, and this late liver-stage arrest. The arrested LINUP mutant allows for expression of an extensive parasite-specific antigenic array during liver stage development, but is severely attenuated for entry to blood stage, subsequent erythrocytic infection and the associated signs, symptoms and pathology of malaria disease. The family of genetically attenuated Plasmodium-species parasites comprising the LINUP disruption, including the double knockout, LARC2 (with deletion of both LINUP and PlasMei2 gene function), are ideal as live immunogens in malaria vaccines and related therapeutics.
A. Amino acid alignment between P. falciparum 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 gray. The predicted and conserved nuclear localization sequence (NLS) is underlined and spans amino acids 132-156.
B. To visualize the localization of P. yoelii LINUP, an mCherry epitope-tagged parasite strain, P. yoelii LINUPmCherry was generated by fusing an mCherry tag to the LINUP C-terminus. The tagged transgenic parasite replaces the endogenous LINUP with the tagged copy. Immunofluorescence assays (IFAs) on P. yoelii LINUPmCherry infected mouse livers using an mCherry antibody, an antibody to the endoplasmic reticulum marker BiP and the DNA strain DAPI, indicate that the protein is expressed after 24 hours of liver stage infection and at both 36 and 48 hours localizes to the nucleus of late liver stages (schizonts). Scale bar size is 5 μm.
C. At 48 hours, LINUP shows partial co-localization with the histone H3 marker acetylated lysine 9 and DAPI, scale bar is 5 μm, which is further indicated in the magnified image to the right where the scale bar size is 2 μm.
A. The schematic depicts the generation of the P. yoelii linup parasite using CRISPR/Cas9-mediated gene editing.
B. Blood stage growth was compared in groups of five Swiss Webster mice for P. yoelii XNL wildtype and P. yoelii linup clones c3 and c5. One million infected red blood cells were injected intravenously into each mouse and parasitemia measured every other day for ten days. Growth rates were comparable suggesting LINUP has no essential role in blood stage replication. Data is represented as mean+/−SEM, n=5 biological replicates. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. P>0.05 is taken as not significant. P. yoelii linup clones c3 and c5 did not have defects in mosquito infection as counts for:
C. oocysts/midgut:
D. oocyst prevalence; and E: salivary gland sporozoites/mosquito were comparable between P. yoelii linup c3 (dark gray) and c5 (light gray) to P. yoelii wildtype (black). Data is represented as mean+/−SD, n=3 biological replicates. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. P>0.05 is taken as not significant.
E. salivary gland sporozoites/mosquito were comparable between P. yoelii linup− c3 (dark gray) and c5 (light gray) to P. yoelii wildtype (black).
Data is represented as mean+/−SD, n=3 biological replicates. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. P>0.05 is taken as not significant.
(A). Comparison of the size of liver stage parasites (based on area at the parasite's largest circumference) between P. yoelii wildtype and P. yoelii linup− at 24, 36 and 48 hours indicate that P. yoelii wildtype liver stage schizonts are significantly larger than P. yoelii linup− at 36 and 48 hours. Data is represented as mean±SD. Each datapoint refers to the mean size of at least 20 parasites for each timepoint. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. *=P.05, ***P<0.001, P>0.05 is taken as ns. Liver stage development was compared at 48 hours using antibodies against:
(B). the parasite mitochondria (mHSP70) and apicoplast (ACP):
(C). the parasite plasma membrane and mature exo-erythrocytic merozoite marker, MSP1; and,
(D). the inner membrane complex protein and mature exo-erythrocytic merozoite marker, mTIP. DNA was stained with DAPI. Scale bar: 10 μm. P. yoelii linup− liver stage parasites are smaller compared to P. yoelii wildtype and display less branching of the mitochondria and apicoplast organelles. P. yoelii linup− expresses the late liver stage proteins MSP1 and mTIP, however there is aberrant segregation of cytomeres and incomplete formation of mature exo-erythrocytic merozoites.
A. The schematic depicts the generation of P. falciparum linup knockout parasites using CRISPR/Cas9-mediated gene editing. Primers used to verify the gene deletion are indicated and the sizes of the PCR amplicons are shown in kilobases:
B. Agarose gel electrophoresis shows the PCR products corresponding to the gene deletion of P. falciparum LINUP in clones B1 and B4. P. falciparum linup did not have defects in mosquito infectivity as counts for:
C. oocysts/midgut:
D. oocyst prevalence; and,
E. salivary gland sporozoites/mosquito were comparable between P. falciparum linup− clones B1 (dark gray) and B4 (light gray) and P. falciparum NF54 (black). Data is represented as mean+/−SD, n=2 biological replicates. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. P>0.05 is taken as not significant.
A. Comparison of the size of liver stage parasites (based on area at the parasite's largest circumference) between P. falciparum NF54 and P. falciparum linup− on days 5 and 7 post-sporozoite infection. While no growth defect was seen between P. falciparum NF54 and P. falciparum linup− liver stage schizonts on day 5, there was a statistically significant difference in the size of late liver stage schizonts on day 7. Data is represented as mean±SD. Each datapoint refers to the mean size of at least 30-50 parasites for each timepoint. Statistical analysis was carried out using two-way ANOVA using Tukey's multiple comparison test. ***P<0.001, P>0.05 is taken as ns.
B. IFA shows that at seven days of infection the wildtype NF54 parasitophorous vacuole membrane protein EXPI has a circumferential staining pattern in the wildtype, as expected. In comparison EXPI expression in mature P. falciparum linup− at day seven of the liver stage is patchy and aberrant.
A. Schematic shows the experimental design of liver stage-to-blood stage transition experiments in FRG NOD huHep mice. One million sporozoites from P. falciparum NF54 and P. falciparum linup− were injected into one and three FRG NOD huHep mice, respectively. Mice were repopulated with human red blood cells as depicted. All mice were euthanized on day seven and 50 μl blood samples were collected from all mice for parasite load based on 18S rRNA qRT-PCR sampling. The remaining blood was transferred to in vitro culture and grown in culture for seven days. Further 50 μl blood samples were collected from in vitro culture samples for analysis of parasite 18S rRNA qRT-PCR samples seven days after transition to in vitro culture (14 days after sporozoite infection).
B. Analysis of parasite load by 18S rRNA qRT-PCR was carried out on extracted RNA from the blood of mice infected with P. falciparum NF54 and P. falciparum linup− P. falciparum linup− sporozoites on day 7, after expected completion of liver stage development and liver stage-to-blood stage transition (Day 7, black bars). There was a severe defect in the P. falciparum linup− transition to blood stage infection as one out of three mice infected with P. falciparum linup− sporozoites did not shown blood stage in the blood, while the other two infected mice had a 10,000-fold reduction in parasite RNA load as compared to wildtype. Blood was transitioned to in vitro culture and parasite load was measured after a further seven days (day 14, gray bars). Wildtype NF54 blood stages replicated during the seven days in culture, whilst no significant replication was seen for P. falciparum linup− blood stages. This shows that the liver stage-to-blood stage transition of P. falciparum linup− did not release viable blood stage parasites that can initiate blood stage replication.
C. Analysis of parasite liver load by 18S rRNA qRT-PCR was carried out on extracted RNA from P. falciparum NF54 and P. falciparum linup− infected livers on day 7 post infection, depicted as copies of P. falciparum 18S rRNA/μg of extracted liver RNA. Parasite liver burden is comparable between P. falciparum NF54 and P. falciparum linup−.
A. The cartoon depicts the generation of the P. falciparum plasmei2 parasite using CRISPR/Cas9-mediated gene editing. Recombinant parasites were cloned by limiting dilution. The cartoon depicts the generation of the P. falciparum linup parasite using CRISPR/Cas9-mediated gene editing. 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. Primers used to verify the gene deletion are indicated. The combination of primers used and the sizes of the PCR products is indicated in Table 2. Agarose gel electrophoresis for lack of P. falciparum PlasMei2
B. lack of P. falciparum LINUP
C. and lack of plasmid DNA
D. in four P. falciparum LARC2 clones-C12, F7, B7, and B5.
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.
A. Exflagellation centers were counted in mature gametocyte cultures of P. falciparum LARC2 on day 15 post set up for four P. falciparum LARC2KO clones B7, C12, D5 and F7 and found to be comparable to P. falciparum NF54. These gametocyte cultures were fed to female Anopheles stephensi mosquitoes in standard membrane feeding assays and on day 8 post feed midguts were dissected:
B. Oocyst prevalence:
C. counts for oocyst/mosquito were comparable to wildtype for P. falciparum LARC2KO clones F7 and B7. These two clones will be used for further phenotypic analysis; and
D. counts for sporozoites/mosquito were comparable to wildtype for P. falciparum LARC2 clones F7 and B7. Clone F7 was used for further phenotypic analysis.
A. The schematic depicts the experimental design. P. falciparum NF54 and P. falciparum LARC2 sporozoites were isolated from salivary glands of infected Anopheles stephensi mosquitoes. To evaluate for blood stage transition of P. falciparum LARC2 in FRG NOD huHep mice, 1×106 aseptic cryopreserved P. falciparum NF54 (PFSPZ) and aseptic cryopreserved P. falciparum LARC2 (PFSPZ LARC2) sporozoites were injected intravenously into four and six FRG NOD huHep mice per group respectively. On day 6 and 7, 400 μl of 70% RBCs were injected intravenously to enable transition of liver stage parasites to blood. Four hours after 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 asexual media, a volume of human RBCs equal to the packed RBC volume was added and blood was transferred to in vitro culture. Fresh media was replaced daily, and cultures were analyzed every 2-3 days by thick smear for presence of parasites for up to 6 weeks.
B. On day 7, parasite densities for mice infected with PfSPZ ranged between 103-105 parasite equivalents/ml by P. falciparum 18S qRT-PCR, which further increased to 109 parasite equivalents/mml after seven days of in vitro culture. In contrast, in mice infected with PFSPZ LARC2, 0-103 parasite equivalents/ml were detected by P. falciparum 18S qRT-PCR and after seven days in culture there was a drastic decline in P. falciparum 18S rRNA signal, indicating that no viable exo-erythrocytic merozoites were 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 in all four mice infected with PfSPZ within 1-3 days of transition to in vitro culture (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.
(C) Livers of infected mice were also harvested on day 7 for extracting RNA and analysis of parasite liver burden by P. falciparum 18S qRT-PCR. Parasite liver load is depicted as log 10 copies 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.
“Additive” as used herein as a noun is a compound or composition added to a sporozoite preparation. Additives include diluents, carriers, excipients cryoprotectants and the like.
“Aseptic” as used herein means absent the introduction of detectable contamination of other microorganisms such as bacteria, fungi, pathologic viruses and the like. Aseptic sporozoite preparations results in a sterile preparation of sporozoites-free of any other type of microorganism or infectious agent. Microbiological assays used to monitor an aseptic methodology assess the presence or absence of contamination. They include, but are not limited to, the Microbial Limits Test, current USP <61>, incorporated herein by reference.
“Attenuate” as used herein means to render a live organism unable to complete its life cycle without killing it. The organism may have a limited capacity to replicate, express proteins, and to develop through some life cycle stages, but arrests development at a particular life cycle stage and is unable to developmentally progress beyond that stage. With regard to the attenuated Plasmodium parasites disclosed herein, they retain the ability to infect host hepatocytes and express stage specific proteins, but are unable to develop beyond liver stage, are unable to transition to the blood stage infections in the blood stream of infected hosts after liver-stage development and are unable to cause the disease pathology of malaria.
“Challenge” as used herein refers to the presentation of an infectious pathogen to a subject that has been previously been provided with a vaccine intended to confer a protective immunity against infection/disease caused by the challenging pathogen. With regard to malaria, the challenge can be by (CHMI, [31, 32] inoculation of infectious Plasmodium sporozoites: by the exposure to Anopheles mosquitoes carrying infectious Plasmodium sporozoites: or field trials in which vaccinated subjects are monitored in a region where malaria parasites are naturally transmitted by infected mosquito bite.
“Conferring protective immunity” as used herein refers to providing to a population or a host (i.e., an individual) 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 the clinical manifestations, pathology, or symptoms of disease in a host are reduced as compared to a non-treated host, or such that the rate at which infection, or clinical manifestations, pathology, or symptoms of disease appear within a population are reduced, as compared to a non-treated population.
As used herein, the term “disrupt” with regard to gene function, means interfering with the gene function such as to inhibit, inactivate, attenuate, or block the gene function or the function of the encoded gene product. The interference or disruption can be accomplished, for example, by altering the gene sequence (e.g., substitution, modification, deletion, addition, knockdown, knock out or knock in) in a manner and/or to degree such that the translated protein, if any, no longer performs its wildtype function. Alternatively, the gene sequence can be deleted.
As used herein, the term “genetically modified” refers to a modification to the genome of the wildtype Plasmodium organism that results in a defined difference from the wildtype genome sequence. The genetic modification is imposed by human manipulation, e.g., by genetic engineering. With regard to the invention disclosed herein, the genetic modification may be one or more insertions or deletions in a gene, knockout of the gene, or other modifications. In these inventions, genetic modification results in functional disruption of the LINUP gene and in certain embodiments, the LINUP gene and the PlasMei2 gene.
“Immune response” as used herein means a response in the recipient to the introduction of attenuated sporozoites generally characterized by, but not limited to, production of antibodies and/or T cells. Generally, an immune response may be a cellular response such as induction or activation of CD4+ T cells or CD8+ T cells specific for Plasmodium species epitopes, a humoral response of increased production of Plasmodium-specific antibodies, or both cellular and humoral responses. With regard to a malaria vaccine, the immune response established by a vaccine comprising live sporozoites includes but is not limited to responses to proteins expressed by extracellular sporozoites, the intracellular liver stages or other stages of the parasite. Mononuclear cells such as dendritic cells will take components of said parasites and present these antigens to relevant immune cells. In the instant invention, upon subsequent challenge by infectious organisms, the immune response prevents development of pathogenic parasites to the asexual erythrocytic stage that causes disease.
As used herein, the term “live” refers to continued metabolic activity in the Plasmodium organism. In some embodiments “live” indicates that the Plasmodium organism is capable of eventually establishing at least a transient infection, for example within hepatocytes (cultured or in vivo). The Plasmodium organism can be in any relevant developmental stage as is practical considering the genetic attenuation. Thus, for example, the Plasmodium organism can be in the intra-mosquito developmental stages, infective sporozoite stage, or intra-hepatocytic (liver) stage.
Sporozoites produced “in vitro”, or “iSPZ” as used herein means sporozoites that have developed external to mosquitoes, i.e, wherein sporogony from gametocyte stage to mature, infectious sporozoite stage is external to mosquitoes [33].
“Metabolically active” as used herein means alive, and capable of performing sustentative functions and some life-cycle processes. With regard to attenuated sporozoites this includes but is not limited to sporozoites capable of invading hepatocytes in culture and in vivo, potentially having a substantial capacity to divide and progress through some developmental stages, and de novo expressing stage-specific proteins.
As used herein, the terms “Plasmodium organism” or “Plasmodium-species” refer to any parasite that belongs to the genus Plasmodium. In some embodiments, the Plasmodium organism has a human host range, for example, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. In some embodiments, the Plasmodium organism is P. falciparum.
“Promoter” as used herein is a region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind, to initiate and regulate transcription of that gene.
“Transgene” as used herein is a gene that is not normally occurring in the organism to which the transgene has been introduced. In some embodiments of the current invention, the transgene, or expression of the encoded polypeptide, serves to disrupt one or more gene function of the organism. In other embodiments, the transgene encodes a foreign antigen, the expression of which elicits an immune response.
“Vaccine” as used herein is a preparation comprising an immunogenic agent and a pharmaceutically acceptable additive, e.g. excipient, adjuvant and/or additive or protectant. The immunogen may be comprised of a whole infectious agent or a molecular subset of the infectious agent (produced by the infectious agent, synthetically or recombinantly). When the vaccine is administered to a subject, the immunogen stimulates an immune response that will, upon subsequent challenge with infectious agent, protect the subject from illness or mitigate the pathology, symptoms or clinical manifestations caused by that agent. A therapeutic (treatment) vaccine is given after infection and is intended to reduce or arrest disease progression. A preventive (prophylactic) vaccine is intended to prevent initial infection or reduce the rate or burden of the infection.
The present disclosure is directed to genetically attenuated malaria parasites that develop to the late liver stage, but are blocked in the transition to blood stage of infection of erythrocytes. In particular, the attenuated malaria parasites comprise genetic alternations that disrupt the functionality of the liver stage nuclear protein (LINUP) gene. In some embodiments, the malaria parasites comprise Plasmodium-species of human host range, e.g. P. falciparum. P. vivax. P. malariae. P. ovale. P. knowlesi. In some embodiments the malaria parasites contain additional genetic modifications wherein other genes critical for the transition from liver to blood stage are interrupted. As a non-limiting example, the malaria parasite of an embodiment additionally comprises genetic modifications wherein both the LINUP and the PlasMei2 gene functionalities are disrupted (this double knockout is variously referred to as “linup/plasmei2 double knockout (KO)”, “LARC2”, “LARC2 KO” and these terms are equivalent). The disclosed attenuated malaria parasites are useful for compositions comprising additives, vaccines for the prevention and amelioration of malaria, methods for inducing and/or stimulating human immune systems against Plasmodium-specific antigens, methods for inducing an immune response in a human subject against one or more Plasmodium antigens, or methods for conferring protective immunity in a human subject against malaria caused by a Plasmodium-species parasite, which are also encompassed by the present disclosure. In some embodiments, the disclosed attenuated malaria parasites are useful for compositions and methods for ameliorating, reducing, preventing, treating, and/or protecting against malaria infection.
As described in more detail below, the inventors discovered a novel protein (LINUP), described as a liver stage nuclear protein, or as a liver-stage-specific protein that localizes to the parasite nucleus. Knockout of the gene or gene function encoding this protein results in life-cycle arrest late in liver stage development. Additionally, the inventors developed a double knockout, referred to as LARC2 (late arresting replication competent double KO), which contains a functional deletion of both the PlasMei2 and LINUP genes. This double knockout is a superior immunogen compared to the previous generations of replication deficient whole parasite based vaccines, because it undergoes near complete development in the liver, to late liver stage, resulting in expansion of parasite antigen biomass and diversity. In addition, LARC2 is more securely attenuated due to genetic manipulation of two genes required for transition to blood stage, and also ensures batch-to-batch homogeneity, and overcomes the need for antimalarial drug cover, which is a major limitation following immunizations with live non-attenuated sporozoite vaccines under drug cover, i.e., chemo-attenuated vaccination (another late liver stage approach described above). Genetically attenuated Plasmodium-species (e.g., P. falciparum) with a genetic knockout of LINUP, and in another embodiment, another liver stage specific gene such as PlasMei2, or in still other embodiments, other genes encoding proteins necessary for transit from liver stage to blood stage, can be used to induce an immune response in a subject, particularly a human subject, against one or more Plasmodium-species antigens, for conferring protective immunity in a subject, particularly a human subject, against malaria caused by a Plasmodium-species parasite, for immunization of a subject, particularly a human subject, as a pre-erythrocytic stage vaccine that provides or boosts immunity against re-infection and/or to treat, prevent, or ameliorate malaria infection. The complete interruption of the Plasmodium-species parasite life cycle at the liver stage also results in the prevention of parasite transmission to other individuals. This is vital in elimination campaigns to eradicate malaria.
In an embodiment, a live Plasmodium-species organism is genetically modified to disrupt a LINUP gene that encodes a Liver Stage Nuclear Protein, thereby preventing the biological function of a protein encoded by the wildtype LINUP gene.
In an embodiment, the live Plasmodium-species linup− parasite arrests life cycle development in late liver stage within a mammalian intermediate host.
In other embodiments, the Plasmodium-species linup− or LARC2 parasite comprises at least one transgene, and in still other embodiments, the transgene is under control of a promoter that results in transcription of the transgene during the sporozoite or liver stage of development. In certain embodiments, one or more transgene encodes a blood stage- or gametocyte-associated antigen. By providing for the expression of one or more blood stage- or gametocyte-associated antigens, the GAP will possess additional immunogenicity and provide protection against blood stage parasites (asexual and sexual). Thus, even if the GAP fully arrests prior to development into a blood stage, it will still be able to stimulate the immune cells against antigens characteristic of blood stage parasites. This provides further protection against blood stage parasites and reduces the risk of clinical symptoms as well as transmission of infection in the gametocyte antigens. In other embodiment, one or more transgenes encode antigens from other pathogens, and thus provide an immune response to these additional pathogens.
In an embodiment, the Plasmodium-species organism is P. falciparum. P. vivax. P. malariae. P. ovale, or P. knowlesi.
In an embodiment, the Plasmodium-species organism is at the sporozoite stage of development.
In an embodiment, the functional LINUP gene of the live Plasmodium-species linup−, prior to genetic modification has at least about 40%, 50%, 60% 70%, 80%, 90%, or 95% nucleotide sequence identity to SEQ ID NO: 35.
In an embodiment, in the live Plasmodium-species linup− parasite, the functional LINUP gene prior to genetic modification encodes a LINUP polypeptide with 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.
In an embodiment a vaccine composition comprises Plasmodium-species linup− parasite at the sporozoite stage, and an additive, where the vaccine composition is prepared aseptically.
In an embodiment, the Plasmodium-species linup− parasite is genetically modified to disrupt one or more additional gene functions, each additional gene function necessary for the liver stage to blood stage transition, and in another embodiment, the additional gene function is PlasMei2. In certain embodiments the functional PlasMei2 gene prior to genetic modification comprises a nucleic acid sequence that is at least at least about 70%, 80%, 90%, or 95% identity to the nucleic acid sequence set forth in SEQ ID NO:34. In certain embodiments, the amino acid sequence of a functional PlasMei2 polypeptide encoded by the PlasMei2 gene comprises is at least at least about 70%, 80%, 90%, or 95% identity to the sequence set forth in SEQ ID NO: 36.
In an embodiment a vaccine composition comprises the Plasmodium-species LARC2 (linup−/plasmei2−) and an additive. In certain embodiments, this vaccine composition is prepared aseptically.
Other embodiments relate to methods for inducing an immune response in a human subject against one or more Plasmodium antigens, comprising administration of one or more doses of the Plasmodium-species linup− vaccine composition to the subject, and still other embodiments relate to methods for inducing an immune response in a human subject against one or more Plasmodium antigens, comprising administration of one or more doses of the Plasmodium-species LARC2″ vaccine composition to the subject. Still other embodiments relate to methods wherein the immune responses generated by the administration of these vaccines ameliorates or protects against infection from a subsequent wildtype Plasmodium challenge. Still other embodiments relate to methods of conferring protective immunity in a human subject against malaria caused by a Plasmodium-species parasite by administration of these vaccines.
In an embodiment, Plasmodium-species LINUP″ sporozoites are produced in vitro, and in an embodiment, Plasmodium-species LARC2″ sporozoites are produced in vitro. Plasmodium-species sporozoites produced in vitro have been disclosed [34], and particularly, genetically attenuated Plasmodium-species sporozoites produced in vitro have been disclosed [35], and both of these references are incorporated herein in their entirety.
Methods of purifying live organisms about 10 microns in size, such as Plasmodium-species sporozoites, e.g. P. falciparum SPZ, are known in the art as exemplified by [36], and particularly genetically attenuated Plasmodium-species SPZ [37]. These methods utilize a series of size exclusion filters of different types and with different pore sizes, assembled in a non-intuitive fashion. The methodology eliminates attendant material from preparations of live, motile parasites. A unique aspect of this method is that the pore size of a size exclusion filter in sequence is not always smaller than the pore size of the size exclusion filter which precedes it. Another inventive aspect is that some filters provide a matrix with a nominal pore size and at least one filter provides a track-etched filter with a precise pore diameter. At least one filter has a pore size close to or slightly smaller than the diameter of the parasite.
Typically in a preparation for purification, the salivary glands from 150 to 400 mosquitoes are dissected. The sporozoites are released from the salivary glands by passage back and forth in a needle and syringe (trituration), and sporozoites from these glands are collectively purified. However, several fold more mosquitoes may be dissected in scaled up preparations, in an embodiment up to 1,000 mosquitoes, in another embodiment up to 5,000 mosquitoes, in another embodiment up to 10,000 mosquitoes. Sporozoites are released from salivary glands by trituration and the triturated salivary gland preparations (pre-purification preparations) are purified by the size exclusion filtration process disclosed herein. Sporozoites are maintained throughout the purification process in an excipient, typically one percent human serum albumin (HSA) in Medium 199 with Earle's salts (E-199).
The triturated dissection product (pre-purification preparation) is received altogether in a single tube at a time. This is the mosquito salivary gland material (SGM) pre-purification preparation. It represents about 100,000 to 1 billion sporozoites, preferably at least 1 million sporozoites and more preferably at least 25 million sporozoites. The measured amount of SGM in the pre-purification preparation is usually between 300 ng and 12,000 ng per 25,000 sporozoites, more typically, between 400 ng and 1,100 ng per 25,000 sporozoites. The pre-purification preparation is then diluted to 10 ml with excipient. Solutions and samples are kept between 15-30° C. for the duration of the purification.
Using a peristaltic pump the diluted pre-purification preparation is pumped across a series of size exclusion filters at a flow rate of at least 1 ml/min but no more than 1000 ml/min, preferably at least 2 ml/min, but no more than 500 ml/min, and more preferably with a flow rate of at least 3 ml/min and no more than 200 ml/min. The corresponding flux across each filter is at least 1 L/hr/m2 but no more than 2000 L/hr/m2, preferably 3 L/hr/m2 to 1500 L/hr/m2, and most preferably at least 125 L/hr/m2 but no more than 250 L/hr/m2. Filters are connected in series, usually with medical grade silicone tubing. Preferably, the initial filter (Filter #1) or the initial two filters (Filters #1 and #2) are matrix filters and are made of polypropylene, however, nylon, mixed cellulose ester and borosilicate glass or other material known to those in the art may be used. Preferably, the penultimate filter (Filter #3) is a membrane filter, most preferably a track-etched polycarbonate filter, although other filters with similar properties known to those in the art may be used. For aseptic procedures, the filters are sterile. In an embodiment, three filters (Filter #1, Filter #2 and Filter #3) are connected in series and sporozoites are captured by dead end filtration on Filter #4. Additional filters may be used. In an embodiment, filter #1 is a membrane matrix with a nominal pore size of at least about 2.5 microns, but no more than about 30 microns, preferably at least about 5 microns, but no more than 20 microns. In one embodiment the filter used has a nominal pore size of about 10 microns with a filtration area of 17.5 cm2. (Polygard®-CN Optiscale-Millipore Cat. No. SNIHA47HH3). In a scaled up embodiment the filtration area is 1800 cm2. The nominal pore size of Filter #2 (also a membrane matrix) is at least about 0.3 microns but not larger than about 1.2 microns. In one embodiment, the nominal pore size is about 0.6 micron with a filtration area of 17.5 cm2 (PolygardR-CN Optiscale filter-Millipore Cat. No. SN06A47HH3)—smaller than the diameter of the Plasmodium sporozoite. In a scaled up embodiment the filtration area is 1800 cm2. In one embodiment, Filter #3 is a track-etched membrane filter with precise pore diameter and consistent pore size, and has a pore size of at least 1.2 microns but not larger 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 with a filtration area of 11.3 cm2. (Isopore membrane, 47 mm in diameter-Millipore Cat. No. RTTP04700) held in a Swin-Loc filter holder (Whatman Cat. No. 420400). In a scaled-up embodiment the filtration area is 127 cm2. Filtered material is captured on Filter #4 in a stirred ultrafiltration cell (Millipore, model 8200) fitted with an Isopore membrane, 90 mm in diameter with a filtration area of 28.7 cm2, and a track-etched pore size of no more than 0.8 microns, preferably no more than 0.6 microns, and preferably no more than 0.2 microns. In one embodiment the pore size is 0.4 microns (Millipore Cat. No. HTTP09030). In a scaled-up embodiment the filtration area is 162 cm2. In another scaled-up embodiment the filtration area is 63 cm2. The system is washed several times with media. When the retentate volume reaches about 40 ml in the stirred cell, the stirred cell container outlet is opened and drained by gravity leaving about 5-10 ml of residual retentate although the retentate volume can be reduced by other methods such as applying pressure from compressed gas such as nitrogen or a mechanical device such as a piston, gravity is the preferred method. This residual retentate is collected and transferred, together with three washes using purification media to a total of about 35 ml, typically in a sterile 35 ml Oak Ridge or similar centrifuge tube (the size of the tube will vary depending on the volume of the preparation). Purified sporozoites in media in the 35 ml Oak Ridge tube are centrifuged at 5,000 g to 25,000 g, preferably at 16,300 g, for 2 minutes to 12 minutes, preferably five minutes, to pellet the sporozoites. The supernatant media is decanted. This step additionally purifies the sporozoite preparation by removing smaller more buoyant materials and soluble materials that remain in the supernatant.
Using the 3 size-exclusion filter methodology, this procedure provides greater than a substantial reduction of attendant material in the purified sporozoite preparation relative to the attendant material in the pre-purification preparation (reduction factor) of from 200 to 10,000-fold. The amount of residual SGM in purified preparations of sterile purified sporozoites routinely is less than 25 ng of attendant material per 25,000 sporozoites (greater than 97% reduction relative to the initial amount of SGM), preferably less than 15 ng per 25,000 (98% reduction) sporozoites, and more preferably less than 1 ng per 25,000 sporozoites (99.9%). The contaminating SGM in each purified preparation described herein is usually reduced several thousand-fold relative to SGM in the initial triturated pre-purified salivary gland material from which each purified preparation is derived. Preferably the purification reduction factor is at least 15-fold, more preferably, the purification factor is at least 1,500-fold and most preferably at least 3,500-fold. The geometric mean of the reduction factor in the 10 campaigns described in Example 1 is 1625, a 99.93% reduction in SGM during the purification process.
This method is effective at reducing contamination and attendant mosquito salivary gland material in a crude sporozoite preparation. The sporozoites may then be cryopreserved as is also known in the art.
In some embodiments, Plasmodium-species sporozoites, and particularly purified Plasmodium-species sporozoites, are prepared aseptically. This methodology is known in the art as exemplified by and further exemplified in the description of the GMP manufacture of aseptic, purified Pf-LARC2 in Example 7, below.
Genetically attenuated Plasmodium-species sporozoites, including such vaccines in which the immunogen is Plasmodium-species P. falciparum linup− and Plasmodium-species P. falciparum-LARC2, are generally administered in a regimen of one to three doses, and usually are administered parenterally, including subcutaneous, intradermal and intravenous administration, preferably by intravenous direct venous inoculation (DVI) methodology, known in the art. A suitable dose of genetically attenuated Plasmodium sporozoites, such as P. falciparum genetically attenuated sporozoites, per inoculation may be between about 10,000 to about 10 million sporozoites, preferably between 100,000 and 1,000,000 sporozoites. In some embodiments of the invention, the vaccine is administered with an adjuvant, for example 7DW8-5 as described in [39], and such administration in conjunction with adjuvant would likely reduce and number of sporozoites required per dose.
Such vaccines are useful for prevention or reduction of severity of malaria, its manifestations, symptoms or its pathology. In an embodiment, compositions and vaccines comprising aseptically prepared genetically attenuated purified sporozoites provide partial, enhanced, or full protection in human and other mammalian subjects not previously exposed to a malaria-causing pathogen, or exposed, but not fully protected. These compositions and vaccines are similarly useful to reduce the chance of developing a disease-producing infection from parasites that causes malaria, including species of Plasmodium. e.g. P. falciparum or P. vivax, and the like, and reduce the chance of becoming ill when one is infected, reduce the severity of the illness, such as fever, when one becomes infected, reduce the concentration of parasites in the infected person, or reduce mortality rates from malaria in populations exposed to malaria parasites. In many cases even partial protection or delay in the time it takes an immunized individual as compared to a non-immunized individual to become infected with the parasites or ill from infection is beneficial. Similarly, a vaccine treatment strategy that results in any of these benefits in about 30% of a population may have a significant impact on the health of a community and of the individuals residing in the community. It is generally contemplated that inoculating a subject according to the methods of the invention with genetically attenuated Plasmodium sporozoites of one Plasmodium species will induce protective immunity against challenge with wildtype 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, thus, eliciting cross-species protection in this manner is also within the scope of the invention.
Also provided are methods for prevention of malaria in a subject. The methods comprise administering to the subject a vaccine comprising a genetically attenuated Plasmodium-species vaccine lacking LINUP gene function, which has been prepared aseptically and comprises substantially purified live genetically attenuated Plasmodium sporozoites in an amount effective to prevent malaria.
The prevention and/or treatment of malaria may be readily ascertained by the skilled practitioner by evaluation of clinical or pathological manifestations associated with malarial infection, for example elevated temperature, headache, fatigue, coma, or percent of erythrocytes parasitized. Thus, according to the methods of the present invention, the subject shows improved or absent clinical signs, symptoms or pathological manifestations of malaria following administration of a vaccine comprising purified live attenuated Plasmodium sporozoites.
In some embodiments, the administering step results in infection of a hepatocyte of the subject, and development of the genetically attenuated Plasmodium parasite through liver stage, providing an array of Plasmodium-specific antigens, and the generated immune response ameliorates or protects against infection from a subsequent wildtype Plasmodium challenge. Accordingly, in some embodiments, the disclosed methods confer protective immunity sufficient to reduce or prevent the symptoms of malaria in at least 60% of subjects, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of subjects, following exposure to wildtype Plasmodium.
Effective and optimal dosage ranges for vaccines and immunogens can be determined using methods known in the art. Guidance as to appropriate dosages to achieve an anti-malarial effect is provided from the exemplified assays disclosed herein. More specifically, results from the immunization pattern described herein and in cited references can be extrapolated by persons having skill in the requisite art to provide a test vaccination schedule. Volunteer subjects are inoculated with varying dosages at scheduled intervals and test blood samples are evaluated for levels of protection against malaria upon subsequent challenge with infective parasites. Such results can be used to refine an optimized immunization dose and dosage regimen (schedule) for effective immunization of mammalian, specifically human, subjects.
An immune response in a subject can be measured by standard tests including, but not limited to the 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 to the art: natural killer cell cytotoxicity assays cell proliferation assays [41] immunoassay's of immune cells and subsets [42, 43] and skin tests for cell mediated immunity [44]. Various methods and analyses for measuring the strength of the immune system have been described, for example, [45].
The vaccines provided comprise aseptic and non-aseptic compositions (preferably aseptic) of purified live attenuated Plasmodium sporozoite substantially free of attendant material, and compositions with a pharmaceutically acceptable diluent, excipient, or carrier. These vaccines are effective in preventing or mitigating malaria upon subsequent challenge with infectious parasites. Methods of formulating pharmaceutical compositions and vaccines are well known to those of ordinary skill in the art [see. e.g., 46].
Comprehended by the invention are vaccine compositions, aseptically prepared or otherwise, comprising purified, live attenuated or non-attenuated Plasmodium sporozoites along with appropriate diluent and buffer. Diluents, commonly Phosphate Buffered Saline (PBS), or Normal Saline (NS), are of various buffer content pH and ionic strength. Such compositions may also include an excipient such as serum albumin, particularly human serum albumin. Serum albumin may be purified from naturally occurring sources such as human blood, or be produced by recombinant DNA or synthesis technologies. Such compositions may also include additives such as anti-oxidants e.g., ascorbic acid, sodium metabisulfite, and/or preservatives or cryopreservatives. Incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes may also be used. (See. e.g., [46] pages 1435-1712 which are herein incorporated by reference).
In order to determine the effective amount of the vaccines, the ordinary skilled practitioner, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Experiments to determine levels for dosages can be ascertained by one of ordinary skill in the art by appropriate human clinical trials in which various dosage regimens are evaluated for their capacity to elicit protection against malaria.
Disclosed vaccines and disclosed methods of using these vaccines may be useful as one component in a vaccine regimen, each component in turn comprising a discrete vaccine to be administered separately to a subject. Regimens may include sequential immunization with attenuated Plasmodium species sporozoites and other types of Plasmodium vaccines, so-called, prime-boost strategies. This may include attenuated sporozoites as a prime, and Plasmodium-related recombinant protein or proteins in adjuvant as a boost or vice versa. This may also include Plasmodium-related DNA vaccines or a recombinant virus, such as adenovirus, that express Plasmodium-related proteins, as a prime and purified, attenuated sporozoites vaccine as a boost, or vice versa. It may also include sequential or mixed immunization with attenuated Plasmodium species sporozoites and some form of erythrocytic stage parasites, including, killed and live attenuated. A vaccine complex comprising separate components may be referred to as a vaccine regimen, a prime/boost regimen, component vaccine, a component vaccine kit or a component vaccine package, comprising separate vaccine components. For example, a vaccine complex may comprise as a component, a vaccine comprising purified, aseptic, live attenuated sporozoites. The complex may additionally comprise one or more recombinant or synthetic subunit vaccine components, including but not limited to recombinant protein, synthetic polypeptide, DNA encoding these elements per se or functionally incorporated in recombinant virus, recombinant bacteria, or recombinant parasite. A vaccine component may also include aseptic attenuated axenic sporozoites that are allowed to develop to the early liver stage extracellularly.
The following are experimental disclosures of the discovery and analysis of LINUP as a viable target for genetic attenuation, as well as the development of double knockout attenuated Plasmodium.
It is generally noted that the use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
Following long-standing patent law, the words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted.
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise,” “comprising,” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, such as in the sense of “including, but not limited to.” Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below,” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. Words such as “about” and “approximately” imply minor variation around the stated value, usually within a standard margin of error, such as within 10% or 5% of the stated value.
Disclosed are materials, compositions, and components that can be used for, in conjunction with, and in preparation for the disclosed methods and compositions. It is understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.
Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties.
To identify novel genes giving rise to attenuation only during liver stage development, the late liver stage P. vivax transcriptome that was isolated from the infected livers of human-liver chimeric FRG NOD huHep mice was utilized. Mice were infected with one million P. vivax Thai field strain sporozoites isolated from Anopheles dirus mosquitoes. The mice were euthanized at day 8 after infection, late in liver stage development and the livers were processed for RNA extraction. Probes specific to 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 one million mapped reads (RPKM). This resulted in the capture of 4397 unique gene transcripts with RPKM values greater than two. To enrich for liver stage specific reads, the P. vivax RPKM reads were expressed as a fold change over the maximal RPKM reads for the orthologous P. falciparum genes expressed during the blood stage of the life cycle. This enabled comparison and contrast of asexual P. vivax liver stage development with asexual P. falciparum blood stage development and specifically isolate genes that were highly expressed only during liver stage development. The top ten genes from this analysis (Table 1) included genes or metabolic pathways already known to be expressed during liver stage development and not during blood stage development.
Plasmodium vivax
Plasmodium
Plasmodium
vivax day 8
Plasmodium
Plasmodium
vivax gene
falciparum asexual
falciparum
Plasmodium
yoelii ortholog
Known genes included LISP1 (30), LISP2 (31-33), PALM (34) and SIAP2 (35). Malonyl CoA-acyl carrier protein transacylase was also detected, a precursor of type II fatty acid biosynthesis, a pathway known to be expressed during P. falciparum liver stage development and critical for P. yoelii and P. berghei liver stage development (36-39). This result gave us assurance that our methodology for the discovery of liver stage specific transcripts was sound. We also had a requirement that orthologs were present in the P. yoelii genome so we could use this rodent malaria model to efficiently analyze spatial and temporal expression as well as essentiality. Of the remaining five genes in the top ten, four had orthologs in the P. yoelii genome and included a putative protoporphyrinogen oxidase, PY17X_0513300, and three hypothetical genes, PY17X_1003700, PY17X_1465200 and PY17X_1369800. Analysis using PlasmoDB of the P. yoelii genes and their P. falciparum orthologs for both protein expression (P. yoelii and P. falciparum) and essentiality in the blood stage (P. falciparum) suggested that although PY17X_051300/PF3D7_102800 was dispensable in P. falciparum asexual blood stages, the protein was expressed in the P. falciparum blood stage gametocyte and the P. yoelii oocyst sporozoite and thus the gene was not studied further. Analysis of PY17X 1369800/PF3D7_1351300 suggested the gene was essential for P. falciparum asexual blood stage replication and thus not studied further. Analysis of
PY17X_1003700/PF3D7_0404600 suggested the gene was dispensable in P. falciparum asexual blood stages, but the protein was expressed in both the P. falciparum blood stage merozoite and sporozoite and thus the gene was not studied further. Of note, analysis of PY17X 1465200/PF3D7_1249700 suggested the gene was dispensable for P. falciparum asexual blood stage replication. Further, there was no evidence of protein expression in P. falciparum and expression in the liver stage of P. yoelii at 40 hours of development. Therefore, PY17X_1465200/PF3D7_1249700 underwent further study.
PY17X_1465200 is a single exon gene coding for a 746 amino acid protein with no known protein features and is conserved across Plasmodium species (
To study the essentiality of LINUP expression on P. yoelii liver stage development, we used CRISPR/Cas9 technology to delete the LINUP gene from the P. yoelii genome (
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 wildtype parent during blood stage growth (
To determine if liver stage development was affected by the deletion of LINUP, P. yoelii wildtype and linup− sporozoites were isolated from infected Anopheles stephensi mosquito salivary glands and injected intravenously into groups of laboratory mice including outbred Swiss Webster mice as well as the susceptible inbred strains BALB/cJ and BALB/cByJ (Table 3).
P. yoelii
P. yoelii
aTwo independently generated clones of Py linup− (c3 and c5) were used for analysis.
bThe strain of mice used for the analysis is indicated. SW: Swiss Webster.
cSalivary gland sporozoites were isolated from infected Anopheles stephensi mosquitoes, and mice were challenged intravenously with the listed number of sporozoites.
dThe number of patent mice per number of mice challenged is indicated. Detection of blood stage patent parasitemia was carrier out by Giemsa-stained thin blood smear. Attenuation was considered if mice remained blood stage negative for 21 days.
eIf mice became blood stage patent, the day to patency is listed, with the number of mice that became patent in parentheses.
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 wildtype sporozoites all demonstrated blood stage infection on days three whereas nine of ten mice demonstrated blood stage infection when infected with P. yoelii linup− sporozoites and there was a severe delay to patency with mice becoming infected on days eight and ten after infection, suggesting that P. yoelii linup− liver stage are severely attenuated (Table 3). Similarly, BALB/cByJ mice infected with 50,000 wildtype sporozoites all demonstrated blood stage infection on day three whereas only nine of twenty mice infected with P. yoelii linup− sporozoites demonstrated blood stage infection and the day to infection ranged from days eight through ten (Table 3). A dose escalation study in BALB/cJ mice revealed further evidence of liver stage attenuation. Mice were infected with 1,000, 10,000 and 50,000 sporozoites. Mice infected with wildtype sporozoites all demonstrated blood stage infection on day four (1,000), days three-four (10,000) and day three (50,000) whereas for P. yoelii linup− sporozoite infection only one of ten mice demonstrated blood stage infection (1,000) on day twelve, seven of twenty demonstrated blood stage infection (10,000) on days seven through twelve, and five of ten became patent (50,000) on days seven through nine (Table 3). Thus, P. yoelii linup− parasites are severely attenuated during liver stage development.
To further study the attenuation of the P. yoelii linup− liver stage, BALB/cByJ mice were infected intravenously with 250,000 P. yoelii linup− sporozoites. Mice were euthanized at time points after infection (24, 36 and 48 hours), livers were removed, perfused, fixed, sliced and subjected to IFA to determine both size and protein/DNA expression patters in comparison to a wildtype infection (
Rodent malaria GAP that arrest during liver stage development are powerful immunogens that can protect from a wildtype sporozoite challenge [26-28]. To determine if immunization of susceptible BALC/cJ mice with P. yoelii linup− sporozoites could protect from challenge groups of mice were intravenously immunized with either 1,000 or 10,000 P. yoelii linup− sporozoites or mock immunized with an equivalent volume of salivary gland extract from uninfected mosquitoes (Table 4). Only mice that did not demonstrate liver stage infection after P. yoelii linup− sporozoite infection were ultimately challenged with wildtype sporozoites. Mice were immunized twice, approximately 33 day's apart and then challenged after a further 34 days with an intravenous injection of 10,000 wildtype sporozoites. Mice immunized with salivary gland extract all became blood stage infected three-four days after challenge (Table 4).
P. yoelii linup− immunization protects from a wildtype sporozoite challenge
P. yoelii linup−
P. yoelii linup−
a
P. yoelii linup− salivary gland sporozoites were isolated from infected Anopheles stephensi mosquitoes, and mice were immunized intravenously with the listed number of sporozoites. The days after the prime that the boosts took place is indicated in parentheses.
bMice were challenged intravenously with wildtype salivary gland sporozoites. The days after the boost the challenge took place are indicated in parentheses.
cThe number of protected mice per number of mice challenged is indicated and the days to patency are indicated in parentheses. Protection was considered complete if mice remained blood stage negative for 21 days after challenge, based on Giemsa-stained thin blood smear.
Six of nine mice immunized twice with 1,000 P. yoelii linup− sporozoites were protected from challenge and the remaining three mice showed a significant delay to blood stage infection, becoming infected on days eight to ten after challenge (Table 4). All mice (thirteen) immunized twice with 10,000 P. yoelii linup− sporozoites were protected after challenge. This result demonstrates that immunization with P. yoelii linup− GAP engenders a highly effective immune response that protects mice from a significant wildtype sporozoite challenge.
P. falciparum NF54 and P. falciparum linup− sporozoites were isolated from salivary glands of infected Anopheles stephensi mosquitoes. For analysis of P. falciparum linup− liver stage, 1×106 P. falciparum NF54 and 1×106 P. falciparum linup− sporozoites were injected intravenously (retro-orbital) into four FRG NOD huHep mice per group. Livers were harvested on days 5 and 7 and used for IFA (
The values for the three mice injected with P. falciparum linup− sporozoites ranged from 0 to 1×104, demonstrating a severe attenuation in the transition (
P. falciparum NF54 and P. falciparum LARC2 sporozoites were isolated from salivary glands of infected Anopheles stephensi mosquitoes. To evaluate for blood stage transition of P. falciparum LARC2 in FRG NOD huHep mice, 1×106 aseptic cryopreserved P. falciparum NF54 (PFSPZ) and aseptic cryopreserved P. falciparum LARC2 (PFSPZ LARC2) sporozoites were injected intravenously into four and six FRG NOD huHep mice per group respectively (
On day 7, parasite densities for mice infected with PfSPZ ranged between 103-106 parasite equivalents/ml by P. falciparum 18S qRT-PCR (
P. falciparum LARC liver stages do not transmit
1Each mouse was injected with either wild type (PFSPZ) or LARC2 (PFSPZ LARC2) sporozoites
Blood stage parasites were detected by Giemsa-stained smears in all four mice infected with PFSPZ within 1-3 days of transition to in vitro culture (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 for mice infected with PfSPZ LARC2 ranged between 0-103 parasite equivalents/ml by P. falciparum 18S qRT-PCR, which suggests that parasite 18S rRNA is released from non-viable and attenuated liver stage parasites of P. falciparum LARC2 into the blood stream. However, viable exo-erythrocytic merozoites are not formed in the P. falciparum LARC2. Livers of infected mice were also harvested on day 7 for extracting RNA and analysis of parasite liver burden by P. falciparum 18S qRT-PCR. Parasite liver load is depicted as log 10 copies of P. falciparum 18S rRNA per μg of total liver RNA (
The production of live aseptic purified and cryopreserved GMP quality PfSPZ-LARC2 Vaccine has been accomplished, and follows the life cycle steps of the genetically attenuated Pf parasite, wherein sporozoites are produced in a biological system—the salivary glands of intact aseptic mosquitoes. This requires simultaneous production of Pf gametocytes from a Master Cell Bank and aseptic mosquitoes from a colony of A. stephensi. Pf were cultured in vitro to produce gametocytes. The P. falciparum infected RBC were mixed with uninfected RBC and Supplemented RPMI. They were then placed into wells of a (6-well) culture plate containing Supplemented RPMI and the culture was expanded. To initiate the production of gametocytes in 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 checked for monitoring the gametocyte growth. The numbers of stage V gametocytes were assessed on days 14 to 19 after initiation of gametocyte production. Cultures were selected for preparation of an infectious blood meal to feed mosquitoes. The gametocytes in an infectious blood meal were fed to aseptic adult mosquitoes that were cultured from aseptic eggs, larvae and pupae.
Aseptic mosquito production involves all stages of the mosquito life cycle, beginning with disinfection of eggs and preparation of solutions and ending when mosquitoes were ready for feeding (U.S. Pat. No. 8,802,919). An artificial membrane was placed across the mouth of a container and autoclaved. The container was kept around 35-37° C. and adult mosquitoes were introduced. Pf gametocyte-infected blood was added to the membrane and the mosquitoes were allowed to feed for 30 minutes on the gametocyte-infected blood meal. After feeding, the mosquitoes were returned to the incubator and maintained at 26° C., 77% relative humidity, 12-hour light: dark cycles.
The infected aseptic adult mosquitoes were then dissected, the salivary glands harvested, the sporozoites are extracted, purified as described. The aseptic purified PfSPF, vaccine formulated, was then cryopreserved. Cryopreservation begins with the addition of 2×CPA to 1:1 ratio. The preparation was dispensed in aliquots of 20 μL into the cryovials. The vials were prepared for cryopreservation by rate freezing, which is a multistep process that cools the vials to cryogenic temperatures below −150° C. The cryovials were eventually transferred to storage in liquid nitrogen vapor phase (LNVP), which is between −150° C. to −196° C.
The assessment of two separate preparations of PfSPZ-LARC2 Vaccine are shown in Table 6.
200 million aseptic, purified PfSPZ-LARC2 were vialed at 106 PfSPZ-LARC2 per vial.
Plasmodium yoelii LINUP knockout
Plasmodium yoelii LINUP mCherry
Plasmodium falciparum LINUP knockout
Plasmodium yoelii gene
Plasmodium yoelii gene SEQ ID NO: 33
Plasmodium falciparum PlasMei2 gene
Plasmodium falciparum PlasMei2 gene SEQ ID NO: 34
Plasmodium falciparum LINUP gene
Plasmodium falciparum LINUP gene SEQ ID NO: 35
Plasmodium falciparum PlasMei2 amino acid
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
This application claims the benefit of U.S. Provisional Application No. 63/234,872, filed Aug. 19, 2021.
This invention was made with Government support under R01 AI125706 awarded by National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/075238 | 8/19/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63234872 | Aug 2021 | US |
