# Patent

## Title
Methods for detecting virulent Plasmodium, for evaluating Plasmodium virulence, and for screening new drugs employing the 3′ UTR of Plasmodium SUB2 and the Plasmodium SUB2 serine protease

## Abstract

Methods for regulating the serine protease of Plasmodium. Recombinant DNA constructs which express the Plasmodium serine protease, especially those comprising a sub2 3′UTR and coding segment which express a SUB2 a serine protease. Recombinant Plasmodium containing such constructs and exhibiting increased virulence. Methods for detecting virulent Plasmodium strains by detecting the presence or amount of sub2 3′UTR sequences, sub2 mRNA or cDNA, SUB2 polypeptide expression, or other Plasmodium proteins, such as AMA1 or MSP1, which have been post-translationally modified by SUB2.

## Background
REFERENCE TO SEQUENCE LISTING
This application contains a sequence listing which forms a part of the disclosure.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention pertains to recombinant Plasmodium expressing the SUB2 subtilisin-like protease which has a modified non-coding 3′UTR (3 prime untranslated region). The invention also related to methods for identifying virulent strains, and/or evaluating the virulence, of Plasmodium, and for screening new anti-malaria drugs are disclosed.
2. Description of the Related Art
Plasmodium is the parasite that causes malaria. The host-parasite relationship between malaria and its host involves a complex equilibrium between parasite multiplication, which is essential for its dissemination, and host responses1. Collateral damage attributable to the host-parasite interaction is responsible for the disease burden, morbidity and mortality in endemic areas where malaria is a major public health problem2.
The virulence of malaria is associated with the multiplication rate of the parasite and the ability of Plasmodium blood states to cytoadhere microvessels in deep organs1,3,4. While virulence factors involved in cytoadherence have been identified4, those involved in high multiplication rate, for which erythrocyte invasion by merozoite is a crucial step, remain poorly understood1,3,4.
Proteases are known to play a crucial role in various infectious diseases and cancer and have been successfully defined as chemotherapeutic targets. The specificity of protease activity permits the design of highly specific inhibitors which block a pathogenic process without considerable toxic side effects. Thus protease inhibitors are selectively toxic agents applicable for therapeutic use.
Biochemical and genetic analysis have shown that proteases and particularly serine proteases play a central role for the liberation and entry of merozoites into the host red blood cell.
Compared to intra-erythrocytic parasite states, the merozoite, as the sporozoite, is briefly free in the host plasma and therefore accessible to external factors including host immune factors like antibodies or to therapeutic drugs. The Merozoite Surface Protein 1 (MSP1) of Plasmodium is considered a promising candidate for a malaria vaccine. MSP1 undergoes a two step maturation process. The final processing of MSP1-42 occurs while the merozoite enters into the host RBC and is achieved by a parasite membrane-bound and calcium-dependent serine protease. Despite MSP1 polymorphism, this sequential processing is precisely conserved, even amongst different Plasmodium species. Even if the biological function linked to this maturation is still unknown, its inhibition using specific monoclonal antibodies or serine protease inhibitors efficiently blocks the RBC invasion.
It has recently been shown that the same parasite serine-protease is responsible for the second maturation step of the vaccine candidate Apical Membrane Antigen-1 (AMA1). Originally, AMA1 was identified to be the target of monoclonal antibodies which prevent RBC invasion by merozoites and more recently hepatocyte invasion by sporozoites. The inhibition of the maturation of AMA1 results in abortive RBC or hepatocyte invasion by the merozoites and the sporozoites, respectively.
Over the last decade, the establishment of Plasmodium genetic tools7, the genome sequencing of the laboratory clone P. falciparum 3D78 and various parasite transcriptome and proteome global projects9,10, have made profound changes in the field of malaria research. Interestingly, global expression profiling shows that 50% of the 5400 parasite genes are stage regulated, while the proportion of proteins predicted to be involved in gene regulation is low, especially when compared to S. cerevisiae8. Only a handful of regulatory elements have been identified11,12 and none is known that controls the multigene family coding for proteins mediating cytoadherence, which are subjected to antigenic variation.
A regulatory role for the respective introns of some malaria genes and 3′UTR (3 prime untranslated region) has been recently proposed13, as previously shown for the 3′UTR of the ookinete Pgs28 gene14. The 3′UTR, and particularly the addition of a polyadenylated tail, have been widely shown to play a central role in the regulation of eukaryotic15 and prokaryotic16 gene expression via the modulation of messenger RNA (mRNA) stability and translation initiation.
In malaria parasites, poly-A addition does not necessarily occur at the eukaryotic canonical AAUAAA (SEQ ID NO: 1) site and poly-A− mRNA have been identified17,18. In the absence of an inducible system to genetically study essential malaria genes, the deletion of 3′UTR was used to decrease gene expression, revealing interpretable phenotypes19,20. To evaluate in vivo the contribution of the crucial merozoite subtilisin-like P. berghei-SUB2 protease5,6 to the parasite life cycle, the inventors aimed to modulate Pbsub2 expression via the modification of its 3′UTR.
BRIEF DESCRIPTION OF THE INVENTION
The inventors have now demonstrated that efficient expression of the essential merozoite subtilisin-like maturase SUB2 strictly depends on functional poly-A sites, whose mutation gives rise to recombinant parasites over-expressing SUB2. The accumulation of SUB2 in these recombinant parasites correlates with more efficient maturation of the surface antigens MSP1 and AMA1, which are both vaccine candidates, and increased virulence of these recombinant Plasmodium organisms. The inventors thus show that non-coding regions involved in the regulation of crucial genes can be important virulence factors. Such genes may represent a dormant reservoir of virulence in Plasmodium and in other parasitic organisms in which invasion or virulence is mediated by enzymes involved in polypeptide/antigen maturation, such as the particular polypeptides/antigens which mediate host cell adhesion or invasion. Such parasitic organisms include Toxoplasma, Babesia, Leishmania and Trypanosomes. 
For the first time, the inventors have now demonstrated that SUB2 is involved in the maturation of the merozoite surface vaccine candidates MSP1-42 and AMA1 and have shown that these maturations are not only crucial for the host cell invasion to succeed, but represent a limiting step on virulence in vivo. Based on these discoveries defining SUB2 as a maturase whose activity is critical for the biological cycle of malaria parasites, they establish that SUB2 is an anti-malaria drug target.
The inventors have discovered that the efficient expression of the essential merozoite subtilisin-like maturase P. berghei-SUB25,6 strictly depends on functional poly-A sites, the mutation of which gives rise to malaria parasites which over-express PbSUB2. A significantly higher parasite multiplication rate in vivo correlates with the accumulation of PbSUB2 since higher amounts of this enzyme provide a more efficient maturation of merozoite surface proteins, such as MSP1 (Merozoite Surface Protein 1) and AMA1 (Apical Merozoite Antigen 1), which are both candidate antigens for vaccine production.
Host cell invasiveness is known to be a key factor of malaria parasite virulence1 which aggravates when the parasite does not show any selectivity for blood cells3,23. Beyond the first direct evidence that SUB2 is involved in the maturation of the merozoite surface vaccine-candidates MSP1-42 and AMA1, the present inventors genetically demonstrate that this maturation is not only crucial for the host cell invasion to succeed, thus confirming previous biochemical and immunological studies21,24, but is also a limiting step in vivo. These discoveries indicate that in vivo, maturation of these proteins contributes to the invasion of host blood cells in an age-independent manner. The present inventors have thus defined SUB2 as a potent malaria virulence factor related to tropism independent host-cells invasion.
Based on these discoveries the present inventors describe new methods for determining and/or evaluating parasite virulence based on the presence of particular motifs in non-coding region sequences, such as a motif found in the 3′ UTR of sub2. The 3′ UTR or 3 prime untranslated region corresponds to a section of mRNA that is past the stop codon for a coding region or exon. Graphically it may be depicted as follows: 5′-UTR—translated RNA—3′UTR.
Since regulation of sub2 gene expression has now been demonstrated to be an important virulence factor, methods of determining and/or evaluating and/or quantifying virulence may be based on the identification of regulatory sequences in the 3′ UTR, the amount of sub2 mRNA (or cDNA corresponding to this mRNA), the amount of SUB2 protein expressed, or on SUB2 activity, for example, as measured by the post-translational processing of AMA1 or MSP1 polypeptides.
Increased or decreased virulence of a modified organism is based on a comparison with the virulence of corresponding unmodified organism or control strain. Likewise, the term “attenuated” means less virulent when compared to the corresponding unmodified or control strain.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Pbsub2 3′UTR characterization and modification following a double crossing-over strategy.
FIG. 1A. Pbsub2 specific 3′RACE was performed on P. berghei segmented schizonts total RNA, fractionated on a 1.5% agarose gel, ethidium bromide stained (Lane 1) or Southern-blotted and probed with Sub2Cter (Lane 2). Lanes 3, 4 and 5 correspond to RT-PCR performed respectively without reverse transcriptase, on genomic DNA and without template. Size markers are indicated. Polyadenylation signals and CA addition sites of the Pbsub2 3′UTR are schematised on the left panel. The position of the nucleotides is relative to the Pbsub2 start codon. In FIG. 1A, the two polynucleotide sequences at the top correspond respectively to SEQ ID NO: 2 nucleotides 306-317 and 343-374 and the two polynucleotide sequences at the bottom correspond to SEQ ID NO: 2 nucleotides 176-187 and 215-231.
FIG. 1B. Schematic representation of the wild type Pbsub2 3′UTR double crossing-over modification strategy and (bottom) the resulting WT-TmDX, single (SPA) or null poly-A sites (NPA) recombinant loci.
The polyadenylation sites (•), the TrimycDuoXpress (TmDX) epitope (hatched box) and the homologous coding (shaded) and untranslated (thickened) regions are indicated. The position of the BsmI (B), ClaI (C), HindIII (H) and SacI (S) restriction sites are indicated.
FIG. 1C. Genomic DNA prepared from WT-TmDX (w.) and SPA-TmDX (S.) cloned parasites was digested as indicated and probed with Sub2Cter or TgDhfr specific probes. The pattern obtained for SPA-TmDX gDNA supports a 0.2 kb truncation of the wild-type Pbsub2 3′UTR (FIG. 1C). Size markers are indicated.
FIG. 2: Comparative analysis of the Pbsub2 mRNA and protein in WT-TmDX1 and SPA-TmDX1 recombinant parasites.
FIG. 2A. Northern blot analyses were performed on total RNA extracted from wild type and recombinant parasites, fractionated and probed with Sub2Cter (upper panels) or Pbrab6 (lower panel) specific probes.
FIG. 2B. Western blot analysis of proteins SDS-extracted from segmented schizonts using anti-c-myc and trans-species specific anti-Plasmodium HSP70 specific monoclonal antibodies.
FIG. 2C. Subcellular localisation of the tagged PbSUB2 protein. Immunofluorescence analyses were performed on air-dried segmented schizonts, incubated with Sub2Cter-GST serum or the anti-c-myc plus the anti-Xpress monoclonal antibodies. Nucleus staining (Propidium Iodide, red) is merged with the antibody labelling (FITC, green).
FIG. 3: Comparative analysis of PbMSP1-42 and PbAMA1 maturation in WT-TmDX1 versus SPA-TmDX1 recombinant parasites.
Western blot analyses of SDS-extracted proteins from mature merozoites using rabbit anti-PbMSP1 polyclonal serum, rat trans-species specific anti-AMA1/28G2 (C) and mouse HSP70/1-C11 (B, D) specific monoclonal antibodies. The equivalent number of parasites per lane, and molecular weight markers are indicated.
FIG. 4: In vivo multiplication rates of the WT-TmDX1 versus SPA-TmDX1 in Balb/c and C57B1/6 mice.
Four BALB/cJ (A panel) or five C57B1/6J mice (B panel) were infected with 5×104 WT-TmDX1 (triangle) or SPA-TmDX1 parasites (square). Parasitaemia monitored using Giemsa stained blood smears are presented from day 2 to day 6 post-injection (dpi). The percentage of WT-TmDX1 (open triangle) or SPA-TmDX1 (open square) parasites in reticulocytes is presented with broken lines on the right panel. Values represents mean±SD of 4-5 mice per group, p<0.05 versus WT-TmDX1 as calculated by Mann Whitney U-test.
FIG. 5: Chromosomal localisation of the Pbsub2 recombinant loci.
The P. berghei chromosomes were prepared from wild type, SPA-TmDX and five WT-TmDX clones and separated by CHEF. Chromosomes 9 to 12 are indicated.
FIG. 5A. The wild type, WT-TmDX1 and SPA-TmDX1 P. berghei chromosomes were probed with Sub2Cter specific probe.
FIG. 5B. The wild type, SPA-TmDX1 and WT-TmDX1, 2, 4, 6 and 7 chromosomes were probed with TgDhfr specific probe. Given the unexpected rearrangement of the chromosome bearing Pbsub2 in the WT-TmDX2 clone, further analyses were performed with WT-TmDX1.
FIG. 6. Alignment of the 3′UTR of Pbsub2, Pysub2 and Pfsub2. In FIG. 6, Pbsub2-3′UTR, Pysub2-3′UTR and Pfsub2-3D7-3′UTR respectively correspond to SEQ ID NOS: 2, 3 and 4. Consesus nucleotides shared by the depicted sequences appear with a black background.
The sequences corresponding to the 3′UTR of Pfsub2-3D7 and Pysub2, were extracted from www.plasmodb.org, and aligned with Pbsub2 (Genebank access number AJ242629) using CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). Nucleic Acids Research, 22:4673-4680). Identical residues appear in a black background and the stop codon of each gene is boxed. “AAUAAA” (SEQ ID NO: 1) poly-adenylation signals and “CA” dinucleotides corresponding to the poly-A tail addition sites are labelled respectively in red and blue. Distal and proximal Pbsub2 “AAUAAA” (SEQ ID NO: 1) poly-adenylation signals described in this issue are indicated. Pfsub2 and Pysub2 putative “AAUAAA” (SEQ ID NO: 1) poly-adenylation signals and “CA” poly-A tail addition sites have been identified using www._softberry.com/cgi-bin/programs/polyah.pl and http://_rulai.cshl.org/sgi-bin/tools/polyadq online software.
FIG. 7 (Supplemental FIG. 3). Measure of the erythrocytic cycle length of WT-TmDX1 and SPA-TmDX1 parasites in vitro.
The WT-TmDX1 and SPA-TmDX1 parasites were synchronised by inoculation of mice with merozoites. The parasites collected 4 hours later were in vitro cultured for 26 hours post-invasion (hpi). The percentage of emerging parasites with 2 to 4 (plain line), 5 to 8 (broken line), and more than 8 nuclei (dots) has been determined every two hours from 18 to 26 hpi, Determination of the in vitro schizogony properties. The synchronised WT-TmDX1 or SPA-TmDX1 parasites were prepared as follow: ten Swiss mice were infected with 2.5×107 parasites on day 0. Two days later, the blood was collected by heart puncture and maturated in vitro as previously described (van Dijk et al. (1995), Science 268, 1358-62). Merozoites were collected and injected into five Swiss mice. Four hours post-injection, the blood was collected and the parasites maturated in vitro for 18 to 26 hours. The parasites were counted according to their nuclei number using Giemsa stained blood smears.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The 3′UTR of Pbsub2 was first characterised based on 3′RACE experiments using total RNA preparations from mature P. berghei schizonts (FIG. 1A). A major 750 bp and a weak 550 bp fragments hybridising to the Pbsub2 C-ter probe were amplified, consistent with the two predicted canonical polyadenylation motifs (AAUAAA) (SEQ ID NO: 1) (FIG. 1A, diagram). The sequence of six clones corresponding to the major 750 bp fragment revealed three different mRNA species with poly-A addition following CA dinucleotides 37, 43 and 62 bp respectively downstream from the distal and principally used AAUAAA (SEQ ID NO: 1) polyadenylation motif (FIG. 1A; Supplemental FIG. 2).
Two constructs were designed to sequentially delete Pbsub2 polyadenylation sites (FIG. 1B) following a double crossing-over event, giving rise respectively to the WT-TmDX6, SPA-TmDX (Single PolyAdenylation site, corresponding to the proximal Pbsub2 polyadenylation site) and the NPA-TmDX (no polyadenylation site) recombinant parasites (FIG. 1B). Despite several attempts, the NPA-TmDX recombinant parasites were not recovered, while SPA-TmDX and WT-TmDX recombinant parasites were reproducibly selected using the same pool of parasites prepared for transfection experiments. The structure of the Pbsub2-locus from in vivo cloned recombinant parasites was assessed by Southern and chromosomal blots (FIG. 1C; Supplemental FIG. 1) and by PCR analyses.
The merozoite PbSUB2 protease being essential for the erythrocytic cycle, since, the inability to delete both Pbsub2 polyadenylation sites shows that the Pbsub2 transcript polyadenylation following canonical AAUAAA (SEQ ID NO: 1) sites is crucial for the correct expression of the PbSUB2 protease.
Plasmid pSPA from which Plasmodium SPA-TmDX can be obtained, has been deposited at the CNCM on May 3, 2005 under accession number I-3423.
Interestingly, the 3′UTRs of P. yoelii and P. falciparum sub2 orthologs display similar polyA-addition motifs, (Supplemental FIG. 2), suggesting a trans-species conserved post-transcriptional modification for the sub2 transcripts.
In eukaryotic cells, the use of canonical AAUAAA (SEQ ID NO: 1) and CA motifs to trigger polyadenylation involves a set of conserved proteins which form a complex after binding to the poly-addition motifs15. Data mining of the P. yoelii and P. falciparum genomes identifies putative orthologs of these proteins: a Poly-A Polymerase III (PAP), a Cleavage and Polyadenylation Stimulation Factor (CPSF), a Cleavage and Stimulation Factor (Cstf) and a Cleavage Factor I (CF1) that are respectively >33%, 50%, >30% and 30% identical to their eukaryotic counterparts (Supplemental Table 1). While P. falciparum putative PAP mRNA expression is constitutive, the CPSF-like, Cstf-like and SUB2 mRNAs are concomitantly expressed during the merozoite biogenesis5,6,9,10. Therefore, although poly-A addition has previously been shown to occur at unusual sites14,18, malaria parasites possess a classical stage regulated eukaryotic polyadenylation machinery which could be involved in post-transcriptional regulation of mRNA expression.
As suggested by the data in Supplemental Table 1, the regulation of mRNA transcription or post-translational regulation of mRNA stability, as observed for the sub2 gene may also represent virulence factors for other Plasmodium proteins. Thus, alteration of mRNA transcription efficiency or mRNA stability for transcripts of these genes could affect virulence of Plasmodium. Many other bloodstream and intracellular parasites also encode enzymes involved in the maturation of antigens involved in attachment or invasion of host cells. Thus, a similar regulation of mRNA transcription or mRNA stability via sites in the 3′UTR of maturation enzymes in organisms such as Trypanosomes, Leishmania, Babesia and Toxoplasma may represent important virulence factors in these organisms as well. Therefore, the methods employing the Plasmodium sub2 gene, sub2 gene mRNA transcript, and SUB2 protein may also be applied to the genes and gene products from parasites other than Plasmodium in an analogous manner.
Quantitative Northern blot experiments revealed a 3 to 4 fold increase of the Pbsub2-mRNA in the SPA-TmDX1 clone compared to wild-type and WT-TmDX1 parasites (FIG. 2A and Supplemental Table 2). Whether this phenotype is a consequence of a modified Pbsub2-mRNA transcription efficiency or stability is unknown, but it correlates with a two-fold increase of the recombinant SPA-TmDX1 PbSUB2-protein (FIG. 2B and Supplemental Table 3). PbSUB2 protein principally accumulates as its intermediate activation form, whose final maturation takes place during its post-reticulum secretion21. Thus, the post-translational processing and localisation (FIG. 2C) of the SPA-TmDX1 PbSUB2-protein are identical to that found in wild-type parasites, indicating that the excess of PbSUB2 is available for further activation to perform its biological role.
To investigate the potential effects of PbSUB2 over-expression during the intra-erythrocytic parasite development, the inventors first compared the kinetics of schizogony by measuring the ratio of the 2-4, 4-8, and >8 nuclei during a comparative in vitro maturation of highly synchronised WT-TmDX1 and SPA-TmDX1 parasites (Supplemental FIG. 3). Their timing of nuclei multiplication, average number of merozoites per schizont and erythrocytic cycle duration were not significantly different. This result is in accordance with the fact that PbSUB2 is a merozoite-specific enzyme which is not detectable in other parasite blood stages5,6. A contrario, when analysing in detail mature SPA-TmDX1 merozoites, it appears that the accumulation of PbSUB2 results in a significant increase of respectively the merozoite surface PbAMA1 15 kDa-C-terminal ((PbAMA1-CT15) and PbMSP1-19 maturation products (FIG. 3 & Supplemental Tables 4 and 5).
The maturation of the merozoite surface MSP1-42 and the AMA1 has been shown to play a crucial role during the invasion process per se. The inventors have now determined that this observation which was obtained in vitro correlates with a virulence phenotype in vivo. As shown by the FIG. 4, SPA-TmDX1 parasites grow statistically faster in vivo than WT-TmDX1. This behaviour is independent from the host genetic background, since the injection of 5.104 parasites to Swiss (not shown), C57B1/6J and BALB/cJ yields to respectively 84% and 46% increase of the multiplication rates in vivo for the PbSUB2 over-expressing SPA-TmDX1 parasites (FIG. 4 and Methods). The SPA-TmDX1 parasites increased growth rate in vivo leads to the death of the infected mice one day before the WT-TmDX1 infected ones. Thus, the 3′UTR-driven accumulation of PbSUB2 protein in SPA-TmDX1 parasites results in a significant increase of the parasite virulence in vivo.
The WT-TmDX1 and SPA-TmDX1 clones derive from P. berghei ANKA parasites, harbouring a marked tropism for reticulocytes22. Thus, the inventors investigated whether the multiplication rate difference between these clones was related to a better invasion of normocytes by SPA-TmDX1 parasites. At day 4 (2.5% parasitaemia), more than 30% of both WT-TmDX1 and SPA-TmDX1 parasites were detected in reticulocytes (FIG. 3B); when SPA-TmDX1 and WT-TmDX1 parasitaemia reached 8.5% and 7.8% on day 5 and 7 respectively, the proportion of parasites in reticulocytes decreased to 2.7% and 1.1% respectively. Based on these observations, PbSUB2 accumulation does not impair P. berghei tropism in vivo. The 2 day quicker consummation of reticulocytes being a consequence of the SPA-TmDX1 clone's higher multiplication rate in toto.
As a more general concern, the increase of P. berghei virulence in vivo following the modification of the Pbsub2 3′UTR indicates that malaria parasite virulence can be potentiated following the mutation of a single gene regulatory element. Preliminary results indicate that the P. falciparum Pfsub2 3′UTR is highly polymorphic and could lead to Plasmodium falciparum parasites over-expressing PfSUB2. Adaptation of parasite gene expression to a selective pressure has been shown to participate in the resistance to anti-malarials20,25. Thus, non-coding regions involved in the regulation of expression of crucial Plasmodium genes should now be considered as potential virulence factors, a situation already reported for some viruses and bacteria26-28.
This large latent reservoir of virulence may not yet have been explored by the parasite but could be revealed under a sub-lethal selective pressure, such as a partial immunity driven by vaccines which reduces pathogen growth rate29,30. Since the selection of such vaccine overcoming parasites is not anticipated in clinical trials, and considering the large population targeted, this observation may lead to dramatic consequences for public health following imperfect intervention strategies against malaria parasites, or other invasive pathogens.
Malaria pathogens include those which infect humans, simians and other animals, for example, Plasmodium berghei, Plasmodium brasilianum, Plasmodium chabaudi, Plasmodium cynomolgi, Plasmodium falciparum, Plasmodium gallinaceum, Plasmodium knowlesi, Plasmodium lophurae, Plasmodium malariae, Plasmodium ovale, Plasmodium reichenowi and Plasmodium vivax. 
The following sequences are incorporated by reference. P. falciparum SUB2 (AJ132006 and NC—004315, geneID: 810927); P. yoelii SUB2 (PY01222, proteinID: EAA20512.1); Toxoplasma gondii SUB2 and SUB1 (AF420596 and AY043483, respectively). Detailed information about the structures and functions of 3′UTR is incorporated by reference to Proudfoot et al., Curr. Biol. 12:R855-7 (2002).
A recombinant Plasmodium according to the invention may comprise a modified regulatory segment within the 3′UTR. Such a modification may increase or decrease the expression of a gene like sub2, which encodes a protein involved in maturation or post-translational processing of other parasite antigens, such as the SER2 subtilisin-like maturase. Recombinant organisms having modifications in the 3′UTR which provide higher amounts of a maturase like SUB2 have been demonstrated to be more virulent compared to the corresponding unmodified strains. Similarly, recombinant strains modified to express less of a maturase like SUB2 (e.g., an attenuated strain) would be expected to be less virulent. An example of a use for a less virulent strain would be as a live vaccine or as a comparative control. More virulent strains can be used in vaccine testing procedures, for example, to test the efficacy of a particular vaccine, to provoke particular humoral or cellular immune responses, or as comparative controls in assays involving Plasmodium strains with other virulence characteristics.
To detect or evaluate the virulence of a Plasmodium strain, the amount of SUB2 protein expressed by that strain compared to a control strain may be determined Alternatively, the amount of sub2 mRNA may be detected or measured as a comparative indication of virulence of the test and control strains. Methods for determining the levels a particular protein, such as SUB2, are well known in the art, for example, SUB2 levels may be determined by ELISA or Western blotting using antiserum to SUB2 epitopes. The mRNA encoding SUB2 may also be determined or quantified by conventional methods.
Methods for determining the amount of mRNA (or corresponding cDNA) encoding a Plasmodium protein such as SUB2, AMA1 or MSP-1 are well known in the art and are also incorporated by reference to Current Protocols In Molecular Biology, Chapter 25 (2005) which is incorporated by reference.
Western blotting and other methods of identifying or quantitating the amounts of a particular protein, such as SUB2, AMA1 or MSP-1 are well known in the art and are also incorporated by reference to Current Protocols in Molecular Biology, e.g. Chapters 10 and 11 (2005) which is incorporated by reference.
For some applications, proteins that are essentially identical or at least 90%, 95%, or 99% similar to SUB2, AMA1 or MSP-1 may be used to construct a recombinant Plasmodium according to the present invention. Generally, a nucleic acid sequence encoding a variant will have 70%, preferably 80%, more preferably 90, 95 or 99% similarity to a native sequence. Such similarity may be determined by an algorithm, such as those described by Current Protocols in Molecular Biology, vol. 4, chapter 19 (1987-2005) or by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Likewise, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
Coding sequences for these proteins may also be determined by the ability of a polynucleotide to hybridize under stringent conditions to the complement of a coding region of a known SUB2, AMA1 or MSP-1 gene. Such hybridization conditions may comprise hybridization at 5×SSC at a temperature of about 50 to 68° C. Washing may be performed using 2×SSC and optionally followed by washing using 0.5×SSC. For even higher stringency, the hybridization temperature may be raised to 68° C. or washing may be performed in a salt solution of 0.1×SSC. Other conventional hybridization procedures and conditions may also be used as described by Current Protocols in Molecular Biology, (1987-2005), see e.g. Chapter 2.
The polynucleotide of the invention which comprises a Plasmodium subtilisin-like maturase SUB2 untranslated region and a regulatory segment differs from the corresponding native sequence. The difference may be in the deletion or addition of a regulatory segment, such as a polyadenylation site, or in the polynucleotide sequence of a regulatory segment.
The deletion, addition or alteration of the regulatory segment provides a different degree of SUB2 protein expression (it modulates protein expression) than that of the corresponding native sequence. The polynucleotide may be conveniently isolated or purified from other nucleic acids or components of Plasmodium. The alteration in the regulatory sequence of the 3′UTR may occur within a polyadenylation site. Such a regulatory segment or polyadenylation site may conform to the native motif, but have 1, 2, 3, 4, 5 or more nucleotides deleted, inserted or substituted compared to the native sequence. Preferably, an altered regulatory segment will contain a high degree of similarity to the native sequence, e.g. 60, 70, 80, 90, 95% similarity, but will function to increase or decrease SUB2 expression compared to the native sequence. Such increases may range from 1, 2, 5, 10, 20, 50, 100, 200% or more compared to the level of expression of SUB2 provided by the unmodified native sequence. Similarly, a modified regulatory sequence may decrease SUB2 expression down to 95, 90, 80, 75% 50, 40, 25, 10, 5% or less of SUB2 expression provided by the unmodified native regulatory sequence. The same parameters apply to regulatory sequences for other non-SUB2 genes involved in antigen or polypeptide processing in Plasmodium or other parasites.
Polyadenylation sites or other motifs in a 3′UTR may be identified using software such as CLUSTALW. Sequences and/or motifs shared by the 3′UTRs of Plasmodium species are also graphically described by SEQ ID NOS: 2, 3, and 4 in FIG. 6.
High-throughput screening of candidate molecules is known in the art. U.S. Pat. No. 6,770,451 describes a method for screening enzyme inhibitors, U.S. Pat. No. 6,368,789 describes a method for identifying telomerase inhibitors and U.S. Pat. No. 6,051,373 describes methods for screening inhibitors of the transcription-enhancing activity of the X protein of hepatitis B virus. The screening methods and libraries disclosed by these patents is incorporated by reference. None of these documents disclose the use of the SUB2 target molecule of the present invention. Combinatorial Chemistry vol. 8, issue 1, pp. 1-5 by N. K. Terrett (January, 2006) also describes a number of different methods and libraries for high throughput screening of anti-malaria agents. These methods and libraries are also incorporated by reference. Large numbers of test compounds may be efficiently screened for their ability to bind and/or inhibit SUB2 activity. These test compounds may have various structures, such as small organic molecules having a molecular mass of about 50 to 2,500 daltons, molecules containing metal ions, carbohydrates, saccharides, peptides having less than 100 residues, polypeptides, antibodies, and molecules or other products isolated from natural sources such as from bacteria, fungi, parasites, plants, and animals.
EXAMPLES
Methods
Parasites. P. berghei parasites were amplified and collected as previously described6. Noteworthily, due to the reactivity of the anti-c-myc-specific monoclonal antibody (mAb) with the host 30 kDa Myc proteins, special care was taken to deplete leucocytes using Plasmodipur filters (Euro-Diagnostica, the Netherlands), before preparing erythrocyte extracts for Western blotting experiments.
Polyadenylation sites mapping. RT-PCR analyses were performed with 1 μg of total RNA as previously described6. The 3′ untranslated region of Pbsub2 was reverse transcribed from 1 μg of total segmented-schizont RNA using the Tun primer (5′TTTTTTTTTTTTTTTTTTTT[ACG][ACGT]3′) (SEQ ID NO: 5). The resulting cDNA was PCR amplified using the Tun and CterBamHI (5′TTTGGATCCCATCATCAAAGTAAACAACGCG3′) (SEQ ID NO: 6) primers (95° C., 20 sec; 45° C., 1 min, 62° C., 2 min for 3 cycles; then 95° C., 20 sec; 50° C., 1 min; 62° C., 2 min for 30 cycles), separated in 1% agarose gels, cloned into the pCR2.1-TOPO vector (Invitrogen), and sequenced using a Sequenase 2 kit (USB corporation).
Generation of transfected constructs. The pSub2-SPA-TmDX and pSub2-NPA-TmDX plasmids were obtained following the same three step procedure used for the pSub2-WT-PA plasmid, with the following modifications6. The Pbsub2-3′UTR fragments used to generate the pSub2-SPA-TmDX and pSub2-NPA-TmDX plasmids were PCR amplified with the reverse primers 5′CCGGATCCATAAAAATATAGTCATACATAC3′ (SEQ ID NO: 7) and 5′CCGGATCCATATTATGCTATATCATTGTGA3′ (SEQ ID NO: 8) respectively. The constructs were entirely sequenced prior to transfection.
Parasite transfection and nucleic acids analyses. Seventy micrograms of each BsmI digested plasmid DNA were transfected into purified schizonts of the P. berghei ANKA strain and pyrimethamine selection of the transformed parasites were performed as previously described6. The pSub2-SPA-TmDX transfected parasites were cloned by limiting dilution as previously described6. Southern and Northern blot analyses were performed as described with the appropriate probe6. The Rab6 probe was PCR amplified using the 5′TTGGGAGAACAAGCAGTTGG3′ (SEQ ID NO: 9) and 5′GTAACCTTTCTAAGATCGGCC3′ (SEQ ID NO: 10) primers, and dATP[α32P] labelled (Megaprime, AP Biotech). The Northern blot bands were quantified using the Quantity One software (Biorad).
Seventy micrograms of each BsmI digested plasmid DNA were transfected into purified schizonts of the P. berghei ANKA strain and pyrimethamine selection of the transformed parasites were performed as previously described.sup.6. The pSub2-SPA-TmDX transfected parasites were cloned by limiting dilution as previously described.sup.6. Southern and Northern blot analyses were performed as described with the appropriate probe.sup.6. The Rab6 probe was PCR amplified using the 5′TTGGGAGAACAAGCAGTTGG3′ (SEQ ID NO: 9) and 5′GTAACCTTTCTAAGATCGGCC3′ (SEQ ID NO: 10) primers, and dATP[α32P] labelled (Megaprime, AP Biotech). The Northern blot bands were quantified using the QUANTITY ONE@ software (Biorad).
Immunodetection. For PbSUB2 quantification, total proteins were extracted from WT-TmDX1 and SPA-TmDX1 parasites using 2% SDS and quantified following the Folin method (Sigma) prior to gel loading. Western blot analyses were performed as described6 using 1:5000 diluted horseradish peroxidase (HRP) coupled c-myc mAb or 1:5000 diluted 1c11 mAb, revealed with HRP coupled secondary antibodies. The 1c11 and c-myc mAb labelling was detected using SuperSignal Pico and SuperSignal Femto reagents respectively (Pierce). The bands were quantified using the QUANTITY ONE® software (Biorad).
Immunofluorescence assays were performed on air-dried thin films of P. berghei infected erythrocytes using 1:50 diluted Sub2Cter-GST sera or 1:100 diluted anti-c-myc and anti-Xpress mAb as previously described6. Primary antibodies were revealed using ALEXAFLUOR GREEN® anti-mouse antibodies (Molecular Probes).
In vivo infection of mice and determination of the average daily multiplication rate.
The average daily multiplication rate (ADMR) was calculated as:

ADMR={[Parasitaemia×(1×1010)]/(5×104)}1/4 

where 1×1010 and 5×104 stand respectively for the erythrocyte total number per mouse and the total number of parasites injected at day 0. The ADMR values obtained are 9.5 and 8.1 for SPA-TmDX1 and 6.5 and 4.4 for WT-TmDX1 parasites in BALB/cJ and C57B1/6J mice respectively.







SUPPLEMENTAL TABLE 1







Description of the putative parasite orthologs involved in the polyadenylation


addition. Data were extracted from www._plasmodb.org, Bahl et al., (2003).















Minimum % of identity




PlasmoDB
Chomosome
with eukaryotic putative


Abbreviation
Name
access number
location
orthologues





CPSF
Cleavage and
PFC0825w
PFC0825w: 3
50% of identity with



Polyadenylation
PY00757

eukaryotic putative



Stimulation factor


orthologues


Cstf
Cleavage Stimulation
PFI1600w
PFI1600w: 9
30% of identity with



factor
PY02603

eukaryotic putative






orthologues


PAP
PolyA Polymerase III
PFF1240w
PFF1240w: 6
33-40% of identity with




PY02044

eukaryotic putative






orthologues


CF1
Cleavage Factor I
PFA0450c
PFA0450c: 1
30% of identity with






A. thaliana CF1





PlasmoDB: the Plasmodium genome resource.


A database integrating experimental and computational data. Nucleic acids research, 31, 212-215).













SUPPLEMENTAL TABLE 2







Quantification of the Pbsub2 mRNA in wild-type, WT-TmDX1 and


SPA-TmDX1 parasites. 











Wild Type
WT-TmDX1
SPA-TmDX1

















1 μg
5 μg
10 μg
1 μg
5 μg
10 μg
1 μg
5 μg
10 μg



















Pbsub2 mRNA
nd
138
347
nd
128
373
47
541
1011


 






  
    
      SPA
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      value
    
    x
  
  ⁢
  Ratio





3.9
2.9

4.2
2.7








The amounts of mRNA are presented in arbitrary units.


The ratios of SPA-TmDX1 over wild type and WT-TmDX1 intensity counts are presented


nd: not determined.













SUPPLEMENTAL TABLE 3







Quantification of the PbSUB2 protein from WT-TmDX1 and SPA-TmDX1 extracts.










WT-TmDX1
SPA-TmDX1














20 μg
40 μg
80 μg
20 pg
40 μg
80 μg
















PbSUB2
115
253
516
 238
 457
627


HSP70
2623
3372
3935
2315
2863
3372


 






  
    PbSUB2
    HSP70
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    1
    )
  




0.044
0.075
0.131
  0.103
  0.160
0.186


 
















  
    
      WT
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
    
      SPA
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    2
    )
  




  2.3
  2.1
1.4





The amounts of PbSUB2 and PbHSP70 proteins are presented in arbitrary units.


The ratio between PbSUB2 and HSP70 corresponding to the corrected amount of PbSUB2 is presented (1).


The ratio between PbSUB2 corrected amounts from WT-TmDX1 and SPA-TmDX1 protein extracts are presented in bold (2).













SUPPLEMENTAL TABLE 4







Quantification of the AMA1 protein from WT-TmDX1 and


SPA-TmDX1 extracts.










WT-TmDX1
SPA-TmDX1











Parasites
6.5 × 107
13 × 107
6.5 × 107
13 × 107














PbAMA1 -CT15
104
606
1140
Out of






range


HSP70
668
1017
751
1603


 






  
    
      PbAMA1
      ⁢
      
        -
      
      ⁢
      CT15
    
    HSP70
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    1
    )
  




0.16
0.60
1.52
/


 













  
    
      SPA
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
    
      WT
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    2
    )
  




9.7
/





The amounts of PbAMA1-CT15 and PbHSP70 proteins are presented in arbitrary units.


The ratio between PbAMA1-CT15 and PbHSP70 corresponding to the corrected amount of PbAMA1 is presented (1).


The ratio between PbAMA1-CT15 corrected amounts from WT-TmDX1 and SPA-TmDX1 protein extracts is presented in bold (2).













SUPPLEMENTAL TABLE 5







Quantification of the PbMSP1-19 protein from WT-TmDX1 and SPA-TmDX1 extracts.










SPA-TmDX1
WT-TmDX1













Parasites
13 × 106
6.5 × 106
1.3 × 106
13 × 106
6.5 × 106
1.3 × 106





PbMSP1-19
7380
5189
1599
3725
1911
464


HSP70
2541
2079
 796
2636
1892
664


 






  
    
      PbMSP1
      ⁢
      
        -
      
      ⁢
      19
    
    HSP70
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    1
    )
  




  2.90
  2.50
  2.01
  1.41
  1.01
 0.70


 














  
    
      SPA
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
    
      WT
      ⁢
      
        -
      
      ⁢
      TmDX1
      ⁢
      
        
      
      ⁢
      Ratio
      ⁢
      
        
      
      ⁢
      
        (
        1
        )
      
    
  
  ⁢
  Ratio
  ⁢
  
    
  
  ⁢
  
    (
    2
    )
  




  2.1
  2.5
 2.9





The amounts of PbMSP1-19 and PbHSP70 proteins are presented in arbitrary units.


The ratio between PbMSP1-19 and PbHSP70 corresponding to the corrected amount of PbMSP1-19 is presented (1).


The ratio between PbMSP1-19 corrected amounts from WT-TmDX1 and SPA-TmDX1 protein extracts are presented in bold (2).


Underlined values correspond to under-estimated ratios due to the saturation of the signal on the film.






Modifications and Other Embodiments
Various modifications and variations of the described products and methods and their methods of use as well as the concept of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not intended to be limited to such specific embodiments. Modifications of the described modes of the invention which would be obvious to those skilled in the microbiological, parasitological, molecular biological, diagnostic, therapeutic, pharmacological and biochemical arts or related fields are intended to be within the scope of the following claims.
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## Claims

1. An isolated or purified polynucleotide that is at least 95% identical to SEQ ID NO: 2, 3 or 4; and that contains a deletion of a polyadenylation site present in SEQ ID NO: 2, 3 or 4.

2. An isolated or purified polynucleotide that is at least 95% identical to SEQ ID NO: 2 and that contains the deletion of a polyadenylation site present in SEQ ID NO: 2.

3. An isolated or purified polynucleotide that is at least 95% identical to SEQ ID NO: 3 and that contains the deletion of a polyadenylation site present in SEQ ID NO: 3.

4. An isolated or purified polynucleotide that is at least 95% identical to SEQ ID NO: 4 and that contains the deletion of a polyadenylation site present in SEQ ID NO: 4.

5. An isolated or purified polynucleotide comprising the polynucleotide of claim 1 and further comprising a polynucleotide encoding a Plasmodium subtilisin-like maturase SUB2.

6. A vector comprising the isolated or purified polynucleotide of claim 1.

7. An isolated host cell comprising the isolated or purified polynucleotide of claim 1.

8. The host cell of claim 7 that is a Plasmodium host cell.