Method for inoculating a vertebrate host against malaria, by administering to the host a live Plasmodium organism that is genetically engineered to disrupt a gene whose expression is up-regulated in liver stage parasites and whose function is not required for entry into hose hepatocytes.

SUMMARY OF THE INVENTION

Here we show by reverse genetics that selected individual genes, exemplified by UIS3 (up-regulated in infective sporozoites gene 3) and UIS4, are essential for early liver stage development: uis3(-) and uis4(-) sporozoites infect hepatocytes but are no longer able to establish blood stage infections in vivo and thus do not lead to disease. The invention thereby provides the first live Plasmodium organisms that are genetically engineered to disrupt liver-stage-specific gene functions.

Surprisingly, immunization with either uis3(-) or uis4(-) sporozoites confers complete protection against infectious sporozoite challenge in a rodent malaria model. This protection is sustained and stage-specific. These findings provide the first genetically attenuated whole organism malaria vaccines.

Thus, the invention provides a method for inoculating a vertebrate host against malaria, by administering to the host a live Plasmodium organism that is genetically engineered to disrupt a liver-stage-specific gene function. The invention further provides a vaccine composition comprising a live Plasmodium organism that is genetically engineered to disrupt a liver-stage-specific gene function. In addition, the invention provides the use of a vaccine composition comprising a live Plasmodium organism that is genetically engineered to disrupt a liver-stage-specific gene function. The invention also provides for production of a vaccine composition, by suspending and packaging the subject engineered Plasmodium organisms in a suitable pharmaceutically acceptable carrier solution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention provides a method for inoculating a vertebrate host against a Plasmodium parasite, by administering to the host a live Plasmodium organism that is genetically engineered to disrupt a liver-stage-specific gene function.

By "Plasmodium parasite" or "Plasmodium organism" is meant any member of the protozoan genus Plasmodium, including the four species that cause human malaria: P. vivax, P. malariae, P. falciparum, and P. ovale. The corresponding "vertebrate host" is a human or other secondary host that is susceptible to infection by the wild-type Plasmodium parasite.

For use as a live anti-malarial vaccine, the Plasmodium parasite is genetically engineered to disrupt a liver-stage-specific gene function. The term "disrupt liver-stage-specific gene function" or "disrupt LS-specific gene function" means interfering with an LS-specific gene function such as to completely or partially inhibit, inactivate, attenuate, or block the LS-specific gene function, for example, by gene disruption or influencing transcription, translation, protein folding, and/or protein activity. The term "liver-stage-specific gene function" or "LS-specific gene function" refers to a function that is required in liver stage parasites to ultimately produce infectious merozoites and establish the erythrocytic stage of the life cycle, but that is not required for entry into host hepatocytes or, preferably, maintenance of the parasite in asexual blood cell stages and production of infective sporozoites in mosquitoes.

Malaria infection is initiated by Plasmodium sporozoites in the salivary glands of mosquitoes. These sporozoites invade hepatocytes of the vertebrate host and differentiate into liver stage (LS) forms. After a few days the LS parasites produce several thousand merozoites that are released from the hepatocytes and invade erythrocytes to start the blood stage cycle that causes malaria disease. According to the invention, the Plasmodium parasite is genetically engineered to disrupt at least one LS-specific gene function such that the genetically engineered parasites remain capable of invading hepatocytes but cannot produce merozoites that can establish blood stage infections. Of course, pursuant to this disclosure, more than one LS-specific gene function can be disrupted (such as by creating, for example, double knock-outs) as such redundancy may ensure an additional degree of protection against parasitemia.

Pursuant to this disclosure, an LS-specific gene function may be identified using routine methodology that is standard in the art. For example, an LS-specific gene function may be identified by assessing the function of genes whose expression is up-regulated in liver-stage parasites ("LS-up-regulated genes"). For example, genes whose expression is up-regulated in liver-stage parasites may be expressed at higher levels in liver-stage parasites than, e.g., in the sporozoite population that emerges from mosquito mid-gut oocysts. Up-regulation of expression of such genes may also be observed in mature, infective salivary gland sporozoites (like in the UIS4 and UIS3 genes discussed in the Examples below). Well-known methods for differential transcriptional profiling, including, but not limited to, subtractive hybridization screens, differential display, and genome-wide microarray analyses, may be used for identifying genes whose expression is up-regulated in liver-stage parasites. Such methods have been previously used to analyze infectivity-associated changes in the transcriptional repertoire of sporozoite-stage parasites (11) and to identify Plasmodium genes that encode pre-erythrocytic stage-specific proteins (12). For example, suppression subtractive hybridization permits selective enrichment of differentially regulated cDNAs of high and low abundance through a combination of hybridization and polymerase chain reaction (PCR) amplification protocols that allow the simultaneous normalization and subtraction of the cDNA populations. Suppression subtractive hybridization has been used to analyze transcriptional differences between non-infective and infective sporozoites and to identity genes controlling infectivity to the mammalian host (11). This procedure has permitted the identification of LS-up-regulated genes, including but not limited to the UIS3 and UIS4 genes disrupted in the Examples below. Suppression subtractive hybridization of Plasmodium salivary gland sporozoites versus merozoites has also been used to identify stage-specific pre-erythrocytic transcripts (12). Differential expression of candidate LS-specific genes may be confirmed using methods that are standard in the art, including dot blots, reverse transcriptase PCR (RT-PCR), immunoblotting, immunofluorescence microscopy, and/or microarray expression analyses.

In some embodiments of the invention, LS-specific gene functions are identified by analyzing the function of LS-up-regulated genes, as further described below. However, not all genes with an LS-specific gene function are necessarily LS-up-regulated genes. Thus, genes whose expression is not up-regulated in LS forms may nevertheless possess an LS-specific gene function.

Interference with a liver-specific function may also be achieved by LS-specific overexpression of an inhibitory factor. This factor may be inserted by reverse genetics methods into a pseudogene, i.e., one that is not essential for parasite survival at any time point during the life cycle (47). The inhibitory factor should not confer toxicity to the parasite but rather act in arresting LS development. Such a factor may include, but is not limited to, inhibitors of cell-cycle progression and/or ubiquitin-mediated proteolysis, and/or factors that interfere with post-transcriptional control of gene-expression.

LS-specific gene functions may be identified by analyzing the phenotype of parasites in which one or more gene functions have been disrupted. Several methods for disrupting gene functions in Plasmodium are well-known in the art and may be used in the practice of the invention. Such methods include, but are not limited to, gene replacement by homologous recombination, antisense technologies, and RNA interference. For example, methods of gene targeting for inactivation or modification of a Plasmodium gene by homologous recombination have been established (13). Such methods were herein successfully used to disrupt LS-specific gene functions, as described in Examples 1 and 2. Antisense technology has also been successfully used for disrupting Plasmodium gene functions. For example, exogenous delivery of phosphorothioate antisense oligonucleotides against different regions of the P. falciparum topoisomerase II gene result in sequence-specific inhibition of parasite growth (14). Similarly, transfection of an antisense construct to the Plasmodium falciparum clag9 gene, which had been shown to be essential for cytoadherence by targeted gene disruption, resulted in a 15-fold reduction in cytoadherence compared to untransfected control parasites (15).

Another exemplary technology that may be used in the practice of the invention to disrupt LS-specific gene functions is RNA interference (RNAi) using short interfering RNA molecules (siRNA) to produce phenotypic mutations in genes. RNAi has been used as a method to investigate and/or validate gene function in various organisms, including plants, Drosophila, mosquitoes, mice, and Plasmodium (see, e.g., 37 44). In Plasmodium, RNAi has been used, for example, to demonstrate the essential role of a PPI serine/threonine protein phosphatase (PfPP1) from P. falciparum (41). RNAi has also been used to inhibit P. falciparum growth by decreasing the level of expression of the gene encoding dihydroorotate dehydrogenase (42) and by blocking the expression of cysteine protease genes (43). In the mouse malaria model, RNAi has been used to inhibit gene expression in circulating P. berghei parasites in vivo (44). These studies have demonstrated the use of RNAi as an effective tool for disrupting gene function in Plasmodium organisms.

The gene disruption approaches described above (for example, gene targeting by homologous recombination, antisense, and RNAi) have been used successfully to investigate the function of virtually all genes in an organism's genome. For example, the availability of sequenced genomes has enabled the generation of siRNA libraries for use in large-scale RNAi studies to screen for genes that are involved in various processes, such as developmental pathways or stages (see, e.g., 45 and 46). Such screens may be used in the practice of the invention to identify LS-specific gene functions in Plasmodium. Assays that may be used for identifying LS-specific gene functions include, but are not limited to, phenotypic analyses such as the phenotypic assays described in Examples 1 and 2. The term "phenotypic analysis" includes all assays with vital recombinant parasites that are generated in a wild type, fluorescent or any other transgenic reporter background. Assays may be performed in vivo, with cultured cells, in in vitro development assays or any other system that provides a read-out for LS development.

The engineered Plasmodium organisms in which an LS-specific gene function has been disrupted are typically grown in cell culture or animals, expanded in the mosquito host, and harvested as sporozoites for use in vaccines (see, e.g., 16).

The subject vaccine compositions are produced by suspending the attenuated live Plasmodium organisms in a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include sterile water or sterile physiological salt solution, particularly phosphate buffered saline (PBS), as well known in the art.

Vaccines according to the invention can be administered, e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, and intravenously.

Dosage is empirically selected to achieve the desired immune response in the host. By "immune response" is meant an acquired and enhanced degree of protective immunity, preferably complete or sterile protection, against subsequent exposure to wild-type Plasmodium sporozoites. In the working examples described below, sterile protection was achieved following three vaccinations with 10,000 live genetically attenuated sporozoites per inoculation.

DETAILED TECHNICAL DESCRIPTION

Background. Radiation-attenuated sporozoites are a singular model that achieves sterile, protective immunity against malaria infection.

Malaria causes more than 300 million clinical cases and more than 1 million death annually. The disease has a severe negative impact on the social and economic progress of developing nations. Transmission of the malaria parasite Plasmodium to the mammalian host occurs when infected mosquitoes bloodfeed and inoculate the sporozoite stage (spz). After entering the bloodstream, spzs are quickly transported to the liver where they extravasate and invade hepatocytes (2). Within hepatocytes, spzs transform into liver stages (LS) (also called exo-erythrocytic forms, EEFs). LS parasites grow, undergo multiple rounds of nuclear division and finally produce thousands of merozoites (17, 18). Merozoites released from the liver rapidly invade red blood cells and initiate the erythrocytic cycle, which causes malaria disease. A protective malaria vaccine would have tremendous impact on global health but despite over a century of efforts, no vaccine has been developed that confers prolonged protection. Yet, we have known for more than 35 years that sterile protracted protection against malaria infection is possible.

Immunization of mice with radiation-attenuated rodent model malaria spzs (gamma-spzs) induces sterile immunity against subsequent infectious spz challenge, thus completely preventing the initiation of blood stage infection from the liver (5). Importantly, based on these findings it was later shown that immunization of humans with gamma-P. falciparum spzs completely protected greater than 93% of human recipients (13 of 14) against infectious spz challenge and that protection can last for at least 10 months (6). Gamma-spzs retain the capacity to infect the liver of the mammalian host and invade hepatocytes (19 20). However, LS derived from gamma-spzs suffer arrested development and thus do not produce red blood cell-infectious merozoites. Although, the inoculated stage is the spz, the main immune target is the infected hepatocyte harboring the LS (21). Protective immunity is spz-dose and radiation-dose dependent: greater than 1000 immunizing bites from P. falciparum-infected mosquitoes exposed to 15,000 20,000 rads of gamma radiation is required to protect the majority of subjects exposed to infectious spz challenge (6). Mosquitoes inoculate between 10 100 spzs during a bite (22 23). Therefore, the total spz dose for complete protection comes to 10,000 100,000. Importantly, immunization with over-irradiated spzs or heat-inactivated spzs fails to induce protection, indicating that the spz must remain viable for some time after inoculation and must progress to a liver stage that induces protection (6, 24). On the basis of observations in the rodent malaria model, protracted protective immunity may depend on sufficient expression of LS antigen (Ag), because treatment with primaquine, a drug that kills LS, aborts the development of protection (21). Importantly, protection induced by P. falciparum gamma-spzs is strain-transcending: inoculation with gamma-spzs of one parasite strain confers protection against heterologous strains (6).

Although we have learned much about spz gene expression in the last few years (25 27), the LS as the putative immunological target(s) of gamma-spzs induced protection have so far completely eluded gene expression analysis because of their inherent experimental inaccessibility. We currently know only one liver stage-specific Ag, liver stage antigen-1 (LSA-1) (28). Thus, the fine Ag specificity of lymphocytes participating in protective immunity remains unknown in humans, because the Ags expressed by LS parasites remain unknown.

Feasibility to create genetically attenuated Plasmodium Liver Stages. To generate genetically attenuated Plasmodium LS that are defective only in LS development a stage-specific gene that plays an essential and exclusive role at this stage needs to be disrupted. The gene should not be essential during the blood stage cycle given that Plasmodium is haploid and transfection is done with asexual blood stages and the mutant parasites are typically maintained as blood stages (13). We previously employed transcription-profiling based on the prediction that infectious Plasmodium spzs residing in the mosquito salivary glands are uniquely equipped with transcripts required for hepatocyte invasion and subsequent development of the LS (11). Next, we screened for transcripts that are specific for pre-erythrocytic and absent from blood cell stages (12). The combined screens identified two abundant salivary-gland-spz-enriched transcripts that are absent from blood stages, termed UIS3 and UIS4 (for upregulated in infectious spzs). Cell biological studies have shown that both encoded proteins locate to the parasitophorous vacuole, the parasite-derived organelle where replication and schizogony takes place (data not shown).

Gene knockouts using insertion and replacement strategies have now revealed that both genes are necessary for LS development (see Examples 1 and 2 below). Both proteins are normally expressed in spzs (data not shown), but uis3(-) and uis4(-) parasites develop normal spzs and these invade hepatocyte normally. However, uis3(-) and uis4(-) LS arrest in intermediate-LS development and do not produce late LS (data not shown). Therefore, both UIS3 and UIS4 have LS-specific gene functions. Remarkably, animals infected by natural bite or intravenously with doses of up to 10,000 spzs do not become patent, confirming that both genes play vital roles in successful completion of the Plasmodium life cycle (see Tables 1 and 2 below). Therefore, we succeeded in generating the first genetically attenuated LS. Based on these discoveries we and others can now advance and test various LS-up-regulated genes identified by microarray analysis for their importance in LS development. We predict that more LS-up-regulated genes will turn out to be essential for LS development (i.e., to possess LS-specific gene functions), especially uniquely expressed genes given the remarkable capacity of the parasite to develop from a single spz to more than 10,000 daughter merozoites. Such LS-up-regulated genes can be similarly disrupted to produce additional live vaccine candidates, as described herein.
 

Claim 1 of 1 Claim

1. A method for inoculating a vertebrate host against malaria, comprising administering to the host a live Plasmodium organism that is genetically engineered to disrupt a gene whose expression is up-regulated in liver stage parasites and whose function is not required for entry into host hepatocytes.

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