Patent Application
20110305667
The present invention relates to the use of a DEAD-box RNA helicase from a yeast, from a mammal or from Leishmania infantum for inducing cytokine production by a peripheral blood mononuclear cell (PBMC) of a mammal and applications thereof.
The helicases are proteins capable of unwinding duplexes of DNA or of RNA by utilizing the energy released by the hydrolysis of nucleotide triphosphates (NTP) (Caruthers and McKay, 2002; Tanner and Linder, 2001). Two classes of helicases have been identified depending on the specificity of the substrate:
On the basis of comparisons of protein sequences, Gorbalenya and Koonin proposed a general classification of helicases in 5 main families (Gorbalenya and Koonin, 1993). Each family is characterized by a characteristic number and sequence of conserved motifs. All the families possess motifs for binding and hydrolysis of NTPs called walker A and B (Walker et al., 1982). The superfamilies 1 and 2 (SF1 and SF2) are the most represented. They include DNA and RNA helicases of bacteria, of archebacteria, of eubacteria, of eukaryotes and of viruses. They are characterized by 7 to 9 conserved motifs including 5 to 7 motifs in common (see
The DExD/H-box proteins, belonging to the SF2 family, form the largest family of RNA helicases identified to date. They are present in all organisms, from bacteria to humans, as well as in certain viruses (de la Cruz et al., 1999; Rocak and Linder, 2004; Schmid and Linder, 1992). These proteins were identified for the first time when Linder et al. (1989) aligned the sequences of eight proteins that are homologs of the translation initiation factor of the eukaryotes eIF4A. The size of these proteins varies between 400 and 1200 residues. Subsequent studies have demonstrated the existence of nine conserved motifs: Q, I, Ia, Ib, II, III, IV, V and VI, which are contained in a central region, called the helicase core (Cordin et al., 2006; Tanner and Linder, 2001; see
In the course of the last ten years, several structures of helicases of different superfamilies have been characterized. All the structures of the DEAD-box proteins as well as those of the helicases of the SF1 superfamily currently available, reveal a common theme, which consists of two globular domains, connected by a flexible linkage (see
The DEAD-box proteins have, in their central part, nine conserved motifs, arranged identically and spaced with a similar number of amino acids. Mutation analyses combined with structure-function studies have made it possible to attribute roles to them.
The DEAD-box proteins are, like the other helicases, capable of fixing and hydrolyzing a nucleotide triphosphate. In the case of the DEAD-box proteins, only ATP can be fixed and hydrolyzed because of the specific interactions between the Q motif and the adenine base (Tanner et al., 2003; Tanner, 2003). The various biochemical studies conducted on these proteins have shown that their affinity for ATP is moderate, with a Km generally between 80 and 1000 μM (Lorsh and Herschlag, 1998a). Moreover, their kinetic constants are variable from 3-6 min−1 (eIF4A: Lorsh and Herschlag, 1998a; Has1: Rocak et al., 2005) to 600 min−1 (Ded1: Iost et al., 1999; DbpA: Kossen and Uhlenbeck, 1999). The significance of these differences is unclear and it is possible that absence of post-translational modifications, absence of a partner or absence of specific substrates accounts for them. The majority of DEAD-box proteins only hydrolyze ATP in the presence of RNA. This activity is sometimes influenced by the nature (sequence and/or length) of the RNA substrate. In fact, stimulation of the ATPase activity of the DEAD-box proteins of E. coli SrmB, RhlE and CsdA depends on the length of the RNA used as substrate (Bizebard et al., 2004). Most of the DEAD-box proteins do not display specificity of the RNA substrate, in vitro. In fact, specificity for a given RNA seems rather to be afforded by motifs or domains located outside of the helicase core. In the case of the DEAD-box protein of E. coli DbpA, its ATPase activity is stimulated by RNA 23S, more precisely by a fragment of 153 nucleotides containing helix 92 of domain V of this RNA (Fuller-Pace et al., 1993; Tsu and Uhlenbeck, 1998; Tsu et al., 2001). The protein DbpA would interact specifically with helix 92 via its C-terminal domain and nonspecifically with the adjacent region via its helicase core (Tsu et al., 2001; Kossen et al., 2002). The DEAD-box proteins are regarded as RNA helicases, although this activity has only been demonstrated for some of them. Like the DNA helicases, the DEAD-box proteins are capable of unwinding duplexes constituted of at least one strand of RNA either from 5′ to 3′ or from 3′ to 5′, when they are tested in vitro. Most of these DEAD-box proteins require the presence of single-stranded RNA regions either at 5′ or at 3′ of the paired region, probably to be charged there (Rocak and Linder, 2004). The protein eIF4A is capable of unwinding short RNA/RNA or RNA/DNA duplexes; in contrast, it is incapable of unwinding DNA/DNA duplexes. Moreover, it is also capable of unwinding flush-ended RNA duplexes, suggesting that it can interact directly with double-stranded RNA (Rogers et al., 2001a). This property is also observed for the DEAD-box protein of E. coli RhlE (Bizebard et al., 2004). Certain DEAD-box proteins, such as eIF4A, p68 and RhlE are said to be bidirectional, because, in vitro, they are capable of unwinding indiscriminately duplexes bearing a single-stranded end at 3′ or at 5′ (Bizebard et al., 2004; Huang and Liu, 2002; Rogers et al., 2001a). The DEAD-box proteins, like the majority of helicases, are not very processive. Only proteins p68 (162 bp duplex, Hirling et al., 1989), p72 (36 to 46 bp, Rossler et al., 2001) and RhlE (Bizebard et al., 2004) display moderately processive activity. The helicase activity depends on the stability of the duplex used. The stability of the duplexes frequently used is ΔG° between −15 and −30 kcal/mol and the length varies between 10 and 25 bp. Rogers et al., using duplexes of variable length and stability, showed that the protein eIF4A is capable of unwinding several base pairs (Rogers et al., 2001a). Recently, Bizebard et al. showed that the length of the single-stranded region, the minimum size of which varies depending on the proteins, is important for helicase activity (Bizebard et al., 2004).
Certain helicase proteins display, in vitro, pairing activity of homologous sequences. Thus, as well as their helicase activity, the two DEAD-box proteins p68 and p72 are capable, in vitro, of catalyzing the pairing of complementary strands, when they are present in excess relative to the substrate (Rossler et al., 2001). By combining these two activities, they can catalyze, in vitro, rearrangements of secondary structures of RNA, which are moreover too stable to be eliminated by their helicase activity alone (Rossler et al., 2001). Flores-Rozas and Hurwitz showed that in the presence of nonhydrolyzable analogs of ATP, the DEAD-box protein II/Gu catalyzes an intramolecular pairing reaction. The concentration of ATP plays a crucial role in this reaction. At low concentration, the RNA helicase II/Gu promotes intramolecular duplex formation, whereas at high concentrations of ATP, the enzyme acts like RNA helicase and eliminates the secondary structures (Flores-Rozas and Hurwitz, 1993). This pairing activity resides outside of the helicase core in the C-terminal domain, rich in Gly-Arg (Valdez et al., 1997). This might also be the case with the DEAD-box proteins p68 and p72, which possess N-terminal and C-terminal domains rich in glycine and arginine (Lamm et al., 1996). The pairing and RNA-helicase activities are separated physically and functionally. Mutations in motifs II and III of the DEAD-box protein II/Gu cancel the RNA-helicase activity without altering the pairing activity. Moreover, deletion of the pairing domain does not inhibit the helicase activity of the enzyme (Valdez et al., 1997; Valdez, 2000). Protein eIF4B, which stimulates the helicase and ATPase activities of protein eIF4A (Rogers et al., 2001b), also possesses pairing activity (Altmann et al., 1995), proving once again that the two activities are physically separate.
Various models have been proposed for the helicase activity (Tanner and Linder, 2001; Caruthers and McKay, 2002; Singleton and Wigley, 2002). The two models most discussed are: “inchworm” and “active rolling”.
The DEAD-box proteins are also involved in all processes of RNA metabolism (transcription, maturation, transport, translation, biogenesis of ribosomes, RNA interference, stability and degradation of RNAs).
In the course of transcription, several helicases become associated with the transcripts. However, they only seem to play an active role during the late stages of splicing or transport (Linder and Stutz, 2002; Silverman et al., 2003). Recent studies have shown that certain DEAD-box proteins participate in the regulation of transcription by interacting with other transcription factors (Gillian and Svaren, 2004; Yan et al., 2003). However, the usefulness of ATPase and helicase activities in the transcription process is not clearly established. However, Wilson et al. showed that the helicase cores of the DEAD-box proteins p68 and p72 suppress transcription whereas their C-terminal region stimulates transcription (Wilson et al., 2004). Recently, Bates et al. (2005) showed that the DEAD-box protein p68 activates the transcription of p53 following a DNA aberration (Bates et al., 2005).
Splicing is a process that takes place in several stages involving two reactions of transesterification and structural rearrangements in the stage of assembly of the spliceosome which requires the energy derived from hydrolysis of NTP. The precise role of RNA helicases in assembly of the spliceosome is not known, but it is generally assumed that they are involved in unwinding the small duplexes formed between snRNA and pre-mRNA, which is the case with the DExH-box protein, Prp 22. A mutant in motif III of this protein has a phenotype that is lethal at temperatures below 30° C. and a slowing of growth at 34° C. and 37° C. In vitro, the mutant protein is capable of hydrolyzing ATP, but is incapable of unwinding RNA duplexes and liberating mRNA from the spliceosome, suggesting that the loss of helicase activity is the cause of the lethal phenotype. A second mutation in motif Ib, which restores the helicase activity and the dissociation of mRNA from the spliceosome, behaves as an intragenic suppressor, suggesting that helicase activity is necessary in this stage (Schwer and Meszaros, 2000). The majority of DEAD-box proteins that are implicated in the process of mRNA splicing are involved in the early stages of assembly of the spliceosome, which is the case for the DEAD-box protein Prp5. Recently, it was reported that this protein is capable of binding to snRNA U1 and U2 (Xu et al., 2004). The DEAD-box protein Prp5 uses energy derived from the hydrolysis of ATP to rearrange the local RNA-RNA or RNA-protein interactions, which permit the snRNA U2 complex to rejoin the snRNA U1 complex. The DEAD-box protein Prp28 uses the energy released by hydrolysis of ATP to destabilize the snRNA U1 complex and replace it with the snRNA U6 complex at the splicing site (Staley and Gutherie, 1999). The DEAD-box protein p68 is involved in dissociation of the snRNA U1 complex from site 5′, necessary for splicing according to an ATP dependent mechanism.
A large proportion of DEAD-box RNA helicases participate in the biogenesis of ribosomes, in which their helicase or RNPase activity might provide fine regulation of the organization of the multiple RNA-RNA or RNA-protein interactions transiently established in the course of biogenesis and maturation of ribosomal RNAs (Luking et al., 1998; Kressler et al., 1999; Rocak and Linder, 2004). Only the DEAD-box protein Has1 is involved simultaneously in the biogenesis of the 40S and 60S subunits (Emry et al., 2004). Mutations in motif I of the protein Has1, as well as its depletion, affect the biosynthesis of rRNA 18S of the 40S subunits, by slowing or inhibiting cleavage at sites A0, A1 and A2.
The transport of mRNA from the nucleus to the cytoplasm through the nuclear pores involves specific proteins which bind to the nascent mRNA. The RNA helicases that are involved in this process could remodel the mRNP complexes before or during passage through the nuclear pores (Silverman et al., 2003). They could also be involved in dissociation of the nuclear factors of the mRNA to permit export of the latter, or preparation of the messenger in the first cycle of translation (Rocak and Linder, 2004). The DEAD-box protein Dbp5 participates in this process. Double hybrid and co-immunoprecipitation experiments have identified several partners, including Gle1, a factor for exporting the RNA associated with the nuclear pore, and GFd1/ymr255, a factor that interacts both with Dbp5 and GLe1 (Tseng et al., 1998; Hodge et al., 1999; Schmitt et al., 1999). The yeast and human Dbp5 proteins also interact with Nup159, a compound of the nuclear pore complex (Zhao et al., 2002). In yeast, the ATPase activity of the DEAD-box protein Sub2 is necessary for its detachment from the mRNA and its replacement with the mRNA transport factor Mex 67 (Strasser and Hurt, 2001).
Nonsense mediated decay (NMD) is a surveillance mechanism that breaks down mRNAs containing a premature termination codon (PTC). Several proteins which form part of the exon junction complex are involved in this mechanism (Reed and Hurt, 2002). Recently, Ferraiuolo et al. (2004) showed that the DEAD-box protein eIF4AIII belongs to the exon junction complex (EJC) and that it binds to RNA during splicing (Chan et al., 2004; Ferraiuolo et al., 2004; Shibuya et al., 2004). The protein eIF4AIII interacts with eIF4G and eIF4B but, in contrast to eIF4AI and eIF4AII, it inhibits translation (Li et al., 1999). Its role in the nucleus has not yet been established but certain authors suggest a role in RNA transport (Palacios et al., 2004) and in nonsense mediated decay (Ferraiuolo et al., 2004; Palacios et al., 2004; Shibuya et al., 2004).
The DEAD-box protein Ded1 is also involved in initiation of translation, probably independently of eIF4A (Chuang et al., 1997; de la Cruz et al., 1997; Iost et al., 1999; Linder, 2003). The precise role of Ded1 in the process of initiation of translation is not yet known, but it might be involved in the process of displacement of the small subunit 40S up to the initiation codon for eliminating secondary structures on the mRNA or random pairings between mRNA and tRNA, or imperfect codon/anticodon interactions (Linder, 2003). A mutation in the helicase core of the protein Ded1 selectively inhibits the translation of polymerase 2A, thus suppressing replication of the RNA2 genome of the brome mosaic virus whereas it assures general translation of the cell. This suggests that the protein Ded1 might have regulatory functions separate from its general role in translation or that the mutant protein has a helicase activity that is weak, but nevertheless sufficient to assure cellular translation but not translation of RNA polymerase 2A (Noueiry et al., 2000).
The use of immunotherapeutic approaches aiming to improve the immune responses of the host to developing tumors has been demonstrated. They include immunomodulating cytokines such as TNF-alpha, the INFs of type I and of type II, IL-2, IL-12, IL-15 and IL-18, which are among the most potent inducers of antitumor activity according to preclinical studies. IL-12 in particular is receiving particular attention because of its central role in the regulation of innate and adaptive immune responses. By itself, it can induce powerful anticancer effects, but can also act in synergy with other cytokines to increase its immunoregulatory and antitumor activities (Weiss et al., 2007). Among the approaches currently being considered, we may mention the use of recombinant IL-12 in experimental models or in clinical trials (Shiratori et al., 2007; Lenzi et al., 2007); the transduction of dendritic cells by a recombinant adenovirus expressing IL-12 and their use as vaccine (He et al., 2008); or even the use of microspheres loaded with DNA coding for IL-12 (Son and Kim, 2007) or with synergistic combinations of cytokines IL-12 and TNF-alpha (Sabel et al., 2007).
The Leishmania are flagellated protozoa, belonging to the order Kinetoplastidae and to the family Trypanosomatidae. They are responsible for leishmaniases.
The protein LeIF was identified by screening a genomic DNA database of promastigotes of Leishmania braziliensis with serum from patients with mucocutaneous leishmaniasis due to L. braziliensis (Skeiky et al., 1995). Sequence comparison with those present in the databases enabled it to be identified as being homologous to the translation initiation factor eIF4A (Skeiky et al., 1995). It is a cytoplasmic protein of 403 amino acids, with molecular weight of 45.3 kDa, whose transcripts are detected both at the promastigote stage and in the amastigote (Skeiky et al., 1998; Salay et al., 2007) and whose gene is present in duplicate in the Leishmania genome (Myler et al., 1999). Proteomic studies have shown that the protein LeIF is overexpressed in the amastigote and promastigote stage (Bente et al., 2003; Nugent et al., 2004); it is highly conserved between the different species of the genus Leishmania (Barhoumi et al., 2006; Skeiky et al., 1998). It has the motifs characteristic of the DEAD-box proteins (Barhoumi et al., 2006). However, in experiments on yeast, it was shown that the protein LeIF of Leishmania does not make up for loss of the protein eIF4A in yeast but interacts with the translation initiation factors (eIF4G) unproductively, generating a negative dominant phenotype (Barhoumi et al., 2006).
The protein LeIF of the species L. braziliensis has the capacity to induce the production of IFN-γ and of TNF-α by PBMCs (peripheral blood mononuclear cells) of patients with cutaneous leishmaniasis, mucosal leishmaniasis or diffuse cutaneous leishmaniasis, and of IL-12 both by PBMCs of patients and of uninfected individuals (Skeiky et al., 1995). This protein is also capable of inducing the secretion of cytokines IL-12, IL-10 and TNF-α by the antigen-presenting cells: macrophages and dendritic cells obtained from normal individuals (Probst et al., 1997). The cytokine inducing activity of the protein LeIF seems to be localized in the N-terminal region (1-226) (Probst et al., 1997). These results were confirmed by the same authors in the murine model. Thus, Skeiky et al. (1998) showed that the recombinant protein LeIF of the species Leishmania major is capable of stimulating the secretion of IFN-γ by the splenic cells of BALB/c mice infected with L. major, of promoting the development of TH1 clones in BALB/c mice immunized with this protein in the absence of adjuvant and finally of stimulating the production of IFN-γ by splenocytes of SCID mice, by an IL-12-dependent mechanism. They also showed that this activity was maintained with the N-terminal part of the protein (1-226) and lost with the C-terminal part (196-403). The protein LeIF is one of the constituents of a second-generation vaccine against Leishmania composed of three fused proteins of Leishmania, which was described as being immunogenic and conferring a significant degree of protection in the mouse immunized with Leishmania major or L. infantum (Coler et al., 2002; 2007), but which does not seem to be protective in mice immunized with L. braziliensis (Salay et al., 2007). This vaccine is still undergoing clinical trials in humans. The protein LeIF has also been used in immunotherapy in a patient with mucocutaneous leishmaniasis (Badaro et al., 2001). It is also capable of increasing the expression of molecules B7-1 and CD54 (ICAM-1) (molecules of co-stimulation involved in interaction between T cells and antigen presenting cells) on the surface of macrophages and of dendritic cells from healthy donors, tested in vitro (Probst et al., 1997).
The present invention set itself the goal of providing novel molecules capable of modulating the production of cytokines by the host cells of mammals, for treating or preventing infectious diseases or notably cancers.
Thus, the inventors used monocytes derived from PBMCs of healthy donors primed or not with IFN-γ for testing the capacity of recombinant proteins to induce the production of the cytokines IL-12 (in particular IL-12p70), IL-10 and TNF-α, so as to demonstrate their immunomodulating properties.
Five DEAD-box recombinant proteins possessing ATPase and RNA helicase biochemical activities, present in yeast or in mammals, were tested for their immunomodulating properties:
Surprisingly, the inventors showed that the DEAD-box proteins and notably hueIF4A, eIF4AIII, yeIF4A, FAL1 and Ded1, even though they each display less than 57% homology with the protein LeIF of L. infantum, also possess immunomodulating activity and are capable of inducing the secretion of the cytokines IL-12, IL-10 and TNF-alpha.
The inventors also tested fragments of the protein LeIF of L. infantum, 26-403, 1-226, 1-237, 1-195, 25-237, 129-226, 129-261, 196-403 and 237-403, as well as the mutant protein LeIFK76A having a substitution of the lysine in position 76 (in the consensus motif I of the protein LeIF of L. infantum) with an alanine, for their immunomodulating properties.
The inventors showed that all of the fragments of LeIF of L. infantum tested induce the production of IL-12p70 by monocytes from healthy individuals in vitro, and that the mutation, in motif I, of the lysine in position 76 to alanine, which cancels the ATPase activity of the protein LeIF, does not affect its activity for inducing the cytokines IL-12p70, IL-10 and TNF-α.
The present invention therefore relates to the use of a yeast or mammalian DEAD-box RNA helicase comprising in its central part, arranged from (1) to (9) from the N-terminal end to the C-terminal end, the nine motifs of amino acids defined by the following consensus motifs: (1) motif Q (SEQ ID NO: 1), (2) motif I (SEQ ID NO: 2), (3) (SEQ ID NO: 3), (4) motif Ib (SEQ ID NO: 4), (5) motif II (SEQ ID NO: 5), (6) motif III, (SEQ ID NO: 6), (7) motif IV (SEQ ID NO: 7), (8) motif V (SEQ ID NO: 8) and (9) motif VI (SEQ ID NO: 9), of wild-type or having a mutation in consensus motif I (SEQ ID NO: 2), or of a fragment of the latter comprising either the six consensus motifs Q (SEQ ID NO: 1), I (SEQ ID NO: 2), Ia (SEQ ID NO: 3), Ib (SEQ ID NO: 4), II (SEQ ID NO: 5) and III (SEQ ID NO: 6) but not the three consensus motifs IV (SEQ ID NO: 7), V (SEQ ID NO: 8) and VI (SEQ ID NO: 9), or the three consensus motifs IV, V and VI (defined previously) but not the six consensus motifs Q, I, Ia, Ib, II and III defined previously, for inducing the production in vitro or ex vivo of at least one cytokine by a peripheral blood mononuclear cell (PBMC), preferably a monocyte, of a mammal.
“DEAD-box RNA helicase” means a protein possessing RNA helicase activity and comprising in its central part, arranged from the N-terminal end to the C-terminal end from (1) to (9), nine motifs of amino acids defined by the following consensus motifs:
(1) motif Q, of amino acid sequence G-a-x-c-P-o-h-i-Q (SEQ ID NO: 1), where “a” represents F, W or Y, “x” represents any amino acid, “c” represents D, E, H, K or R, “o” represents S or T, “h” represents A, F, G, I, L, M, P, V, W or Y, and “i” represents I, L or V;
(2) motif I, of amino acid sequence A-x-o-G-o-G-K-T (SEQ ID NO: 2), where “x” represents any amino acid, and “o” represents S or T;
(3) motif Ia, of amino acid sequence P-T-R-E-L-A (SEQ ID NO: 3);
(4) motif Ib, of amino acid sequence T-P-G-R-i (SEQ ID NO: 4), where “i” represents I, L or V;
(5) motif II, of amino acid sequence D-E-A-D (SEQ ID NO: 5);
(6) motif III, of amino acid sequence S-A-T (SEQ ID NO: 6);
(7) motif IV, of amino acid sequence i-i-F-C/h-x-T-x-b-c (SEQ ID NO: 7), where “i” represents independently I, L or V, “h” represents A, F, G, I, L, M, P, V, W or Y, “x” represents independently any amino acid, “b” represents H, K or R, and “c” represents D, E, H, K or R;
(8) motif V, of amino acid sequence T-D/N-x-x-A-R-G-i-D (SEQ ID NO: 8), where “x” represents independently any amino acid, and “i” represents I, L or V; and
(9) motif VI, of amino acid sequence H-R-i-G-R-z-G-R (SEQ ID NO: 9), where “i” represents independently I, L or V and where “z” represents T, G or S,
said motifs being spaced apart by a number of amino acids defined according to the motifs in question.
The RNA helicase activity of a protein can be determined by any method known by a person skilled in the art. As nonlimiting examples, it is possible to use the methods described by Rogers et al., 1999 and Cordin et al., 2004.
According to an advantageous embodiment, said RNA helicase possesses ATPase activity. The ATPase activity of a protein can be determined by any method known by a person skilled in the art. As nonlimiting examples, it is possible to use the methods described by Cordin et al., 2004; Rocak et al., 2005 or Tanner et al., 2003.
The DEAD-box RNA helicase can be:
According to another advantageous embodiment, said RNA helicase has a mutation at the lysine in position 7 of the above motif I. Preferably, the mutation is a substitution of said lysine with any other amino acid, preferably with alanine.
According to a preferred embodiment of the present invention, said yeast belongs to the genus Saccharomyces, and preferably to the species Saccharomyces cerevisiae.
According to another preferred embodiment of the present invention, said mammalian DEAD-box RNA helicase is a human DEAD-box RNA helicase.
Advantageously, the DEAD-box RNA helicase of yeast is selected from the proteins yeIF4A (SEQ ID NO: 12), FAL1 (SEQ ID NO: 13) and Ded1 (SEQ ID NO: 14), and the mammalian DEAD-box RNA helicase is selected from the proteins hueIF4A (SEQ ID NO: 15) and eIF4AIII (SEQ ID NO: 16).
Other yeast or human DEAD-box proteins can also be employed; examples of such proteins are mentioned above. We may notably mention the proteins P68 (mammalian DEAD-box protein; Iggo R D and Lane D P, EMBO J., 1989) and Has1 (yeast DEAD-box protein; Rocack et al., N.A.R., 2005).
According to another advantageous embodiment of the present invention, said cytokine is selected from the group comprising IL-12 (in particular IL-12p70), TNF-alpha and IL-10.
According to a particular embodiment of the present invention, said peripheral blood mononuclear cell is activated beforehand by interferon gamma (IFN-γ) to induce production of IL-12.
As a nonlimiting example, IL-12p70 can be produced by monocytes pre-activated with IFN-γ, preferably for 12 hours, then stimulated with the yeast or mammalian DEAD-box RNA helicase as defined above, preferably for 24 hours.
The present invention also relates to the use of a yeast or mammalian DEAD-box RNA helicase of the wild type or having a mutation in consensus motif I or of a fragment of the latter, as defined above, for preparing a medicinal product intended for treating or preventing infectious diseases or cancers.
Said medicinal product is notably useful as a vaccination adjuvant for inducing an immune response of the Th1 type.
Said immune response of the Th1 type preferably consists of production of at least one of the cytokines IL-12 and TNF-alpha.
More particularly, the infectious diseases are parasitic infections, preferably infections induced by an intracellular parasite and more preferably leishmaniases.
The present invention also relates to a pharmaceutical composition comprising a yeast or mammalian DEAD-box RNA helicase of the wild type or having a mutation in consensus motif I, or of a fragment of the latter, as defined above, and at least one pharmaceutically acceptable vehicle.
The pharmaceutically acceptable vehicles are those used conventionally and are selected depending on the method of administration used for the treatment envisaged.
The present invention also relates to products containing (i) a yeast or mammalian DEAD-box RNA helicase of the wild type or having a mutation in consensus motif I, or of a fragment of the latter, as defined above and (ii) IFN-γ, for simultaneous, separate or sequential use in the treatment or prevention of cancer and of infectious diseases, preferably parasitic infections, and more preferably leishmaniases.
The present invention also relates to a fragment of the protein LeIF of Leishmania infantum of sequence SEQ ID NO: 11, selected from:
The present invention also relates to a fragment of the protein LeIF of Leishmania infantum as defined above, preferably the fragments of sequence SEQ ID NO: 17, 19, 20, 21 and 26, or of the protein LeIF of Leishmania infantum of the wild type (SEQ ID NO: 11) or mutated in the consensus motif I for use as a medicinal product.
According to another advantageous embodiment, the protein LeIF of L. infantum has a mutation at the lysine in position 7 of the above motif I, corresponding to the lysine residue in position 76 of the protein. Preferably, the mutation is a substitution of said lysine with any other amino acid, preferably with alanine.
The protein LeIF of mutant Leishmania infantum having a substitution of the lysine in position 7 of motif I defined above with alanine is designated LeIFK76A. Its amino acid sequence is shown in the sequence SEQ ID NO: 27.
According to another advantageous embodiment of this use, the medicinal product is intended for treating or preventing infectious diseases or cancers.
The medicinal product can moreover be used as a vaccination adjuvant for inducing an immune response of the Th1 type, which preferably consists of production of at least one of the cytokines IL-12 (in particular IL-12p70) and TNF-alpha.
The present invention also relates to products containing (i) a fragment of the protein LeIF of Leishmania infantum as defined above, preferably the fragments of sequence SEQ ID NO: 17, 19, 20, 21 and 26, or the protein LeIF of Leishmania infantum as defined above and (ii) IFN-γ, for simultaneous, separate or sequential use in the treatment or prevention of cancer and of infectious diseases, preferably parasitic infections, more preferably infections induced by an intracellular parasite and more preferably leishmaniases.
The present invention also relates to a fragment of the protein LeIF of Leishmania infantum as defined above, preferably the fragments of sequence SEQ ID NO: 17, 19, 20, 21 and 26, or the protein LeIF of Leishmania infantum as defined above for inducing the production in vitro or ex vivo of IL-12 by a peripheral blood mononuclear cell (PBMC), preferably a monocyte, of a mammal, preferably a human.
The present invention also relates to a method of modulating the production of cytokines, characterized in that it comprises stimulation of the production of cytokines IL-12, TNF-α and IL-10, by cells of a host and notably of monocytes, said method comprising the simultaneous, separate or sequential administration of the DEAD-box proteins or their fragments, as defined above, and of IFN-γ.
In addition to the foregoing embodiments, the invention further comprises other embodiments, which will become clear from the following description, which refers to examples illustrating the immunomodulating properties of yeast or mammalian DEAD-box RNA helicases, or of fragments of the protein LeIF of L. infantum, as well as the appended drawings, in which:
I. Material
I.1. Donors
Human monocytes were isolated from blood donations (Tunis Blood Transfusion Center), obtained on anticoagulant (citrate-phosphate-dextrose CPD) from healthy adult volunteers.
I.2. Recombinant Proteins
II. Methods
II.1.1 Isolation of Peripheral Blood Mononuclear Cells (PBMC)
The blood, obtained from healthy donors, on CPD, is centrifuged at 1800 rev/min for 20 min. After removal of the plasma, the blood is diluted in RPMI 1640 medium supplemented with 100 U/ml of penicillin, 100 μg/ml of streptomycin and 2 mM of L-glutamine (RPMI/PS/GLU). The plasma is decomplemented at 56° C. for 30 min, then clarified by centrifugation at 3000 rev/min for 10 min at room temperature. The mononuclear cells (PBMCs) are isolated on a density gradient constituted of Ficoll hypaque (Amersham). The blood is deposited on the Ficoll, then centrifuged at 1600 rev/min for 20 min at room temperature. The red blood cells and the polynuclear cells pass through the Ficoll and are found in the pellet, whereas the PBMCs remain at the interface of the Ficoll. The ring of PBMC is taken, diluted in RPMI/PS/Glu and washed three times by centrifugation at 1600 rev/min for 10 min at room temperature. The cells are counted on a Malassez plate.
II.1.2. Purification of the Monocytes from the PBMCs
Sterile culture flasks are treated with a 2% gelatin solution by incubation for 2 h at 37° C., then the excess gelatin is aspirated and the flasks are placed in a stove at 56° C. overnight. These flasks can be stored at room temperature for some days until they are used.
The decomplemented autologous plasma (AP) is deposited in gelatinized culture flasks. This plasma is rich in fibronectin, a protein that attaches to the gelatin and permits adherence of the monocytes via the fibronectin receptor CD49 expressed on the surface of these cells. After removing the plasma from this suspension, the PBMCs at 2.106/ml are resuspended in RPMI/PS/Glu supplemented with 5% autologous plasma, and are then put back in the flasks. These cultures are incubated for 1 hour at 37° C. in the presence of 5% CO2. The nonadherent cells are removed by washing gently three times, and the adherent cells are detached after incubation for 15 min at 37° C. with a buffer containing 50% of a solution of PBS 10 mM EDTA and 50% of RPMI/10% FCS or AP. The detached cells are washed to remove all traces of EDTA. A fraction of the cells purified in this way is taken for determining the purity of the monocyte preparation, by flow cytometry.
II.1.3. Determination of the Purity of the Monocytes by FACS
106 cells are recovered from the monocyte preparation by adherence, washed and resuspended in PBS 1% BSA, 0.01% NaN3 then distributed at a rate of 2.105 cells/tube in hemolysis tubes. Antibodies (Becton Dickinson) labeled with fluorescein (FITC) or with phycoerythrin (PE) and specific for the surface markers CD14 (present on the majority of monocytes), CD19 (membrane marker of B lymphocytes), and CD3 (present on T lymphocytes) are added at a rate of 5 μl/tube and incubated for 30 min at 4° C., protected from the light. The nonspecific fluorescence of each antibody, which can be effected through the Fc of the IgGs, is monitored by incubation of the cells with an antibody of the same isotype as the specific antibody, used for identifying the various cellular populations, and labeled with the same fluorochrome as this antibody. The cellular suspensions are then washed twice, the pellets are resuspended in a fixing solution (PBS-paraformaldehyde (PFA) 0.3%) and then analyzed by FACS.
II.2. Induction of Secretion of Cytokines and Assay by Sandwich ELISA
II.2.1. Stimulation in Vitro
The human monocytes are resuspended at a rate of 106 cells/ml in RPMI/PS/Glu 3% AP and distributed in 24-well culture plates (Nunc), at a rate of 5×105 monocytes/well (106 cells/ml). The cells in culture are stimulated either with LPS (1 μg/ml) or with the various recombinant proteins (10 μg/ml), for assay of IL-10 and of TNF-α. After incubation at 37° C. for 18 hours in the presence of 5% CO2, the cells are centrifuged at 3000 rpm for 10 min and the culture supernatants are recovered and stored at −80° C. until they are used.
For assay of IL-12, the monocytes are primed with IFN-γ (3000 U/ml) for 12 hours before adding LPS (1 μg/ml) or the various recombinant proteins (10 μg/ml). After incubation for 24 h, the supernatants are collected and stored at −80° C. until they are used.
To verify absence of contaminants of bacterial origin (LPS) in the preparations of recombinant proteins, control cultures were added in the same experimental conditions. For this, LPS (1 μg/ml) and the various recombinant proteins (10 μg/ml) are incubated in the presence of proteinase K, at a final concentration of 100 μg/ml, at 42° C. for 30 min. Afterwards, the enzyme is inactivated at 75° C. for 10 min, then these preparations are used for stimulating the monocytes. Polymixin B, which has the effect of blocking the action of LPS, is added directly to the cells, to a final concentration of 10 μg/ml.
II.2.2. Assay of Cytokines by ELISA (Enzyme Linked Immuno Sorbent Assay)
The levels of cytokines secreted (IL-10, TNF-α and IL-12) in the culture supernatants are quantified by ELISA of the sandwich type. A first antibody or capture antibody, adsorbed on the plastic of a microtitration plate (Maxisorp Nunc), binds specifically to the cytokine to be assayed. A second antibody or detection antibody coupled to an enzyme becomes fixed to the capture antibody/cytokine complexes. This fixation is detected by an enzymatic reaction, which transforms a chromogenic substrate to a colored product. The levels of cytokines secreted in the culture supernatants are determined from a standard range recorded with known concentrations of a standard cytokine.
The ELISA test is performed according to the following stages:
II.2.3. Statistical Analyses
The results were analyzed using the statistical analysis software S plus (version 6.2) by applying the paired t-test. This test compares the difference of the means with zero. It is calculated according to the following equation
where
d: the mean of the difference
S2: the variance of the samples
n: sample size
t: Student t.
III. Results
III.1. Evaluation of the Purity of the Monocytes
Human monocytes were isolated from the PBMCs of healthy individuals by adherence on a gelatin support treated with autologous plasma. A fraction (106) of the cells recovered was analyzed by flow cytometry, to estimate their purity and their viability. For this, the cells were incubated with antibodies coupled to a fluorochrome and that recognize the surface antigens: CD14 (marker of monocytes), CD3 (marker of T cells) or CD19 (marker of B cells). The amount of specific fluorescence emitted by the labeled cells is correlated with the expression of these markers.
Immunofluorescent labeling made it possible to estimate the percentage of the monocytes and that of the contaminating, T and B, lymphocytes.
III.2. The Yeast Proteins yeIF4A, FAL1 and Ded1 and the Mammalian Proteins hueIF4A and eIF4AIII Induce the Secretion of Cytokines IL-12p70, IL-10 and TNF-α by Human Monocytes in Vitro.
The monocytes purified from the PBMCs from 5 healthy donors were activated, or not, by IFN-γ then stimulated by the recombinant proteins LeIF (control), yeIF4A, FAL1, hueIF4A, eIF4AIII and Ded1 to final concentrations of 10 μg/ml.
The results presented in
The proteins yeIF4A, FAL1, hueIF4A and eIF4AIII induce levels of IL-12p70 (1391.3±266.98; 2338.37±738.34; 1820.28±681.35 and 2790±1467.5 pg/ml, respectively) comparable to those induced by the soluble or insoluble protein LeIF (3805.9±1069.1 and 3921.6±1094 pg/ml; p>0.05, cf. Table 1). However, the protein Ded1 induces the secretion of levels of IL-12p70 (497.6±79.19 pg/ml) that are significantly lower than those induced by soluble or insoluble protein LeIF (p<0.05, Table 1).
Table 1 below shows statistical analysis of the differences observed for the levels of production of IL-12p70 in the culture supernatants of the monocytes stimulated with the yeast proteins yeIF4A, FAL1 and Ded1 and mammalian eIF4AI and eIF4AIII. The p values determined by the paired t test are shown.
The results presented in
The proteins FAL1, hueIF4A, eIF4AIII and Ded1 induce the production of levels of IL-10 and of TNF-α comparable to those induced by the soluble or insoluble protein LeIF (p<0.05). However, the yeast protein yeIF4A induces the production of levels of IL-10 and of TNF-α significantly lower than those induced by the soluble or insoluble protein LeIF (p<0.05, Table 2).
Table 2 below shows the statistical analysis of the differences observed in the levels of IL-10 and of TNF-α in the culture supernatants of the monocytes stimulated with the recombinant proteins yeIF4A, FAL1, hueIF4A, eIF4AIII and Ded1. The p values determined by the paired t test are shown.
All these results show that the homologous proteins of the protein LeIF: yeast yeIF4A, FAL1 and Ded1 and mammalian hueIF4A and eIF4AIII, are also capable of inducing the production of cytokines IL-12p70, IL-10 and TNF-α by human monocytes in vitro.
III.3. The Activity Inducing the Production of Cytokines by the Various Recombinant Proteins is Proteinase K Sensitive and Polymixin B Resistant.
To verify absence of contaminants of bacterial origin (LPS) in the preparations of recombinant proteins used, the levels of IL-10 produced in cultures of monocytes stimulated by the recombinant protein or by LPS in the presence or absence of proteinase K (endopeptidase that degrades the proteins) or of polymixin B (which has the effect of blocking the action of LPS) were compared. The results presented in
These results show that the various antigenic preparations used are not contaminated with bacterial endotoxins.
With the aim of identifying the minimum region responsible for the induction of cytokines within the protein LeIF, several fragments of this protein were produced. On the basis that the regions NH2 of LeIF of L. braziliensis and L. major have been described as being responsible for the immunomodulating activity of these proteins, whereas a loss of activity was observed for the central regions and COOH (Probst et al., 1997; Skeiky et al., 1998), the NH2 (1-226; SEQ ID NO: 18), central (129-261; SEQ ID NO: 23) and COOH (196-403; SEQ ID NO: 24) parts of the protein LeIF of L. infantum were constructed. Two other fragments within region NH2 were also constructed: 1-195 (SEQ ID NO: 17) and 129-226 (SEQ ID NO: 22).
Isolation of mononuclear cells (PBMC), induction of secretion of cytokines and assay by sandwich ELISA were carried out in the same conditions as those described in Example 1. Briefly, monocytes purified from PBMCs of 6 healthy individuals were primed for 12 h at 37° C. by IFN-γ, then stimulated, either by the various proteins or fragments at final concentrations of 10 μg/ml or by LPS at 1 μg/ml, for 24 h for production of IL-12p70. For production of IL-10 and TNF-α, the monocytes were stimulated for 18 h with the various recombinant proteins or with LPS. The levels of IL-12, IL-10 and TNF-α are determined by sandwich ELISA.
1. Analysis of IL-12p70 Inducing Capacity
In contrast to the two fragments 129-261 and 129-226, the levels of IL-12p70 induced by stimulation with the fragments NH2 (1-226) (3313.43±1375.7 pg/ml); (1-195) (3532±1485.8 pg/ml) and COOH (196-403) (4764.2±1935.5 pg/ml) are significantly higher than those produced by the monocytes that were not stimulated (24.78±10.76 pg/ml, p<0.05, Table 3) or stimulated with the recombinant proteins NH2 (1-226), 1-195 or COOH (196-403) alone (202.91±94.55, 205.93±89.43 and 267.19±101.87 pg/ml, respectively; p<0.05) or IFN-γ alone (21.56±11.64 pg/ml) (p<0.05, cf. Table 3).
The levels of IL-12p70 induced in the culture supernatants of monocytes activated by IFN-γ and stimulated by the whole protein LeIF (5375.63±1683 pg/ml) are significantly higher than those induced by all the fragments (p<0.05, cf. Table 3) and comparable to those induced by LPS (2678.3±783.51 pg/ml, p>0.05, cf. Table 3).
In contrast to what was reported in the literature (Probst et al., 1997), the COOH part (195-403) of the protein LeIF of the species L. infantum induces the production of IL-12p70 (4764.2±1935.5 pg/ml) by monocytes activated by IFN-γ. These levels of IL-12p70 are comparable to those induced by the NH2 part (1-226) (3313.43±1375.7 pg/ml, p>0.05, Table 3) or the 1-195 fragment (3532±1485.8 pg/ml, p>0.05, Table 3). Since the 129-261 fragment induces low levels of IL-12p70, the inducing activity within the COOH part (196-403) might be localized in the minimum region corresponding to positions 261 to 403.
Within the NH2 region, it was shown that, in contrast to the results obtained with the 129-226 fragment, the 1-195 fragment induces significant levels of IL-12p70, which suggests that within this NH2 region the inducing activity is localized at the level of the 1-129 fragment.
2. Analysis of IL-10 Inducing Capacity
The results illustrated in
The NH2 (1-226) and 1-195 fragments induce the secretion of levels of IL-10 that are significantly higher than those induced by stimulation by LPS (1361.43±261.9 pg/ml for NH2 and 1331.17±228.35 pg/ml for 1-195; p<0.05, cf. Table 4) and by fragments 129-261 (960.26±229.99 pg/ml, p<0.05, Table 10) and 129-226 (631.09±134.59 pg/ml, p<0.05, cf. Table 4).
However, the whole protein LeIF is still more effective than all the fragments analyzed as well as LPS in induction of the production of IL-10 (2329.94±322.02 pg/ml for LeIF and 664.8±106.71 pg/ml for LPS; p=0.0032, cf. Table 4).
Table 4 below shows statistical analysis of the differences observed for the levels of production of IL-10 in the culture supernatants of the monocytes stimulated with the various recombinant proteins. The p values determined by the paired t test are shown.
3. Analysis of TNF-α Inducing Capacity
TNF-α was assayed in the culture supernatants of monocytes purified from PBMCs from 6 healthy donors.
The fragments COOH (195-403), NH2 (1-226) and 1-195 induce the secretion of levels of TNF-α comparable to those induced by the protein LeIF (p>0.05, Table 5). Once again, the 129-261 and 129-226 fragments induce the lowest levels of TNF-α (5341.45±1281.1 and 5543.69±1185.7 pg/ml, respectively).
Table 5 shows statistical analysis of the differences observed in the levels of TNF-α in the culture supernatants of the monocytes stimulated with the soluble protein LeIF and its various domains. The p values determined by the paired t test are shown.
The effect of substitution in motif I, of the lysine in position 76 with alanine, on the cytokine inducing capacity of the protein LeIF was evaluated. This mutation cancels the ATPase activity of the protein LeIF (Barhoumi et al., 2006).
Isolation of the mononuclear cells (PBMC), induction of the secretion of cytokines and assay by sandwich ELISA were carried out in the same conditions as those described in Example 1. Briefly, the monocytes purified from the PBMCs from 3 individuals primed or not with IFN-γ for 12 h, are stimulated by the proteins LeIF (SEQ ID NO: 11) or LeIFK76A (SEQ ID NO: 27) at final concentrations of 10 μg/ml, or by LPS at 1 μg/ml. The culture supernatants are collected after incubation for 24 h for assay of IL-12p70 or 18 h for assay of IL-10 and TNF-α. The levels of the cytokines are determined by sandwich ELISA.
The results presented in
They are comparable to those induced by the soluble recombinant protein LeIF (2564.46±1237.1 pg/ml).
Table 6 shows statistical analysis of the differences observed for the levels of production of IL-12p70 in the culture supernatants of the monocytes stimulated with the proteins LeIF and LeIFK76A. The p values determined by the paired t test are shown.
The results presented in
Table 7 shows statistical analysis of the differences observed in the levels of IL-10 and of TNF-α in the culture supernatants of the monocytes stimulated with the proteins LeIF and LeIFK76A. The p values determined by the paired t test are shown.
All these results show that substitution, in motif I, of the lysine in position 76 with alanine, which cancels the ATPase activity of the protein LeIF, does not affect its activity for inducing the cytokines IL-12p70, IL-10 and TNF-α.
In order to characterize the cytokine inducing activity of the recombinant proteins LeIFΔ25 (protein LeIF of L. infantum after deletion of the first 25 amino-terminal residues that are the most divergent between LeIF and its yeast and mammalian homologs; SEQ ID NO: 21), D1 (25-237; SEQ ID NO: 20), D1+25 (1-237; SEQ ID NO: 19) which corresponds to the NH2 part and D2 (237-403; SEQ ID NO: 25) which corresponds to the COOH part of the protein LeIF of L. infantum, monocytes purified from the PBMCs from five healthy individuals by these various recombinant proteins at final concentrations of 10 μg/ml were stimulated.
The stimulation conditions are identical to those described in Example 1. Briefly, the monocytes purified from the PBMCs from five healthy individuals, primed for 12 h with IFN-γ (3000 U/ml), or not, are stimulated by the soluble protein LeIF and its various domains at final concentrations of 10 μg/ml or by LPS at 1 μg/ml. The culture supernatants are collected after 24 h of incubation for assay of IL-12 or 18 h for assay of IL-10 and of TNF-α. The levels of IL-12, IL-10 and TNF-α are determined by sandwich ELISA.
The results presented in
It should be noted that deletion of the amino-terminal residues 25 does not affect the cytokine IL-12p70 inducing activity of the protein LeIF. In fact, the difference between the levels of IL-12p70 induced by the soluble protein LeIF (3805.9±1069.1 pg/ml) and the protein LeIFΔ25 (2921.95±905.78 pg/ml) is not significant (p>0.05, cf. Table 8). The domains D1+25 (1-237) and D1 induce the production of levels of IL-12p70 comparable to those induced by the protein LeIFΔ25 and significantly higher than those induced by the domain D2 (1482.99±596.89 pg/ml; p<0.05, Table 8).
The difference between the levels of IL-12p70 induced by the domain D1 and those induced by the domain D2 of the protein LeIF may be due to the difference in the number of DEAD-box motifs between the two domains. In fact, domain D1 contains 6 motifs (Q, Ia, Ib, I, II and III) and domain D2 contains the 3 motifs: IV, V and VI. This suggests that the DEAD-box motifs might play a role in the activity of inducing cytokine IL-12p70 by the protein LeIF.
Table 8 shows statistical analysis of the differences observed in the levels of IL-12p70 in the culture supernatants of the monocytes stimulated with the soluble protein LeIF and its various domains. The p values determined by the paired t test are shown.
The results presented in
The protein LeIFΔ25 induces the production of levels of IL-10 (1951.15±408.4 pg/ml) comparable to those induced by the protein LeIF (2067.68±324.69 pg/ml, p=0.4515, cf. Table 9), by the domain D1+25 (1-237) (1477.88±381.92 pg/ml; p=0.0786, Table 9) or by the domain D1 (25-237) (1408.91±269.75 pg/ml; p=0.0786, cf. Table 9). The deletion of the 25 aminoterminal residues does not affect the IL-10 inducing activity of the protein LeIF.
Finally, it should be noted that the domain D2 (237-403) of the protein LeIF induces production of a level of IL-10 (1130.55±184.9 pg/ml) significantly lower than that induced by the protein LeIF (S) or LeIFΔ25 (p<0.05, cf. Table 9) and comparable to that induced by the domain D1+25 (1-237) or D1 (25-237) (p>0.05, cf. Table 9).
Table 9 shows statistical analysis of the differences observed in the levels of IL-10 in the culture supernatants of the monocytes stimulated with the soluble protein LeIF and its various domains. The p values determined by the paired t test are shown.
The results presented in
With the exception of the domain D2, the three other proteins LeIFΔ25, D1+25, D1 induce levels of TNF-α comparable to those induced by the soluble protein LeIF (9305.21±1656.3 pg/ml; p>0.05, cf. Table 10).
Table 10 shows statistical analysis of the differences observed in the levels of TNF-α in the culture supernatants of the monocytes stimulated with the soluble protein LeIF and its various domains. The p values determined by the paired t test are shown.
All these results show that all the domains of the protein LeIF are endowed with activity for inducing IL-12p70, IL-10 and TNF-α. However, a significant drop in this activity is observed for the domain D2. Moreover, deletion of the first 25 aminoterminal residues, which are unique to the protein LeIF of Leishmania, does not seem to affect the activity of the whole protein.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 08290522.5 | Jun 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2009/000665 | 6/5/2009 | WO | 00 | 3/14/2011 |
