EP0880589A2 - Recombinant protein containing a c-terminal fragment of plasmodium msp-1 - Google Patents

Abstract

The invention relates to a recombinant protein fabricated in a baculovirus system, of which the essential constitutive polypeptide sequence is that of a C-terminal fragment of 42 kilodaltons (p42) of the surface protein 1 (protein MSP-1) of the merozoite form of a parasite of the Plasmodium type, particularly Plasmodium falciparum, which is infectious for humans, said p42 fragment being particularly deleted of its region II and, if necessary, also of its region III. Said recombinant protein is applicable to the production of vaccines against malaria.

Description

 RECOMBINANT PROTEIN CONTAINING A C-TERMINAL FRAGMENT OF MSP-1 TDE PLASMODIUM

The invention relates to new active principles of vaccines.

10 derivatives of the major surface protein of merozoite forms of an infectious plasmodium for mammals, in particular humans, more generally known by the designation MSP-1

This protein has already been the subject of numerous studies. It is synthesized in the schizont stage of parasites of the Plasmodium type,

15 in particular Plasmodium falciparum, and is expressed in the form of one of the major constituents of the surface of the merozoites both during the hepatic stage and during the erythrocytic stage of malaria (1, 2, 3, 4). Due to the predominance and conservation in all known Plasmodium species of this protein, it has been suggested

20 that she could represent a candidate for the constitution of malaria vaccines (5, 6)

The same was also true for fragments of this protein, particularly natural cleavage products whose formation is observed, for example during the invasion by the parasite of

25 erythrocytes of the infected host Among these cleavage products, the C-terminal fragment having a molecular weight of 42 kDa (7.8) is noted, which is in turn cleaved again into an N-terminal fragment having a conventional apparent molecular weight of 33 kDa and a C-terminal fragment having a conventional apparent molecular weight of 19 kDa (9)

™ which normally remains attached to the parasite's membrane after modifications of which it is itself the object, via groups of the glycosylphosphatidylinositol (GPI) type (10, 1 1)

It is still found at the early ring stage of the intraerythrocytic development cycle (15, 16), hence the observations that have been made that this 19 kDa fragment could play a role not yet known, but undoubtedly essential in the reinvasive processes. . From this flow the hypotheses already formulated in the past that this protein could constitute a particularly effective target for possible vaccines. It will be understood that the references often made in the following to proteins p42 and p19 derived from a certain type of Plasmodium are understood to relate to the corresponding C-termmal cleavage products of the MSP-1 protein of this Plasmodium, or, by extension, to products containing substantially the same amino acid sequences, obtained by genetic recombination or by chemical synthesis according to conventional techniques, for example by synthesizer of the “Applied System” type or by synthesis on solid phase of the “Merπfield” type. For the convenience of language, references to "recombinant p42" and "recombinant p19" refer to "p42" and "p19" obtained by techniques comprising at least one step of genetic engineering

Faced with the difficulty of obtaining large quantities of parasites for P. falciparum and the impossibility of cultivating P vivax in vitro, it has become evident that the only way to produce a malaria vaccine requires recourse to techniques allowing the use of peptides. or recombinant proteins However, MSP-1 is very difficult to produce entirely because of its large size of around 200 kDa, a fact which led researchers to take an interest in the C-terminal part, the function of which, again unknown, is probably the most important On the title, recombinant proteins relating to the C-terminal part of MSP-1 of P.falciparum, which have been produced and tested in monkeys (12, 40, 41), we will mention

. a p19 fused with a glutathione-S-transferase produced in E coh (40),

• a p42 fused with a glutathione-S-transferase produced in . a p19 fused with a polypeptide derived from a tetanus toxoid and carrying T helper cell epitopes produced in S cerevisiae (12),

. a p42 produced in a baculovirus system (41)

A composition containing the fusion protein p19 with a glutathione-S-transferase produced in E coli in association with alum or liposomes did not have any protective effect on any of the six Aotus nancymai monkeys vaccinated (40)

A composition containing the fusion protein p42 with a glutathione-S-transferase produced in E coli in association with a complete adjuvant of Freund did not exert a protective effect in the two types of Aotus monkeys (A nancymai and A vociferans) to which they had been administered The protein p19 produced in S cerevisiae exerted a protective effect in two Aotus monkeys type A nancymai (12) On the other hand it did not exert a protective effect in two Aotus monkeys type A vociferans Some researchers ( Chang et al) have also reported immunization tests carried out in rabbits with a recombinant protein p42 produced in a baculovirus system and containing an amino acid sequence in common with P falciparum (18) Thus the latter authors indicate that this recombinant p42 behaves in rabbits in much the same way as the whole recombinant protein MSP-1 (gp195) This protein p42 in association with it's a Freund's complete adjuvant has been the subject of a vaccination trial in a non-human primate susceptible to infection by P falciparum., Aotus, lemunnus grisemembra (40) The results show that 2 out of 3 animals were completely protected and the third, although showing a parasitaemia similar to the controls, had a longer latent period II is nevertheless risky to conclude that the antibodies thus induced in humans are protective against the parasites themselves Remember that there is no There are currently no very satisfactory experimental models in primates for P vivax and P falciparum The Saimin model, which was developed for P falciparum and

P vivax, and the Aotus model for P falciparum, are artificial systems, requiring the adaptation of parasite strains and often the spienectomy of animals to obtain significant parasitaemias. Consequently, the vaccination results from these models can only have limited predictive value for humans

Besides that one can therefore wonder about what would be a real rate of vaccination likely to be possibly obtained with such recombinant proteins, the presence in p42 from Plasmodiums of the same species, and more particularly in p33 corresponding, hypervaπable regions could make random in many cases the immunoprotective efficiency of the antibodies induced in people vaccinated with a p42 from a Plasmodium strain against infection by other strains of the same species (13 ) The significant polymorphism of the central regions of p42 could even play a significant role in the immune escape, often observed for this type of parasites

However, it turns out that the p42s originating from various infectious Plasmodiums for humans comprise hypervaπable regions mainly concentrating in their regions III, and even more in their respective regions II see the publication by Longacre, S (13) in which the p42 sequences of P cynomolgy, P vivax (Belem) and P vivax (Sal-I) were aligned. The “consensus” sequence of FIG. 4 attached, added to the sequences of p42 of these three parasites testifies to this.

Reference will be made to the article by Longacre, S. (13) in which are exposed the conditions under which said regions were determined. It is noted that the attached Figure 4 is nothing other than a reproduction of Figure 1 of Longacre's article. The reader is therefore invited to refer to the legend of this figure. This also shows the relative sizes of the four regions of p42 (region IV corresponding to the sequence of p19) expressed in percentages with respect to the size of the coding sequence of p42 as a whole.

As also follows from FIG. 4 of the present application, the percentages of homology are high between the sequences of regions I of P.cynomolgi and P vivax: 84% in region I, 86% in region IV On the other hand, this percentage of homology decreases sharply in region III (69%) and even more in region II (47%).

A first object of the invention is therefore to provide active ingredients of vaccines derived from the p42 better able to protect ^the host against the immune escape he was above question II is evident that the fragmentation of the p42 which has just been considered can also be extended to P falciparum, the parasite mainly responsible for acute forms of malaria in humans, and this all the more so as the locations of the zones separating regions I, II, III and IV of the constitutive sequences of P.cynomolgi and of the two varieties of P vivax were determined by analogy with corresponding sites, previously identified in P.falciparum, as described by (34) (35)

The invention relates more particularly to vaccine compositions against a parasite of the Plasmodium type infectious for humans, containing as active ingredient a recombinant protein glycosylated or not, whose essential constitutive polypeptide sequence is that

• either p42 from which region II has been deleted and, where appropriate, all or part of region III, • or a part of this fragment as soon as it is also capable of inducing an immune response capable of '' inhibit parasitaemia in vivo by the corresponding parasite,

• either of a peptide immunologically equivalent to this fragment p42 or to the abovementioned part of this fragment, and this recombinant protein further comprising conformational epitopes unstable in a reducing medium and constituting, preferably, the majority of epitopes recognized by human antisera trained against the corresponding Plasmodium

The invention therefore relates more particularly to a recombinant protein, glycosylated or not derived from p42 and containing both the essential parts of region I and region IV defined above for the constitution of immunogenic compositions, in particular vaccines

The above-mentioned recombinant protein, glycosylated or not, optionally also contains the conserved part of region III which is located on the C-terminal side of p33, close to p19, in particular that which extends between amino acids 255 to 273, or more particularly between amino acids 255 to 270 (see numbering of the consensus sequence in FIG. 4)

In other words, all or part of the region III that is poorly preserved can be delated from the N-terminal part of region III.

For the convenience of language, reference will often be made in what follows to a “partially deleted p42” to designate the modified p42, as defined above.

The presence in this active principle of the above conformational epitopes could play an important role in the protective efficacy which it is likely to confer on the vaccinated host. particularly found in the active principles having moreover the other characteristics defined above, when they have been produced in a baculovirus vector system. If necessary, it is mentioned below that by the expression "system of baculovirus vector ”means the whole constituted by the baculovirus type vector itself and the cell lines, in particular insect cells transfectable by a baculovirus modified by a sequence to be transferred into these cell lines with the result of the expression of this sequence transferred Preferred examples of these two partners of the baculovirus system have been described in the article by Longacre et al

(19) It is the same system which was used in the examples which follow It goes without saying that variants, both of baculovirus and of cells infectable by this baculovirus can be used in place of that which has been chosen The unstable nature in a reducing medium of these conformational epitopes can be demonstrated, in particular by the test described later in the examples, in particular in the presence of β-mercaptoethanol

From this point of view, recombinant proteins derived from the recombinant p42 produced by S Longacre et al (14) can be used in such compositions It is recalled that S Longacre et al succeeded in producing a recombinant p1 9 from MSP-1 of P vivax in a baculovirus vector system containing a nucleotide sequence coding for the p19 of Plasmodium vivax, in particular by transfection of cultures of insect cells [line of Spodoptera frugiperda (Sf9)] with baculovirus vectors containing, under the control of the polyhedπne promoter, a sequence coding for the peptide fragments defined below, the sequences of which were placed in the following order in the baculovirus vector used 5'-term fragment of 35 base pairs of the polyhednne signal sequence, from which the methionine codon for initiating the expression of this protein had been mutated (in ATT),

A 5′-terminal nucleotide fragment coding for a peptide of 32 amino acids corresponding to the N-terminal part of MSP-1, including the signal peptide of MSP-1,

• either a nucleotide sequence coding for p19, or a sequence coding for p42 of the MSP-1 protein of Plasmodium vivax, these sequences being also, as the case may be, either provided (“anchored” forms) or lacking (soluble forms) ) 3 ′ end regions of these nucleotide sequences whose extreme C-terminal expression products are reputed to play an essential role in the anchoring of the final protein p19 on the membrane of the parasite,

• 2 TAA stop codons For p42, the sequences derived from the C-term region of

MSP-1 therefore extended from amino acid Asp 1325 to amino acid Leu 1726 (anchored form) or amino acid Ser 1705 (soluble form) and for p19, the sequences extended from l amino acid Ile 1602 to amino acid Leu 1726 (anchored form) or to amino acid Ser 1705 (soluble form), it being understood that the complete amino acid sequences of p42 and p19 whose initial and terminal amino acids have shown in the above stem from the Belem P vivax isolate gene which has been sequenced (20)

Similar results have been obtained by using in the same vector systems nucleotide sequences coding for p19 and p42 of Plasmodium cynomolgi P cynomolgi has a double interest it is a parasitic species very close to P vivax, which is very infectious for the macaque very close to P vivax II can also infect humans We also have access to natural hosts of P cynomolgi, rhesus monkeys and toques monkeys, to test the protective efficacy of P.cynomolgl's MSP-1 in natural systems. The rhesus monkey is considered to be one of the most representative species of immune reactions in humans.

In particular, excellent results have been obtained in vaccination trials carried out in the toque monkey with two recombinant polypeptides: soluble p42 and p19, derived from P. cynomolgi, produced respectively in a baculovirus system and purified on a column of affinity with monoclonal antibodies recognizing the corresponding regions of the native MSP-1 protein The following observations were made ^• the six monkeys immunized with only p19 (three monkeys) and the 19 and p42 together (3 monkeys) all testified to almost sterile immunity after challenge infection The results obtained in the three monkeys immunized with p42 were less significant Two of them behaved like the previous ones, but if the third showed less parasitaemia than immune controls with PBS buffer in the presence of Freund's adjuvant (3 monkeys) or non-immunized (3 monkeys), it was not patent oins

The results of the particularly effective tests carried out in the macaque with recombinant polypeptides produced in a baculovirus system using a p42, in association with a recombinant p19 of P.cynomolgi, establish that recombinant polypeptides containing respectively recombinant p42 from other Plasmodiums must behave in the same way These tests are "more telling" for malaria in humans than the results of tests carried out with P. vivax or P.falciparum in their "artificial hosts".

The recombinant baculovirus proteins, derived from a C-terminal part of MSP-1 (p42), and more particularly the partially deleted p42s, have a very significant antimalarial protective effect in a natural system, which constitutes the evaluation model for the most representative protective effect of MSP-1 for humans The protective effect obtained could be all the better if the partially deleted p42 is devoid of the hypervaπable region of the N-terminal part of p42, the effect of which can be deleterious in natural situations in which the vaccinated subject is confronted with significant polymorphism

However, the deletion of region II and of all or part of region III normally leads to better results. It is clear that a person skilled in the art would have no difficulty in producing fusion proteins between regions I and IV of p42. corresponding, or even between a region I and a region IV originating respectively from two p42 originating from two different varieties of Plasmodium II naturally goes without saying that these fusion proteins may also contain linking elements still corresponding to parts of region II , preferably from region III, preferably chosen from among the best conserved. For example, mention will be made of the C-terminal polypeptide sequence of p33, when it is present, comprises less than 50 amino acid residues, or even even less than 35, or even less than 10 amino acid residues

However, the polypeptide sequence of the partially deleted p42 protein may not contain the entire sequence coding for p19 (or of the region IV), naturally provided that the latter retains the capacity to induce protective antibodies against parasite In particular the aforesaid “part of fragment has a molecular weight of 10 to 25 kDa, in particular of 10 to 15 kDa Preferably, this part of polypeptide fragment contains at least one of the two regions EGF (abbreviation of the English expression "Epidermal Growth Factor")

Naturally, the same observations apply to region I of the recombinant protein according to the invention. It is clear that a person skilled in the art is able to differentiate between the active fragments and those which would cease to be so, in particular of experimentally by producing modified vectors containing, for example, inserts comprising parts of p42 and in particular of deleted p42, of different lengths, respectively produced from the coding sequence for p42, if appropriate, partially deleted, by reaction with appropriate restriction enzymes, or alternatively exonucleolytic enzymes which would have been kept in contact with the fragment coding for the initial p42, if necessary, partially deleted for variable times; the capacity of the expression products of these inserts in corresponding eukaryotic cells, in particular insect cells, transformed by the corresponding modified vectors, to exert a protective effect, which can then be tested, in particular under the experimental conditions which will be described more away, about the examples. In particular, the expression products of these inserts must be capable of inhibiting a parasitaemia induced in vivo by the entire corresponding parasite.

Likewise, the invention includes all immunogenic or even vaccinating compositions in which the essential constitutive polypeptide sequence of the active principle would consist of a peptide capable of inducing an immunological response of cell and / or humoral type equivalent to that produced by the protein. partially deleted as defined above, since the addition, deletion or substitution in its sequence of certain amino acids by others would not result in a significant modification of the capacity of the peptide thus modified hereinafter called "immunologically equivalent peptide" - also to inhibit the above parasitaemia

Partially deleted p42 can naturally also be associated, whether on the N-terminal side or on the C-Terminal side or via a peptide bond, with another fragment of plasmodial protein having a vaccinating potential such as for example a protein (“Duffy Bmding Protein” by P vivax (29) or EBA-175 by P. falciparum (30) and (31) whose region is specifically rich in cysteine), provided that is not altered, on the contrary amplified, its capacity to inhibit a parasitaemia normally introduced in vivo by the corresponding parasite

The fragment coding for the partially deleted p42 or a part thereof, may also contain, upstream from its N-terminal end, an even different peptide sequence, for example a C-terminal fragment of the signal peptide of the protein MSP- 1. This sequence preferably comprises less than 50 amino acids, for example from 10 to 35 amino acids

These observations extend in the same way to partially deleted p42 from other Plasmodium, in particular

P.falciparum, the dominant species of parasites, responsible for one of the most serious forms of malaria

However, the techniques recalled above for the production in a baculovirus system of a recombinant p42 originating from P. vivax or P. cynomolgi are difficult to transpose as such to the production of a recombinant p42 from P. falciparum with a satisfactory yield. , if only to obtain appreciable quantities allowing the carrying out of immunoprotection tests.

The invention also provides a method which largely remedies this difficulty. It also becomes possible to obtain much higher yields of p42 of P.falciparum - and other plasmodiums when similar difficulties are encountered - by putting in place implements a synthetic nucleotide sequence of substitution for the natural nucleotide sequence coding for the p42 of Plasmodium falciparum in an expression vector of a baculovirus system, this synthetic nucleotide sequence coding for the same p42, but being characterized by a proportion of nucleotides G and C higher than in the natural nucleotide sequence

It is clear to those skilled in the art that this result can be obtained by taking advantage of both its ability to synthesize DNAs by nucleotide synthesis and to choose from the possibilities offered by the code. genetic, to substitute, whenever the genetic code authorizes it, synthetic codons having higher C and / or G contents than the natural codons which, within the native sequences coding for the corresponding native p42, code for the same amino acid. It goes without saying that the same observations extend their effects to p42 whose sequences have been partially deleted as defined above.

In other words, the invention stems from the discovery that the expression in a baculovirus system of a nucleotide sequence coding for a p42, partially deleted or not, was apparently linked to improved compatibility of the successive codons of the nucleotide sequence to express with the "cellular machinery" host cells transformable by baculoviruses, like what is observed for the natural nucleotide sequences normally contained in these baculoviruses and expressed in infected host cells, hence the bad expression, if not sometimes the complete absence of expression of a nucleotide sequence native to P falciparum; hence also a possible explanation for the more efficient expression observed of p42 of P. vivax in a baculovirus system by Longacre et al. (19) and, as the inventors have also observed, of the sequence of P.cynomolgi from the corresponding native p42 nucleotide sequences, due to their much higher relative contents of nucleotide G and C than that of the native nucleotide sequences encoding p42 of P.falciparum. The invention therefore also relates, more generally, to a modified vector of the recombinant baculovirus type containing, under the control of a promoter contained in this vector and capable of being recognized by cells transfectable by this vector, a first nucleotide sequence coding for a signal peptide exploitable by a baculovirus system, characterized by a second nucleotide sequence downstream of the first, also under the control of this promoter and coding for a peptide sequence nevertheless comprising in its own constituent sequence, that:

• either a peptide fragment coding for p42 or a p42 from which region II and, where appropriate, all or part of region III have been deleted; • either of part of this peptide fragment when the expression product of the second sequence in a baculovirus system is also capable of inhibiting a parasitaemia normally induced in vivo by the corresponding parasite;

• either of an immunologically equivalent peptide derived from the above C-terminal peptide fragment (p19) or from the said part of peptide fragment by addition, deletion or substitution of amino acids not causing a significant modification of the capacity of this peptide immunologically equivalent to inducing an immunological response of the cellular and / or humoral type similar to that produced by this peptide fragment p19 or to the abovementioned part of this fragment, and said nucleotide sequence having, where appropriate, a content of nucleotides G and C of between 40 and 60%, preferably at least

50% of the totality of the nucleotides of which it is made up. This sequence can be obtained by construction of a synthetic gene in which the natural codons have been changed by codons rich in G / C without their translation being modified (maintenance of the peptide sequence)

In this case, said nucleotide sequence, provided by synthetic DNA, can have at least 10% of codons modified with respect to the sequence of the natural gene or cDNA while retaining the characteristics of the translated natural sequence, that is to say -to say the maintenance of the sequence in ammo-acids

However, it is not excluded that this content of nucleotides G and C could be further increased, since the resulting modifications in the amino acid sequence of the recombinant peptide -or immunologically equivalent peptide-product would not lead to a loss of the immunological or even protective properties of the recombinant proteins formed, in particular in the tests which will be illustrated below

These observations apply naturally to other Plasmodium infectious to humans, in particular since native nucleotide sequences coding for the corresponding p42s, possibly partially deleted, would have T and A nucleotide contents which are difficult to reconcile with a efficient expression in a baculovirus system The sequence coding for the signal used may be that normally associated with the native sequence of the Plasmodium concerned. It may also be from another Plasmodium, for example P vivax or P cynomolgi or from another organism. if it is capable of being recognized as a signal in a baculovirus system The sequence coding for p42 or a part thereof within the vector considered is, if appropriate, devoid of the anchoring sequence of the protein native to the parasite from which it originates, in which case the expressed protein is generally excreted in the culture medium (soluble form) The invention also relates to the vectors in which the coding sequence contains the 3 'terminal end sequence coding for the hydrophobic C'-terminus sequence of p19 and which is normally involved in induction of the anchoring of the native protein to the cell membrane of the host in which it is expressed This 3'-terminal end region may moreover be heterologous with respect to the sequence coding for the rest of the corresponding p42, for example correspond to the 3'-terminal sequence originating from P vivax or from another organism since it codes for an anchoring sequence for all of the recombinant protein produced at the membrane of the cell host of the baculovirus system used As an example of such anchor sequences, the GPI of the CD59 antigen expressible in insect cells of the spodoptera frugiperda type (32) or the GPI of a human protein CD14 (33)

The invention naturally also relates to recombinant proteins, these proteins comprising conformational epitopes recognized by human sera formed against the corresponding Plasmodium

In general, the invention also relates to any recombinant protein of the type indicated above, as soon as it comprises conformational epitopes such as produced in the baculovirus system, in particular those which are found to be unstable in a reducing medium.

The invention naturally relates to said recombinant proteins, whether in the so-called soluble form or in the form provided with an anchoring region, in particular to the cellular hosts used in the baculovirus system. The invention also relates to any conjugation product between a partially deleted p42 or p42 as defined above, on the one hand, and a carrier molecule - for example a polylysine - alanine - usable for the production of vaccines, on the other hand, by intermediary of covalent bonds or not The vaccinating compositions using them also form part of the invention

The invention also relates to the vaccine compositions using these recombinant or conjugated proteins, including, moreover, the proteins derived from Plasmodium vivax.

Also forming part of the invention are the compositions in which the aforementioned recombinant proteins are associated with an adjuvant, for example an alum. The recombinant proteins comprising the extreme C-terminal region permitting their anchoring to the membrane of the cells in which they are produced are advantageously used in combination with lipids capable of forming liposomes and suitable for the production of vaccines Without having to be limited thereto, recourse may be had to the lipids described for this purpose by example in the work entitled “Liposomes technological, biological and pharmacological aspects” by J Delattre et al, INSERM edition, 1993

The presence of the anchoring region in the recombinant protein, whether it is a homologous or heterologous anchoring region with respect to the vaccinating part proper, is likely to favor the production of cytophilic antibodies, in particular of the lgG _2a and lgG _{2b type} in mice which could have a particularly high protective activity, to the point that one could dispense with associating the active principles of vaccines thus constituted with adjuvants other than the lipids used for the constitution of the liposomal forms II would be an important advantage there, since the liposomes can be lyophilized under conditions allowing their storage and their transport, without cold chains being then essential Other characteristics of the invention will become apparent during the following description of examples of recombinant proteins involving recombinant proteins whose activ sequences es, either contain those of p42, or are limited to that of the corresponding p19 proteins Without these examples being strictly speaking always directly covered by the claims which follow, they nonetheless contribute to establishing the operational nature of the invention which is the subject of the present application

Construction description PfMSP1 _p ι ₉ S (soluble) (soluble p19 from P.falciparum)

The recombinant construction PfMSP1 _p ι ₉ S contains DNA corresponding to the 8 base pairs of the leader sequence and the first 32 amino acids of MSP1 of Plasmodium vivax from MeU to Asp ₃₂ (isolate Belem, Del Portillo et al 1991 PNAS 88 , 4030) FOLLOWED by a GluPhe, due to the EcoRI site linking the two fragments. The whole is followed by the synthetic gene, described in FIG. 1, coding for Plasmodium. IX

falciparum MSP1 _p ι ₉ from Asnι ₆ i ₃ to Serι ₇₀₅ (isolate Uganda-Palo Alto, Chang et al 1988 Exp Parasitol. 67, 1). Construction is completed by two TAA stop codons. This construction gives rise to a recombinant protein which is secreted in the culture supernatant of infected cells.

In the same way and for purposes of comparison, a recombinant construct was produced under conditions similar to those used for the production of p19 above, but by working with a coding sequence consisting of a direct copy of the DNA. correspondent of the P falciparum (FUP) strain described by Chang et al,

Exp Parasit 67, 1, 1989 The copy of this natural gene (ranging from asparagine 1613 to sequence 1705) was formed by PCR from the native gene

The sequences of both the synthetic gene (Bac19) and the "native gene" (PF19) are shown in Figure 1A.

It is noted that 57 codons out of the 93 codons of the native coding sequence for p1 9 originating from P falciparum have been modified (as regards the third nucleotide in 55 of them and the first and third nucleotides in the two remaining codons ) New codons were added at the 5 'end to introduce the signal peptide under the conditions which have been indicated above and to introduce an EcoRI site for cloning, on the one hand, and similarly two were added. stop codons not present in p falciparum p19 for the purpose of obtaining expression termination signals The individualized letters placed respectively above the successive codons correspond to the respective successive amino acids The asterisks ( ^* ) refer to the stop codons The vertical lines underline the nucleotides which are the same in both sequences Construction description PfMSP1 _p ι ₉ A (anchored GPI) (p19 anchored by P.falciparum)

The construction PfMSP1 _p19 A has characteristics of the previous except that the synthetic sequence (Figure 1 B) codes for the MSP1 _p ι ₉ of Plasmodium falciparum (Uganda-Palo alto isolate) from Asn _{! 613} to llβι ₇₂₆ followed by two stop codons of TAA. This construction gives rise to a recombinant protein which is anchored in the plasma membrane of infected cells by a structure of the glycosyl phosphatidyl inositol (GPI) type.

Figure 1C is representative of the sequence of the recombinant protein PfMSP1 pι ₉ S before cutting the signal sequence

Figure 1 D is representative of the sequence of the recombinant protein PfMSP1 _p ι ₉ S after cutting the signal sequence

The amino acids underlined in FIGS. 1 C and 1 D come from the EcoRI site used to join the nucleotide sequences derived from the N-terminal part of MSP1 of P vivax (with signal sequence) and of MSP1 _p19 of P.falciparum

Figure 2 - The recombinant antigen PfMSP1 _p ι ₉ purified by immunoaffinity was analyzed by immunoblotting after SDS-PAGE in the presence (reduced) or absence (not reduced) of B-mercaptoethanol. The samples are loaded on gel after heating to 95 ° C. in the presence of 2% SDS. Under these conditions only bonds of the covalent type

(disulfide bridges) can resist disintegration The left blot was revealed with a monoclonal antibody which reacts with a linear epitope of natural p19 The right blot was revealed with a mixture of 13 human antisera from subjects with immunity acquired from malaria due to Plasmodium falciparum These results show that the recombinant baculovirus molecule reproduces epitopes well conformational in the form of polymers which are recognized in majority by human antiserum

Figure 3 - The soluble PvMSP1 _P42 recombinant antigen (Longacre et al. 1994, op. Cit.) Was incubated for 5 hours at 37 ° in the presence of protein fractions derived from P. falciparum merozoites and separated by isoelectric focusing. the samples were then analyzed by immunoblotting in the presence (reduced) or absence (not reduced) of B-mercaptoethanol Fractions 5 to 12 of isoelectric focusing, as well as two total extracts of merozoites made in the presence (Tex) or absence (T) of detergent were analyzed The immunoblot was revealed with monoclonal antibodies specific for MSP1 _p42 and _pl9 of P. vivax The results suggest that there is a proteolytic activity in the merozoites of P.falciparum which can be extracted in detergent . The digestion of p42 in certain fractions seems to cause a polymerization of the digestion products (p19), this polymerization is probably linked to the formation of disulfide bridges since in the presence of B-mercaptoethanol, the high molecular weight forms disappear in favor of a molecule of around 19 kDa (Tex-R) The polymerization of p19 observed in these experiments could therefore be an intrinsic property of this molecule in vivo

Construction description PcMSP1 _p ι ₉ S (soluble) (p19 soluble from P.cynomolgi)

The DNA used for the above-mentioned construction was obtained from a clone of the Plasmodium cynomolgi ceylonesis strain (22-23) This strain was maintained by successive passages in its natural host (Macaca smica) and cyclic transmissions through mosquitoes (27) Blood parasites were obtained from infected monkeys at the mature schizont stage when the parasitaemias reached a level of 5% They were then purified according to the methods described in (25) The DNA was then extracted as described in (26 ) A 1200 base pair fragment was then produced using the PCR reaction using the oligonucleotides underlined in FIG. 4 and originating from P vivax The 5 'oligonucleotide included an EcoRI restriction site and the oligonucleotide 3 'two synthetic TAA stop codons FOLLOWED by a Bglll restriction site This fragment was introduced by ligation and via these EcoRI and Bglll sites into the plasmid pVLSV ₂₀ o already containing the signal sequence of the protein MSP-1 of P vivax (19) The new plasmid (pVLSV ₂₀ oC ₄₂ ) was used for the analysis of DNA sequences

The sequences of P cynomolgi and corresponding sequences of P vivax have been aligned The black arrows designate the presumed primary and secondary cleavage sites They have been determined by analogy with the known sites in P falciparum (27, 28) Vertical lines and horizontal arrows locate the limits of the four regions which have been studied Region 4 corresponds to the sequence coding for p19 of P cynomolgi Glycosylation sites are boxed and the conserved cysteines are underlined In the lower part of Figure 4 are indicated identity percentages between the two isolates of P vivax and P cynomolgi

The PcMSP1 _p ι ₉ S recombinant construct contains DNA corresponding to the 8 base pairs of the “leader” sequence and the first 32 amino acids of MSP1 from Met Plasmodium vivax! a Asp ₃₂ (Belem isolate, Del Portillo et al 1991 PNAS 88, 4030) FOLLOWED by a GluPhe, due to the EcoRI site making the connection of the two fragments The whole is followed by the sequence coding for the MSP1 _p ι ₉ of Plasmodium cynomolgi ( Ceylon strain) from Lys ₂₇₆ to Ser ₃₈ o Construction is terminated by two TAA stop codons This construction gives rise to a protein ^• >?

recombinant which is secreted in the culture supernatant of infected cells

Purification of the recombinant protein PfMSP1 p19 by immunoaffinity chromatography with a monoclonal antibody specifically recognizing p19 of Plasmodium falciparum.

The chromatography resin was prepared by binding 70 mg of a monoclonal antibody (obtained from a G17 12 hybπdome deposited at the CNCM (Paris, France) on February 14, 1997 under the n ° l-1846, this G17 hybπdome 12 was constructed from myeloma X63 Ag8 653 producing

IgG 2a / k recognizing p19 from P falciparum) to 3g of active CNBr-Sepharose 4B (Pharmacia) by standard methods detailed in the instructions for use provided by Pharmacia Culture supernatants containing soluble pMSMS1 p19 were incubated in batch with the chromatography resin for 16 hours at 4 ° C. The column was washed once with 20 volumes of 0 05% NP40, 0 5M NaCl, PBS, once with 5 volumes of PBS and once with 2 volumes of 10 mM sodium phosphate, pH 6 8 Elution was carried out with 30 ml of 0 2 M glycine, pH 2 2 The eluate was neutralized with 1 M sodium phosphate, pH 7 7 and then concentrated by ultrafiltration and dialysis against PBS For the purification of the anchored PfMSP1 p19, all the washing and elution solutions contained an additional 0 1% 3- (Dιmethyl-dodecylammonιo) -propane sulfonate (Fluka)

Plasmodium vivax recombinant MSP1 vaccination trial (p42 and p19) in the squirrel monkey Saimiri sciureus.

This vaccination trial was carried out in male Saimin sciureus boliviensis 2 to 3 years old, not splenectomized Three monkeys were injected 3 times by intramuscular route at 3 weeks apart with a mixture of approximately 50 to 100 μg each, PvMSP1 _p42 and _p ιg soluble recombinant (19), purified by immunoaffinity. The complete and incomplete Freund's adjuvant was used as follows - 1 ^st injection: 1: 1 FC A / FIA; ^2nd injection 1: 4 FCA / FIA; ^3rd injection: FIA. These adjuvant compositions were subsequently mixed 1: 1 with the PBS antigen. The five monkeys

5 controls received the glutathione-S-transferase (GST) antigen produced in E.coli according to the same protocol. The challenge infection was carried out by injecting 2.10 ⁶ infected red cells with a suitable strain of Plasmodium vivax (Belem) 2 5 weeks after the last injection Protection was evaluated by determining the daily parasitaemias in

I O all animals by examining stained smears with giemsa.

The curves in Figure 5 are representative of the variation in parasitaemia measured in number of red blood cells parasitized per microhtre of blood (on the ordinate axis on the logarithmic scale) as a function of the time elapsed after infection (in days ) Curve A corresponds to

15 mean values observed in the three vaccinated monkeys, curve B, the mean values in five control monkeys

From the examination of the figure follows a very strong reduction in parasitaemia under the effect of vaccination

20 Vaccination test of recombinant MSP1 of Plasmodium cynomolgi (p42 and p19) in the toque monkey, Macaca sinica.

Fifteen captured monkeys were used as follows: (1) 3 animals injected with 100 mcg PcMSP1 _p42 soluble, 3 animals injected with 35 micrograms (1 ^st injection) or 50 ug (2 ^nd and 3 ^rd injections) PcMSP1 soluble _p19; (3) 3 5 animals injected with a mixture of PcMSP1 _p42 and _p , ₉ ; (4) 3 animals injected with the adjuvant plus PBS, (5) 3 animals not injected. The complete and incomplete Freund's adjuvant was used according to the protocol described above. The injections were made intramuscularly 4 weeks apart. The challenge infection was made by injecting 2.10 ⁵ red blood cells infected with Plasmodium cynomolgi 4 weeks after the last injection Protection was assessed by determining daily parasitaemias in all animals by examining parasitaemias with giemsa Parasitaemias were classified as negative only after counting 400 smear fields Parasitaemias are expressed as a percentage of parasitized red blood cells

Figures 6A-6G are illustrative of the results obtained In each of them appear the parasitaemias (expressed as percentages of parasitized red cells on the ordinate axis on the logarithmic scale) observed in the test animals as a function of the times after infection (in days on the x-axis)

The results concern

In FIG. 6A, unvaccinated control animals,

FIG. 6B relates to animals which had received a saline solution additionally containing the Freund's adjuvant, • FIG. 6C is a superposition of FIGS. 6A and 6B, with the aim of showing the relative results resulting from the administration of Freund's adjuvant to animals (the variations are obviously not significant),

FIG. 6D provides the results obtained after vaccination with p42,

FIG. 6E relates to animals vaccinated with only p19,

Finally, FIG. 6F relates to the animals vaccinated with a mixture of p19 and p42

P42 certainly induces a certain level of protection But as shown in FIGS. 6E and 6F, the protection conferred by the recombinant p19 according to the invention is considerably improved

It can be hypothesized that the improvement in protection results from a secondary cleavage of p42 which is accompanied by the release of free cysteine which subsequently forms intermolecular disulfide bridges giving rise to very p19 multimers. characteristics of this form in the recombinant proteins of the three species tested

The figures used to develop the graphs (6A-6F) are specified in Figure 6G

P.cynomolgilsïnge toque vaccination trial; second challenge infection in monkeys vaccinated with p19 alone and controls (Figures 8)

Six months later without further immunization, the 3 monkeys who received MSP-1 p19 alone with FCA / FIA (Figure 6E) and the 3 monkeys who received saline containing Freund's adjuvant (Figure 6B) as well as 2 new naive unvaccinated monkeys were re-infected by challenge by injection of 1 1 0 red blood cells infected with Plasmodium cynomolgi Protection was assessed by determining daily parasitaemias in all animals by examining smears with giemsa Parasitaemias were classified as negative only after counting 400 smear fields Parasitaemias are expressed as a percentage of parasitized red blood cells (the figures used to draw charts 8A-C are specified in Figure 8D) The six immunized animals which had undergone a challenge infection six months previously had no detectable parasitaemia except for 1 animal in each group which presented 0 008% parasitaemia ant 1 day (Figures 8A and 8B) The two naive controls show a classical parasitaemia with a maximum of 0 8% and for 21 days (Figure 8C) So the 3 animals vaccinated with MSP-1 p19 were also protected six months more later than the 3 controls who presented a classic complete infection after the first challenge infection, despite an absence or a very weak parasitaemia after the first challenge infection These results suggest that the duration of protection of p19 is at least six months Vaccination trial with p19 in combination with alum in the P.cynomolgils ^' inge toque system (Figures 9)

The previous positive protection results were obtained using Freund's complete (FCA) or incomplete (FIA) adjuvant

However, the only adjuvant currently accepted in humans is alum. For this reason, we have carried out a vaccination trial with M cp-1 p1 9 of P cynomolgi in the toque monkey in the presence of alum as an adjuvant. Six captured monkeys were used as follows (1) 3 animals injected with 4 doses of 50 mg MSP-1 p19 recombinant P cynomolgi with 20 mg of alum (2) 3 animals injected 4 times with physiological water and 10 mg of alum Injections were done intramuscularly 4 weeks apart Test infection was done

5 by injecting 2 10 red cells infected with P cynomolgi 4 weeks after the last injection Protection was assessed by determining the daily parasitaemias in all animals by examining smears with giemsa The parasitaemias were classified as negative only after counting 400 fields of smears Parasitaemias are expressed as a percentage of parasitized red blood cells The results of this experiment are as follows 2 of the 3 monkeys immunized with recombinant p19 in alum had approximately 30 times less total parasitaemia during the duration of the infection (Figures 9A and 9B) that the 3 control monkeys immunized with physiological water and alum (Figure 9D) after the challenge infection The third monkey immunized with p19 (Figure 9C) was not very different from the controls For l vaccination trial

Plasmodium cynomolgi p1 9 in the toque monkey, Macaca sinica, described in Figure 9, the figures used to draw the graphs (9A-9D) are specified (Figure 9E) Although these results are a little less spectacular than the previous ones (Figures 6, 8) is the first time significant protection is observed for recombinant alum MSP-1

Figure 10 Vaccination test with a recombinant p19 of Plasmodium falciparum in the squirrel monkey

Twenty Saimm sciureus guyanensis monkeys (squirrel monkey) of approximately 3 years reared in captivity were used as follows (1) 4 animals injected with 50 mg of Pf MSP-1 p19 soluble in the presence of adjuvant

Freund as follows 1 ^st injection 1 1 FCA / FIA, 2 ^nd injection 1 4 FCA / FIA 3 FIA injection These adjuvant compositions were then mixed with the antigen in PBS 1 1, (2) 2 control animals received the Freund's adjuvant as described for (1) with PBS only, (3) 4 animals injected with 50 mg of Pf MSP-1 p19 soluble in the presence of 10 mg of alum (Alu-Gel-S, Serva), (4) 2 control animals received 10 mg of alum with PBS only, (5) 4 animals injected with approximately 50-100 mg Pf MSP-1 p19 anchored GPI reconstituted into liposomes as follows 300 mmol of cholesterol and 300 mmol of phosphatidyl cholme were dried under empty and resuspended in 330 mM N-octylglucoside in PBS with 1 4 mg of Pf MSP-1 p19, GPI This solution had been dialyzed against PBS with Bio-Beads SM-2 adsorbent (Bio-Rad) and the liposomes thus formed were concentrated by centrifugation and resuspended in PBS The first injection was made with fresh liposomes maintained at 4 ° C. and the 2 ^nd and 3 ^rd injections were made with liposomes having been frozen for preservation, (6) 2 animals injected with control liposomes made in the same way in the absence of the p19 antigen, GPI, as described for (5), (7) 2 animals injected with physiological water Three injections were made intramuscularly 4 weeks apart The challenge infection was made by injecting 1 10 red blood cells infected with Plasmodium falciparum The protection was evaluated by determining the daily parasitaemias in all the animals by examining the smears with giemsa. The parasitaemias are expressed as a percentage of parasitized red blood cells. The results of this vaccination test are set out in Figures 10, A- G

Groups immunized with p19 by Freund's adjuvant or by liposome demonstrated parasitaemias similar to the control groups after a challenge infection (an animal (number 29) vaccinated with p19 with Freund's adjuvant died a few days after infection with test for reasons unrelated to vaccination (heart attack)) Irregularities in the administration of the antigen in these 2 groups (poor Freund emulsion, frozen liposomes) do not allow a complete evaluation of the significance of these results In the alum group 2 animals showed total parasitaemias for the duration of the infection approximately 4 times less important than the controls, I animal approximately 3 times less important and 1 animal was similar to the controls This experiment is a little difficult to interpret because of variability in controls, likely due to the parasite strain used for the challenge infection that did not would not have been sufficiently adapted to the non-spenectomized Saimin model developed only recently in Cayenne Nevertheless, the real, albeit imperfect, effect with alum is encouraging since our antigens seem to be the only recombinant versions MSP-1 of P falciparum which, for the moment, have demonstrated a certain effectiveness in association with alum

Vaccination trial with a recombinant p19 of Plasmodium falciparum in the squirrel monkey (same trial as figure 10).

Monkeys bred in captivity were injected with 1 ml of moculum by the intramuscular route 2 times at 4 weeks apart as follows (1) 4 animals injected with 50 μg of soluble PfMSP1 p19 in the presence of Freund's adjuvant as follows l® ^re injection January 1 FCA / FIA, ^2nd ignition 1 4 FCA / FIA, and mixed by the sequence 1 1 with the antigen in PBS, (2) 4 animals injected with 50 micrograms of PfMSP1 ρ19 soluble in the presence of 10 mg of alum, (3) 4 animals injected with approximately 50 μg of PfMSP1 p19 anchored GPI reconstituted in liposomes compounds 1 1 in cholesterol molar and phosphatidyl cholme The animals were bled 17 days after the second injection

The red blood cells originating from a squirrel monkey with 30% parasitemia due to P falciparum (with mostly ripe forms) were washed in PBS and the residue was diluted 8 times in the presence of 2% SDS and

2% dithiothreitol and heated to 95 ° before being loaded onto a 7% polyacrylamide gel (separation gel) and 4% (stacking gel) (top of the gel) After transferring the immunoblot analysis to nitrocellulose ( immunoblot) was done with antisera as follows (1) pool of antisera of the 4 monkeys vaccinated with PfMSP1 p19 soluble in adjuvant of

Freund diluted to the twentieth, (2) pool of antisera of the 4 monkeys vaccinated with PfMSP1 p19 soluble in adjuvant alum diluted to the twentieth, (3) pool of antisera of the 4 monkeys vaccinated with PfMSP1 p19 anchored in liposomes diluted to the twentieth, (4) ) the monoclonal antibody, which reacts with a linear epitope of PfMSP1 p19, at 50 μg / ml, (5) pool of SHI90 antisera originating from about twenty monkeys repeatedly infected with P falciparum and which have become refractory to any subsequent infection of P falciparum, diluted to five hundredths, (6) pool of antisera of naive monkeys (never exposed to P falciparum) diluted to twentieth The results show that the 3 pools of antisera of monkeys vaccinated with PfMSP1 p19 react significantly and specifically with very high molecular weight complexes (found diffuse in the stacking gel) and present in parasite extracts containing more mature forms These results support the hypothesis of the presence of a specific aggregation of MSP1 p19 m vivo comprising epitopes which are reproduced in the recombinant molecules PfMSP1 p19 synthesized in the baculovirus system, in particular those in the form of an ohgomer

FIG. 7 also illustrates these results. It relates to immunoblots produced on gel. The first three columns of the gel illustrate the m vivo response of monkeys to injections of p19 [(1) with Freund's adjuvant, (2) with l alum, (3) in the form of a liposome) and in particular the existence of high molecular weight complexes confirming the hypothesis of m vivo aggregation of p19 in the form of an oligomer, specific to the stage of maturation (when p42 is cut in p19 and p33)

This vaccination trial also includes a third injection identical to the previous ones. The injection with Freund's adjuvant only includes FIA

Figure 7B The data for this figure are derived from the P falciparum I squirrel monkey vaccination trial (Figure 10 below) The figures correspond to the individual monkeys noted in Figure 10 The techniques and methods for this figure are the same as for Figure 7 except that the individual antiserum of each monkey noted was tested after three injections on the day of the challenge infection and the SHI antiserum was diluted 1,250 The results show that the antiserum of the 4 monkeys vaccinated with p19 and alum react significantly and specifically with very high molecular weight complexes while monkeys from other groups vaccinated with p19 and Freund's adjuvant or liposomes show only a low reactivity with these complexes Since the monkeys vaccinated with p19 and alum were also the best protected, this reactivity with high molecular weight complexes seems to indicate a protective effect, and this a despite that one monkey in the alum group was not protected from controls and another was only partially protected The invention naturally relates to other applications, for example those set out below in relation to some of the examples, which have no limiting character.

Therapeutic

The recombinant molecules according to the invention can be used to produce specific antibodies which can be used by passive transfer for the purpose of therapy adapted to severe malaria due to P falciparum with risk of mortality.

Diagnostic

The recombinant molecules PvMSP 1 p42 and PvMSP1 p19 and according to the invention, derived from baculovirus can and have been used to produce specific monoclonal antibodies muπns These antibodies, in combination with polyclonal anti p42 antisera from another species such as rabbit or goat, may be the basis of a semi-quantitative diagnostic test for malaria and capable of distinguishing between malaria due to P falciparum, which can be fatal, and malaria due to P vivax, which does not is generally not lethal The principle of this test would be to trap and quantify any MSP1 molecule containing the p42 part in the blood

In this context, the advantages of recombinant p42, and in particular partially deleted recombinant p42, are as follows (i) they are both well conserved within the same species and sufficiently divergent between different species to allow production easily species-specific reagents, under conditions whereby antibodies derived from different Plasmodiums can be produced, in particular against P falciparum and P vivax which do not give rise to cross-reactions, (n) since the recombinant p42 molecules derived from baculovirus seem to reproduce more the native structure of the corresponding native proteins, the antibodies produced against these proteins would be well suited for diagnostic use The microorganisms identified below were deposited according to rule 6 1 of Treaty of Budapest on 01 February 1996, under the following numbers

Identification references Registration numbers PvMSP1 p19A -1659

PvMSP1 p19S -1660 PfMSP1 p19A -1661 PfMSP1 p19S -1662 PcMSP1 p19S -1663

The invention also relates to the use of these antibodies, then preferably fixed beforehand on a solid support (for example for affinity chromatography), for the purification of peptides of the p19 type initially contained in a mixture. The purification then involves a bringing this mixture into contact with the antibody, dissociation of the antigen-antibody complex and recovery of the purified p19 peptide

When it is said in the claims which follow that p42 is deleted from a part or parts which are the least well conserved of region III, these are preferably regions having at least 10 amino acids and the degree of which storage in P vivax, P cynomolgi and P falciparum is less than 70% (less than seven of 10 identical amino acids, when aligned

The polyclonal and monoclonal antibodies of the present invention presented as recognizing p42, are preferably those which recognize more specifically regions other than region IV, to exclusion of region IV itself Preferably they recognize region I of p42

The invention also relates to the hybπdomes secreting specific antibodies selectively recognizing the p42 of an MSP-1 protein of the merozoite form of a parasite of the Plasmodium type infectious for humans other than Plasmodium vivax and does not recognize

Plasmodium vivax

In particular, these hybπdomes secrete antibodies which do not recognize the p42 of Plasmodium vivax or which specifically recognize the p42 of Plasmodium falciparum

The invention also relates to a hybπdome characterized in that it produces a monoclonal antibody which specifically recognizes the p42 of P vivax and of P cynomolgi A hybπdome F10-3 was constructed from the myeloma X63 Ag8 653 producing IgG 2b / k recognizing the p42 of P vivax

The invention also relates to vaccine compositions, also comprising mixtures of proteins or fragments, in particular mixtures of the type - p42 of P falciparum and p42 of P vivax,

- p42 of P falciparum and p19 of P falciparum,

- p42 of P vivax and p19 of P vivax,

- p42 of P falciparum, p19 of P falciparum and p19 of P vivax, p42 of P vivax

In all of the foregoing compositions, p42 is, where appropriate, devoid of its most hypervaπable regions.

For example, its region which corresponds to that of the fragment p19 normally included in p42, is itself partially deleted, this region comprising at least one of the two EGF regions normally contained in this p19.

Or it is devoid of region II, or even also of the N-terminal region of region III or all of region III. The invention is not limited to the production of human vaccines

It applies just as much to the production of veterinary vaccine compositions using the corresponding proteins or antigens derived from infectious parasites for mammals and produced under the same conditions. It is indeed known as infections of the same type, babesiosis, also appear in cattle, canines and horses one of the antigens of the Babesia species presents a strong conformational homology (in particular the two domains "EFG-like" and richness in cysteism) and functional with a protein part of MSP-1 [(36 ), (37) and (38)]. Examples of veterinary vaccines using a soluble antigen against such parasites have been described (39)

It goes without saying that the p42s used in these mixtures can also give rise to all the modifications mentioned above, when they were taken into consideration in isolation.

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270-273 (36) G Bourdoiseau et al (May 1995) "Bovine babesiosis", Le point veterinaire, vol 27, n ° 168 (37) P Bourdeau et al (May 1995) "Canine babesiosis a Babesia canis", The veterinary point, vol 27, n ° 168

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Claims

1. Recombinant protein whose essential constitutive polypeptide sequence is that ^•

• either a 42-kilodalton C-terminal fragment (p42) of surface protein 1 (protein MSP-1) of the merozoite form of a parasite of the Plasmodium type and infectious to humans, and from which have been deleted region II and, where appropriate, part or parts of region III, in particular the parts which are less well preserved,

• or a part of this fragment when it is also able to inhibit a parasitaemia normally induced in vivo by the corresponding parasite;

Either a peptide capable of inducing an immunological response of the cellular and / or humoral type equivalent to that produced by this fragment p42 or to the abovementioned part of this fragment, and this recombinant protein comprising if necessary conformational epitopes unstable in reducing medium and constituting the majority of epitopes recognized by human antisera formed against the corresponding Plasmodium

2. Recombinant protein according to claim 1, characterized in that its region which corresponds to that of the fragment p19 normally included in p42, is itself partially deleted, this region comprising at least one of the two EGF regions normally contained in this p19

3. Recombinant protein according to claim 2, characterized in that the molecular weight of this part of fragment p19 is between 10 and 25 kDa, in particular between 10 and 15 kDa.

4. Recombinant protein according to claim 1, characterized in that it contains both the essential parts of the sequence polypeptide of region I and region IV of p42 and that it lacks region II

5. Recombinant protein according to claim 4, characterized in that its polypeptide sequence is that of p42 from which region II has been deleted.

6. Recombinant protein according to claim 5 from which the N-terminal region of region III or all of region III has also been deleted.

7. Recombinant protein according to any one of claims 1 to 6, characterized in that it also comprises a glycosylphosphatidylmosttol group (GPI) of the type of those which allow anchoring of the p19 fragment whose sequence is normally included in that of p42 to the cell host, in particular a eukaryotic cell, preferably an insect cell infectable by a baculovirus, in which said recombinant protein is expressed

8. Recombinant protein according to any one of claims 1 to 7, characterized in that it is devoid of the extreme hydrophobic C-terminal part which intervenes in the induction of the anchoring of this recombinant protein to the cell membrane of the host in which it is expressed, in particular in a eukaryotic cell, preferably an insect cell infectable by a baculovirus

9. Recombinant protein according to claim 8, characterized in that it is water-soluble

10. Recombinant protein according to any one of claims 1 to 9, characterized in that it contains the aforementioned partially deleted sequence of the p42 of the protein MSP-1 of Plasmodium falciparum or said part of the corresponding fragment

11. Recombinant protein according to any one of claims 1 to 9, characterized in that it contains the sequence partially deleted above from p42 of the protein MSP-1 of Plasmodium cynomolgi or said part of the corresponding fragment

12. Recombinant protein according to any one of claims 1 to 9, characterized in that it contains the aforementioned partially deleted sequence of the p42 of the protein MSP-1 of

Plasmodium vivax or said part of the corresponding fragment

13. Recombinant protein according to any one of claims 1 to 12 characterized in that it is conjugated to a carrier molecule usable for the production of vaccines 14. Vaccinating composition against a parasite of the type

Infectious plasmodium for humans, containing, as active principle, the recombinant protein according to any one of Claims 1 to 13

15. Specific antibody selectively recognizing p42 of an MSP-1 protein of the merozoite form of a parasite of the Plasmodium type infectious for humans other than Plasmodium vivax and does not recognize Plasmodium vivax

16. Specific antibody according to claim 15, characterized in that it does not recognize Plasmodium vivax

17. Specific antibody according to claim 15, characterized in that it specifically recognizes the p42 of P falciparum

18. Specific antibody according to claim 15, characterized in that it specifically recognizes the p42 of P vivax

19. Differential diagnostic method making it possible to distinguish between a parasitic infection due to P wvax, on the one hand, and a parasitic infection due to another plasmodium, on the other hand, characterized by the bringing into contact of an infected biological sample. by a plasmodium with an antibody according to claim 18, on the one hand, and with an antibody according to claim 16 or 17, on the other hand, and the detection according to the case, of the production or not of an immunological reaction

20. Modified vector of the recombinant baculovirus type containing, under the control of a promoter contained in this vector and capable of being recognized by cells transfectable by this vector, a first nucleotide sequence coding for a signal peptide exploitable by a baculovirus system, characterized by a second sequence downstream of the first, also under the control of this promoter and at least part of which codes for the peptide sequence ^•

Or a 42-kilodalton C-terminal peptide fragment (p42) of surface protein 1 (protein MSP-1) of the merozoite form of a parasite of the Plasmodium type, infectious for humans, and the where appropriate, region II has been deleted and, where appropriate also, part or parts of region III, in particular the less well-preserved parts thereof,

Either a part of this peptide fragment when the expression product of the second sequence in a baculovirus system is also capable of inhibiting a parasitaemia normally induced in vivo by the corresponding parasite,

Either of a peptide capable of inducing an immunological response of the cellular and / or humoral type equivalent to that produced by this peptide fragment p42 or to the abovementioned part of this fragment, and said nucleotide sequence further having a G content and C between 40 and 60%, preferably at least 50% of the totality of the nucleotides of which it consists

21. Modified vector according to claim 20, characterized in that the said second polypeptide sequence conforms to that defined in any one of claims 2 to 9

22. Modified vector according to claim 20, characterized in that the second nucleotide sequence is a synthetic sequence

23. Modified vector according to any one of claims 20 to 22, characterized in that the first nucleotide sequence codes for a signal peptide from Plasmodium vivax and normally associated with the MSP-1 protein of this Plasmodium

24. Modified vector according to any one of claims 20 to 23, characterized in that the second nucleotide sequence is devoid at its 3 'terminal end of the hydrophobic C-term terminal sequence which is involved in the induction of anchoring of this recombinant protein to the cell membrane of the host in which it is expressed, in particular in an insect cell infectable with a baculovirus 25. Vector modified according to any one of claims

20 to 24, characterized in that it consists of a modified baculovirus

26. Organism, in particular insect cell of the Sf9 type, transfectable and transfected with the modified vector according to any one of claims 20 to 24

27. Synthetic DNA containing a first nucleotide sequence, at least part of which encodes the peptide sequence

Either a 42-kilodalton C-terminal peptide fragment (p42) of the surface protein 1 (protein MSP-1) of the merozoite form Plasmodium falciparum, infectious for humans and from which, if appropriate, have been deleted region II and, where appropriate also, part or parts of region III, in particular the least well-preserved parts thereof,

Either a part of this peptide fragment when the expression product of this DNA in a baculovirus system is capable of inhibiting a parasitaemia normally induced in vivo by the corresponding parasite

Either of a peptide capable of inducing an immunological response of cell and / or humoral type equivalent to that produced by this peptide fragment p42 or to the abovementioned part of this fragment, and said nucleotide sequence further having a content of nucleotides G and C of between 40 and 60%, preferably at least 50% of the total of the nucleotides of which the above-mentioned synthetic DNA is made up.

28. Synthetic DNA according to claim 27, characterized in that its first nucleotide sequence is devoid at its 3 'terminal end of the sequence coding for the hydrophobic C-terminal end region normally involved in the induction of anchoring of the protein p19, the sequence of which is included in that of p42, to the cell membrane of the host in which it is expressed, in particular in an insect cell infectable with a baculovirus

29. Synthetic DNA according to claim 27 or claim 28, characterized in that the first nucleotide sequence is preceded by a signal nucleotide sequence coding for a signal peptide normally associated with a MSP-1 protein of Plasmodium, homologous or heterologous vis- opposite the main sequence

30. Synthetic DNA according to claim 29, characterized in that the signal sequence comes from P vivax

31. Synthetic DNA according to any one of claims 27 to 30, characterized in that the above first nucleotide sequence includes a 3'-terminal sequence coding for a polypeptide region for anchoring to the cell membrane, said anchoring region binding to the recombinant protein expressed on the surface of the membrane of the cellular host transformed with a vector containing said synthetic DNA, said 3 ′ sequence being homologous to that of the main nucleotide sequence, or heterologous, in particular that derived from P vivax

32. Synthetic DNA according to claim 31, characterized in that the 3'-terminal sequence is derived from P vivax.

33. Synthetic DNA according to any one of claims 27 to 31, characterized in that it lacks said 3'-termιnale sequence

34. Hybπdome secreting monoclonal antibodies having the specificities of the antibodies according to any one of claims 15 to

18

35. Process for the separation of a peptide p42 of given specificity from a mixture of peptides characterized by bringing this mixture of peptides into contact with a corresponding antibody, according to any one of claims 15 to 18, preferably fixed beforehand on an insoluble support, by the subsequent dissociation of the antigen-antibody compound formed and by the recovery of the purified p42 peptide

36. Use of the protein according to any one of claims 1 to 12 for the preparation of an immunogenic composition capable of inducing an immune response against a Plasmodium infection

37. A vaccine composition comprising, as active principles, a mixture of a protein according to any one of claims 1 to 13 and either of the corresponding p19, or of another recombinant protein, of p42 or p19 type, originating of a parasite homologous to that from which the above protein is derived

38. vaccine composition according to claim 37 characterized in that its mixture of active principles is chosen from the following mixtures

- p42 of P falciparum and p42 of P vivax,

- p42 of P falciparum and p19 of P falciparum,

- p42 of P vivax and p19 of P vivax,

- p42 of P falciparum, p19 of P falciparum and p19 of P vivax, p42 of P vivax. p42 being if necessary devoid of its most hypervaπable regions

39. Hybπdome according to claim 34, characterized in that it was deposited at the CNCM under the number 1-1846, on February 14, 1997.